Molecular mechanism of CD98 function
A thesis submitted by
Jae Youl Cho
for the degree of
Doctor of Philosophy
in the University of London
2001
Department of Immunology and Molecular Pathology
University College London
Cleveland Street
London WIT 6JF V ProQuest Number: U643096
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CD98 is expressed on both haematopoietic and non-haematopoietic cells and has been implicated in a variety of different functional role in cell physiology and immunobiology such as amino acid transport, oncogenic transformation and cell adhesion and fusion. Little is known about molecular mechanism of CD98 function, including interaction with other molecules and signalling pathways induced by CD98 activation.
The first study examined the functional interactions between CD98 and other adhesion molecules such as CD29 and CD 147 on the surface of the promonocyte line U937, using a quantitative assay of cell aggregation and blocking monoclonal antibodies to adhesion molecules. Two antibodies to CD 147, an immunoglobulin superfamily member whose function has remained unclear, were also potent inhibitors of the aggregation induced via the ligation of both CD98 and CD29. CD98 blocking mAbs also diminished CD29-induced homotypic aggregation. The results suggest that CD98,
CD29 and CD 147 are functionally associated within a multi-molecular unit which regulates cell aggregation.
To dissect signalling pathways induced by CD98 activation, CD98-mediated U937 homotypic aggregation was further carefully evaluated with pharmacological and biochemical approaches. CD98 ligation induced a selective membrane translocation of the novel PKCô isoform, as an essential step in mediating U937 homotypic aggregation.
CD98-induced PKCô activation mediated activation of MAPK, ERKl/2 and p38, as well as tyrosine phosphorylation. Collectively, CD98 activation may be selectively linked to PKCÔ activation.
The CD98-induced aggregation was negatively regulated by PMA-responsive PKC isoforms because PKC inhibitors and activators enhanced or inhibited CD98-induced intercellular adhesion. PMA also induced the translocation of PKC a, p, and y, suggesting that the PMA-responsive PKC isoforms were conventional isoforms of PKC.
Together, these data provide strong evidence that PMA-responsive conventional PKC may play a key role in the negative regulation in CD98-induced signalling and homotypic aggregation. To my parents, with love Acknowledgements
I gratefully acknowledge the contributions of many people to the completion of this project.
First of all, I would like to greatly thank Professor Benny Chain for his special and superb supervision throughout my research in UCL, I was highly impressed with his constant and tireless encouragement during the turbulent period of my life. Through his intellectual insights and unique personality that I have encountered over the years I have had a chance to get a real feeling of what a genuine scientist is like.
Also, my special thanks go to Professor David R. Katz who has guided and supported me with this project all along. Without him, this thesis would never have existed. His studies being continued until late hours in the Department were the most valuable encouragement to me only to keep up myself working hard.
My thanks are also due to all the people, previous and present, who I have had the pleasure of working with in the laboratory. They include: Paul Free, Ariel Rad, Gabriel
Pollara, Claire Swetman, Remi Creusot, Luci Maccormac, Philippa Newton, Brian de
Souza, Charles Alderman, Christina Madsen, Peter Bunyard, Lucine Lopeze, Nini
Kvirkvelia. They have helped me with reagents and experimental protocols, showing me how to use some instruments, giving me a lot of encouragement and equally having been a source of challenge. I will never forget the valuable and pleasant time when we have shared ideas over a couple of pints of Guiness. My deepest appreciation and thanks go to my parents, Young Hyeon Cho and Cheong Ja
Lee, as well as my parents-in-law, Woo II Kim and Seong Jeom Ha, along with my sister and brother-in-law. He Young Cho and Tae Ho Kim, and my brother and sister-in- law, Jae Phil Cho and Eun Ha Choi, and particularly, my lovely wife, Ae Ra Kim and my son, Seok Ho, for their constant support and encouragement throughout my studies and life in London.
Finally, I am most grateful to God - more than words can possibly say - for having given me a chance to study in UCL and an endurance to complete my PhD studies safely. I have completed this thesis with the strength HE has given me. Contents
Page
Title ...... 1
Abstract ...... 2
Dedication ...... 4
Acknowledgements ...... 5
Contents ...... 7
List of Tables ...... 18
List of Figures ...... 20
Abbreviations ...... 24
Chapter 1 General Introduction : CD98 is a central component within a multi-molecular complex
Abstract ...... 27
1.1 Introduction ...... 28
1.2 Other names of CD98 : 4F2 or FRP-1 ...... 30
1.3 Structure of CD98 ...... 30
1.3.1 Heavy chain (CD98HC) ...... 32
1.3.2 Light chain (CD98LC) ...... 35
7 1.4 Regulation of CD98 expression ...... 40
1.5 Tissue distribution ...... 40
1.6 Ligand for CD98HC ...... 43
1.7 The function of CD98 in physiological and pathological processes ...... 44
1.7.1 Involvement in transport system ...... 44
1.7.1.1 Amino acid transport ...... 46
1.7.1.2 Na^/Ca^^ transport ...... 50
1.7.2 CD98 and the control of cell survival, growth and
Differentiation ...... 50
1.7.2.1 Regulation of cell proliferation ...... 50
1.7.2.2 Programmed cell death (apoptosis) ...... 51
1.7.2.3 Regulation of differentiation ...... 52
1.7.2.3.1 Differentiation of Lin'hematopoietic precursors ...... 52
1.7.2.3.2 Induction of osteoclast-like cell ...... 52
1.7.2.3.2.1 Molecular mechanism of CD98-
osteoclastogenesis ...... 53
1.7.2.3.2.2 Possible regulation of CD98-osteoclastogenesis .... 53
1.7.3 A potential role for CD98 in oncogenesis ...... 55
1.7.3.1 Mechanism of CD98-induced tumorigenicity ...... 56
1.7.3.2 Other possible functions in tumor cells ...... 57
1.7.4 Role of CD98 as an adhesion molecule ...... 57
1.7.4.1 Involvement in cell adhesion ...... 57
1.7.4.1.1 Cell-to-extracellular matrix adhesion ...... 58 1.7.4.1.2 Cell-to-cell adhesion ...... 58
1.7.4.1.3 Homotypic aggregation induced by CD98 ligation ...... 59
1.7.4.2 Adhesion molecule involvement ...... 60
1.7.5 Regulation of cell fusion ...... 61
1.7.6 T cell co-stimulation ...... 63
1.8 CD98-associating proteins ...... 64
1.8.1 CD98 as an integrin function regulator ...... 65
1.8.1.1 What are integrins ? ...... 65
1.8.1.2 Regulation of integrins is of therapeutic interest ...... 66
1.8.1.3 Direct inhibition of integrin function: receptor
antagonism ...... 66
1.8.1.4 Indirect inhibition of integrin function ...... 67
1.8.1.5 Biochemical evidence : molecular mechanism ...... 68
1.8.1.6 Functional evidence that CD98 associates with integrins ...... 68
1.9 Role of CD98 as a signal transduction molecule ...... 70
1.9.1 Engagement or ligation of CD98 ...... 72
1.9.2 Intracellular changes upon CD98 ligation ...... 72
1.9.2.1 Tyrosine phosphorylation ...... 72
1.9.2.2 Serine/threonine phosphorylation ...... 74
1.9.2.3 G-protein or G-like protein involvement ...... 74
1.9.2.4 MAPK/transcription factor activation ...... 75
1.9.3 Possible view of CD98 and signal transduction ...... 76
1.10 Conclusion ...... 77
1.11 Aims ...... 78 Chapter 2 Materials and Methods
2.1 Materials ...... 81
2.2 Cell culture ...... 85
2.3 Quantitative homotypic cell aggregation assay...... 85
2.4 Cytofluorometric analysis ...... 86
2.5 Subcellular fractionation for PKC translocation assay ...... 86
2.6 Western Blotting ...... 87
2.7 Immunoprécipitations ...... 88
2.8 Purification of CD98 mAh (AHN-89) ...... 89
2.9 Preparation of F(ab’)2 and Fab fragments from purified AHN-18 ...... 89
2.10 Biotin conjugation of AHN-18 ...... 90
2.11 Protein determination (Bradford method) ...... 90
2.12 MTT assay (colorimetric assay) for measurement of cell viability ...... 90
2.13 Statistical analysis ...... 91
10 Chapter 3 The functional interactions between CD98, pi-integrins, and CD 147 in the induction of U937 homotypic aggregation
Abstract ...... 93
3.1 Introduction ...... 94
3.2 Results ...... 96
3.2.1 Aggregation-inducing activity of CD98 antibodies
is heterogeneous, and is not correlated with binding activity ...... 96
3.2.2 CD98-induced aggregation does not lead to cell death ...... 100
3.2.3 CD98-induced aggregation, but not CD29 induced
aggregation is resistant to EDTA but sensitive to deoxyglucose ...... 103
3.2.4 CD98-induced aggregation and CD29-induced aggregation
are associated with distinct patterns of tyrosine phosphorylation ...... 103
3.2.5 Reciprocal cross-inhibition by antibodies to CD98 and CD29 ...... 105
3.2.6 Inhibition of CD98-induced homotypic aggregation
by antibodies to (32 integrins ...... 108
3.2.7 Inhibition of CD98-induced homotypic aggregation
by antibodies to CD 147 ...... 108
3.3 Discussion ...... 116
11 Chapter 4 CD98 regulates mitogen activated protein kinases and adhesion via the selective activation of protein kinase Cô
Abstract ...... 124
4.1 Introduction ...... 125
4.2 Results ...... 128
4.2.1 Protein tyrosine phosphorylation induced by CD98
ligation is not essential for U937 homotypic aggregation ...... 128
4.2.2. Protein kinase C activity is necessary for CD98-induced
aggregation ...... 128
4.2.3 CD98 ligation selectively induces PKCÔ membrane
translocation ...... 133
4.2.4 CD98 induced aggregation requires downstream activation of
ERK and p38 MAPkinases ...... 133
4.2.5 CD98 induced activation of PKCÔ is independent of diacylglycerol,
and leads to PKCÔ phosphorylation ...... 135
4.3 Discussion ...... 140
12 Chapter 5 Conventional PKC plays a critical role in negative regulation of CD98-induced homotypic aggregation
Abstract ...... 148
5.1 Introduction ...... 149
5.2 Results ...... 152
5.2.1 Comparative kinetic analysis of CD98-induced
homotypic aggregation ...... 152
5.2.2 PKC inhibitors enhance CD98-mediated
homotypic aggregation ...... 154
5.2.3 PKC activators inhibit CD98-induced homotypic
aggregation ...... 154
5.2.4 PMA-mediated inhibition is abrogated by
conventional PKC inhibitors ...... 157
5.2.5 PMA induces translocation of conventional PKC isoforms
to the plasma membrane ...... 157
5.2.6 PMA does not inhibit CD98-induced ERK activation ...... 161
5.2.7 PMA does not block adhesion molecule expression
during CD98 - induced aggregation ...... 161
5.2.8 The relationship between PMA-responsive PKC
and other signalling pathways ...... 166
13 5.2.9 The effect of other intracellular signalling enzyme
inhibitors on PMA inhibition ...... 168
5.3 Discussion ...... 170
14 Chapter 6 Conclusions
6.1 Role of CD98 in the regulation of integrin function ...... 176
6.2 CD98 induces intracellular signaling ...... 178
6.2.1 Positive signaling ...... 180
6.2.2 Negative signaling ...... 182
6.3 Proposed signaling pathway in U937 cells ...... 182
6.4 CD98-induced signaling in mammalian cells ...... 185
6.5 Functional role of CD98 ...... 185
6.6 Future direction ...... 186
6.6.1 CD98 ligation and the reorganization of cell surface
molecules such as integrins and CD 147 ...... 186
6.6.1.1 Lateral mobility of p 1 and p2 integrins and CD 147
in the membrane following CD98 ligation ...... 189
6.6.1.2 Molecular complex of CD98 and adhesion molecules
in lipid rafts ...... 189
6.6.2 Signalling pathways ...... 189
6.6.3 New cellular models with which to analyse CD98 structure
and functions in cell aggregation and T cell costimulation ...... 190
6.6.3.1 myc-tagged variants of CD98 ...... 190
6.6.3.2 CD98-negative variants of U937 cells ...... 190
6.6.3.3 Human/mouse chimaeric model ...... 191
15 Chapter 7 References
References ...... 193
16 List of Tables
Chapter 1
Table 1.1 Summary of heavy chain of CD98 ...... 33
Table 1.2 Summary of light chain of CD98 ...... 37
Table 1.3 Differential biological activities of anti-CD98 mAh ...... 54
Chapter 2
Table 2.1 List of metabolic inhibitors ...... 81
Table 2.2 List of enzyme inhibitors ...... 81
Table 2.3 List of non-labeled Antibodies to surface molecules ...... 83
Table 2.4 List of direct labeled antibodies ...... 84
Table 2.5 List of antibodies to signaling enzymes ...... 84
Chapter 3
Table 3.1 Lack of correlation between cell surface binding and aggregation-inducing activity of CD98 mAbs ...... 99
Chapter 5
Table 5.1 Effect of PMA on surface level of
U937 adhesion molecules ...... 165
17 Chapter 6
Table 6.1 Effect of nutrient-deprived culture conditions on CD98-induced aggregation ...... 179
18 List of figures
Chapter 1
Fig. 1.1 Schematic representation of heterodimers formed by LCs and CD98HC or rBAT ...... 29
Fig. 1.2 CD98-mediated regulation of cellular function ...... 45
Fig. 1.3 The interaction of CD98 ligand with CD98 and the
formation of molecular complex within the cell membrane
initiates signal transduction ...... 71
Chapter 3
Fig. 3.1 A CD98 mAbs differ in their ability to bind to the
surface of U937 cells, and their ability to induce
homotypic aggregation ...... 97
Fig. 3.1 B CD98 mAbs differ in their ability to bind to
the surface of U937 cells, and their ability to induce
homotypic aggregation ...... 98
Fig. 3.2 Time and dose dependency of aggregation induced
by CD98-AHN-18 ...... 101
Fig. 3.3 CD98-AHN-18 mAb does not induce apoptosis of
U937 cells, or inhibit cell division ...... 102
Fig. 3.4 Metabolic inhibitor profile for CD98-, CD29- and
19 CD43-induced aggregation ...... 104
Fig. 3.5 Ligation of CD98 or CD29 induces rapid and distinct patterns of tyrosine phosphorylation ...... 106
Fig. 3.6 Cross-inhibition of U937 homotypic aggregation between CD98 and CD29 mAbs ...... 107
Fig. 3.7 A Functional interactions between CD98 and
P2 integrins (CD 18) ...... 110
Fig. 3.7 B CD98 ligation does not alter cell surface
levels of CD 18 or CD29 ...... I l l
Fig. 3.8 A CD 147 mAbs block aggregation induced via CD98 ...... 112
Fig. 3.8 B CD 147 mAbs block aggregation induced via CD98 ...... 113
Fig. 3.9 CD 147 regulates tyrosine phosphorylation
induced via CD98 or CD29 ligation ...... 114
Fig. 3.10 Sensitivity of CD98 induced aggregation to blocking antibodies added at different times ...... 115
Fig. 3.11 Model of possible functional complex containing
CD98 (heavy and light chains, pi-integrins (a/p chains)
and CD 147 (heavy and light chains) ...... 122
Chapter 4
Fig. 4.1 CD98-induced aggregation is independent of tyrosine phosphorylation ...... 130
Fig. 4.2 CD98-induced U937 aggregation requires
20 protein kinase c activity ...... 131
Fig. 4.3 Rottlerin, a selective inhibitor of novel PKCô, blocks
CD98-induced aggregation and protein tyrosine phosphorylation ...... 132
Fig. 4.4 CD98-ligation induces a selective membrane translocation of PKCÔ ...... 134
Fig. 4.5 CD98-induced aggregation requires PKCô-dependent ERK and p38 activation ...... 137
Fig. 4.6 CD98-induced aggregation and PKCô translocation is independent of diacylglycerol formation ...... 138
Fig. 4.7 CD98 regulation of PKCÔ phosphorylation ...... 139
Fig. 4.8 Model of CD98-induced signaling ...... 147
Chapter 5
Fig. 5.1 CD98-induced aggregation shows a kinetically different pattern from CD29-and CD43-induced aggregation ...... 153
Fig. 5.2 Conventional and novel PKC inhibitors potentiate
CD98-induced intercellular adhesion ...... 155
Fig. 5.3 PKC activators down-regulate CD98-mediated aggregation ...... 156
Fig. 5.4 A Conventional and novel PKC inhibitors toward
ATP-binding site abolish PMA-mediated inhibition of
CD98-induced homotypic aggregation ...... 158
Fig. 5.4 B and C Conventional and novel PKC inhibitors
21 toward ATP-binding site abolish PMA-mediated inhibition of CD98-induced homotypic aggregation ...... 159
Fig. 5.5 PMA induces translocation of conventional PKC
isoforms to the plasma membrane ...... 160
Fig. 5.6 PMA up-regulates CD98-mediated phospho-MAPK/ERK
expression ...... 162
Fig. 5.7 A Effect of PMA on the surface level of U937
adhesion molecule ...... 163
Fig. 5.7 B and C PMA down-regulates the surface level of U93 7 CD98 ...... 164
Fig. 5.8 A and B Relationship between PMA-inhibition and other
enzyme activation in U937 homotypic aggregation ...... 167
Fig. 5.8 C Relationship between PMA-inhibition and other
enzyme activation in U937 homotypic aggregation ...... 169
Fig. 5.9 Model of PKC-mediated negative regulation of
CD98-induced cell adhesion ...... 174
Chapter 6
Fig. 6.1 Signal transduction steps which regulate CD98-induced
U937 aggregation ...... 184
Fig. 6.2 The interaction of CD98 ligand with CD98 and the
formation of molecular complex within the cell membrane
initiates signal transduction ...... 187
22 Fig. 6.3 CD98-mediated regulation of cellular function ...... 188
23 Abbreviations
aa : amino acids
APC: antigen presenting cells
BCH: 2-(-)-endoamino-bicycloheptane-2-carboxylic acid cPKC : conventional PKC
Cys : cysteine
DAG: diacylglycerol
DC: dendritic cells
DOG: dioctanolyglycerol
DONS : dominant negative suppression
DTT: dithiothreitol
ERKl/2 : extracellular signal-regulated kinases 1 and 2
FAK: focal adhesion kinase
PCS : fetal calf serum
FITC: fluorescein isothiocyanate
FRP: fusion regulatory protein h: hour
HC: heavy chain hCD98 : human CD98
HIV: human immunodeficiency virus hsp70 : heat shock protein 70
ICAM-1 : intercellular adhesion molecule-1 kD: kilodalton
24 LC: light chain
LFA-1 : lymphocyte function-associated molecule-1 mAb : monoclonal antibody
MAPK : mitogen-activated protein kinase mCD98 : murine CD98
MFI: mean fluorescent channel number min : minute
NDV: Newcastle disease virus
nPKC : novel PKC
OD: optical density
PBS : phosphate-buffered saline
PI3-K : phosphatidylinositol 3-kinase
PKA: protein kinase A
PKC: protein kinase C
PLD: phospholipase D
PMA : phorbol-12-myristate-13-acetate
PMSF : phenylmethylsulfonyl floride
PPTase : protein phosphatase
PTK: protein tyrosine kinase
TRAP: tartrate-resistant acid phosphatase
ISP: thrombospodin
UC: ulcerative colitis
25 Chapter 1
General introduction
CD98 is a central component within a multi-molecular complex
26 Introduction
Abstract
CD98 is a heterodimeric glycoprotein composed of a 85 kD glycosylated heavy chain
(CD98HC) and a 35 kD non-glycosylated light chain (CD98LC). CD98 activation is linked to an extraordinary diversity of normal biological functions, such as transport of amino acids or Na'^/Ca^'^, T cell costimulation, cell aggregation and fusion, and cell differentiation, survival or death. These effects are mediated by various CD98-induced signalling pathways, which involve tyrosine and serine/threonine phosphorylation, and changes in actin cytoskeleton, upon the formation of a molecular complex including integrins and CD 147. The impairment of CD98 function is associated with several diseases, including lysinuric protein intolerance and oncogenic transformation. This chapter will summarise and review publications which address the role of function of
CD98 in terms of biochemical, molecular biological, immunobiological and pathological aspects.
27 Introduction
1.1 Introduction
Human CD98 is a type II transmembrane glycoprotein that is extensively expressed on most tissues, and on both normal and tumor cells. The protein is composed of two chains (Fig. 1.1); a glycosylated heavy chain (CD98HC, 529 amino-acid (aa), 85 kD)
(Hemler and strominger, 1982; Teixeira et ah, 1987; Quackenbush et ah, 1987) and a non-glycosylated light chain (CD98LC, 507 aa, 40 kD) (Tsurudome et ah, 1999;
Mastroberardino et ah, 1998). The heavy chain is known both to control membrane trafficking of the light chain and to maintain the membrane topology of the heterodimer
(Nakamura et ah, 1999). The light chain is reported to function as an amino acid transporter (Nakamura et ah, 1999; Segawa et ah, 1999). These two proteins are covalently linked by structurally important disulfide bond between cysteines from the heavy and light chains on the membrane surface (Kubota et ah, 1994).
To date, the physiological role of CD98 remains unclear, but CD98 has been implicated in several different cellular functions, including involvement as an amino acid or ion transporter (Michalak et ah, 1986; Segawa et ah, 1999; Nakamura et ah, 1999; Kanai et ah, 1998), controlling cell growth, survival, differentiation and oncogenic transformation (Kara et ah, 1999; Kara et ah, 2000; Shishido et ah, 2000b; Papetti and
Herman, 2001; Warren et ah, 1996), functioning as an adhesion molecule to regulate cell-to-cell adhesion (Warren et ah, 1996; Ohgimoto et ah, 1995, 1996; Tabata et ah,
1994), and induction of T cell proliferation and activation as a costimulatory molecule
(Woodhead et ah, 2000; Stonehouse et ah, 1999; Diaz et ah, 1997b; Friedman et ah,
1994). Because the wide variety of cellular functions of CD98 cannot be simply explained by its role in amino acid transport, this suggests that the molecule may
28 COOH
COOM
CD98 HC rBAT
CD98 LCs b°+AT
COOM cytosol COOM D C NHg
Fig. 1.1 Schematic representation of heterodimers formed by LCs and CD98HC or rBAT (Verrey et al., 1999). Introduction
participate in a variety of cellular activities in many different guises as suggested previously (Helmer et al., 1982).
This chapter reviews our knowledge about CD98, including its structure and its biological functions. It specifically addresses several unresolved questions about CD98 such as how can CD98 have such an extraordinary diversity of biological functions, what is the relationship between pi integrins (CD29) and CD98 as one of the integrin function-regulating proteins, is amino acid transport (light chain) functionally linked to the other biological activities mediated by CD98HC, how is CD98 activated and what enzymes and associated proteins are involved in CD98-induced signaling pathways ?
1.2 Other names of CD98 : 4F2 or FRP-1
CD98 was originally identified as an activation antigen of lymphocytes, the 4F2 antigen
(Haynes et al., 1981). Recently, it has also been shown that fusion regulatory protein
(FRP)-l, which is a newly defined cell surface molecule, is identical to CD98/4F2 by biochemical data such as N-terminal amino acid sequences, molecular mass and covalent modification (Ohgimoto et al., 1995). Therefore, three names (CD98/4F2/FRP-
1) are now simultaneously cited in CD98-related works (in this thesis, however, CD98 will be used as a general name).
1.3 Structure of CD98
As shown in Fig. 1.1, CD98 is a heterodimeric protein of approximately 125 kD which is composed of a glycosylated heavy chain (CD98HC, 85 kD) and a non-glycosylated light chain (CD98LC, 40 kD). These two proteins are covalently linked by a disulfide
30 Introduction
bond to maintain a stable membrane bound form. Specific monoclonal antibodies
(mAbs) produced by immunisation with the stable heterodimer mostly bind to the
CD98HC, suggesting that the glycosylated heavy chain could be more structurally immunogenic than the light chain.
The cloning of the genes for CD98HC and LC allowed predictions of possible structures and the membrane topology of CD98 (Kanai et al., 1998; Mastroberardino et al., 1997;
Rossier et al., 1999; Markovich and Refeer, 1999; Tsurudome et al., 1999; Broer et al.,
1997; Nakamura et al., 1999; Kanai et al., 1998; Segawa et al., 1999). Currently, there are no studies of the structural properties of CD98, such as a crystal structure or any data on structural regulation of CD98 function. Predictive amino acid sequences have, however, suggested sites of potential modifications such as phosphorylation or glycosylation. N-linked glycosylation plays an important role in the cyclical appearance of CD98 and its association with 45-kD light chain because tunicamycin (a glycosylation inhibitor)-treated cells do not displayed a 125 kD heterodimer but only
CD98HC of 85 kD (Papetti and Herman, 2001).
The heterodimeric structure of CD98 and its characteristics are analogous to the structural regulation of the Na^,K^-ATPase (Verry et al., 1999). This ion-exchange pump is composed of two subunits, a catalytic multitransmembrane subunit (a-chain) and a type II glycoprotein (P-subunit). Thus, CD98HC and the p-subunit of Na"^,K^-
ATPase seem to be required for stabilisation of the complex, its maturation and to allow transport of CD98LC or the a subunit of the ATPase from the endoplasmic reticulum to the plasma membrane (Glitscher and Ruppel, 1991; Verry et al., 1999). A similar
31 Introduction
situation also exists for rBAT (Fig. 1.1), a surface glycoprotein, which is able to covalently associate with type amino acid transporters (Tsurudome and Ito, 2000;
Verry et al, 1999).
1.3.1 Heavy chain (CD98HC)
The heavy chain of human CD98 was first cloned from a genomic X library by Teixeira et al (1987) and the murine form was cloned soon after (Parmaccek et al., 1988). The cDNA was about 1.8 kb in length including a poly (A) tail and open reading frame, and encoding 529 amino acids (aa) with a 81-aa cytoplasmic domain and a 425-aa ectodomain, as summarised in Table 1.1
Even though the crystal structure of CD98HC is currently unknown, a number of structural features have been predicted, based on the DNA sequences and hydropathicity analyses. These include an internal transmembrane spanning region, 4
(human) or 7 (murine) potential glycosylation sites in the C-terminal extracellular membrane domain, and a N-terminal cytoplasmic domain having about 81 amino acids
(Warren et al., 1996; Parmacek et al., 1989; Lumadue et al., 1987; Quackenbush et al.,
1987; Teixeira et al., 1987) The CD98HC has two structurally important cysteine residues at residues 109 and 330. Cys 109 forms a disulfide bond linking CD98HC to the light chain (Teixeira et al., 1987; Pfeiffer et al, 1998). The highly conserved nature of the Cys 109 in many different species confirms its importance for structural stability.
Cys 330 is supposed to be involved in forming a homodimer between heavy chains
(Ohta et al, 1994; Papetti and Herman, 2001) and particularly for stabilising the overall structural conformation (Shishido et al, 2000b).
32 Table 1.1 Summary of heavy chain of CD98.
Reference Teixeire et al., 1987 Quackenbush et al., 1987 Ludadue et al., 1987 Parmacek et al., 1989 Warren et al., 1996
Origin human human human murine murine
mRNA size 2.1 kb 1.854 kb 1.8 kb 1.8 kb 1.8 kb
Number of AA 529 495 - 526 529 526 533
M.W. (estimated) 58 kD 58 kD 58.817 kD
NH2"terminal lacks lacks signal peptide
Cys for disulfide Cys 109 bond
T ransmembrane- 1 2 1 1 1 spanning region
Potential 4 7 glycosylation sites
Cytoplasmic N-terminal 81 50-81 75 AA size
Tissue high level : testis, lung, brain distribution kidney, spleen low level : liver, cardiac muscle Remarks pi : 5.55
AA : amino acid, M.W. : molecular weight, Cys : cysteine. Introduction
Interestingly, a comparison of the human and murine heavy chain cDNA sequences indicates a marked level of homology with regard to the general structure, and amino acid sequences (Parmacek et al., 1989; Warren et al., 1996). Thus, 77.5% nucleotide sequence and almost 80% amino acid sequence are identical between murine and human, suggesting that the protein has an important and conserved function in the normal physiology of cells or tissues. In particular, the highest levels of identity between two species were observed within the intracytoplasmic (86% identity at the amino acid level) and the transmembrane (100% identity at the amino acid level) regions (Parmacek et al., 1989), indicating the structural importance of these domains.
cDNA sequence analysis (Warren et al (1996)) also identified potential modification sites of CD98. These are two potential cAMP/cGMP-kinase phosphorylation (R/T-Xaa-
S/T) sites, eight potential protein kinase C (PKC) phosphorylation (S/T-Xa-R/K), and eight potential Casein kinase II phosphorylation (S/T-Xaa-D/E) sites. However, there are no consensus sites for tyrosine phosphorylation, nor are there protein tyrosine kinase
(PTK) or other catalytic motifs in the murine heavy chain (mCD98HC) cDNA.
Intriguingly, the large COOH-terminal extracellular domains of the CD98HC are similar to those of glycosidases, which show a globular tertiary structure (Wells and Hediger,
1992). Catalytic activity has not been shown yet, and it remains unclear whether this function may or may not be related to amino acid transport.
CD98HC has an important role in maintaining overall structure of CD98 complex. The
CD98HC transports the light chain from the Golgi apparatus to the plasma membrane
34 Introduction
(Nakamura et al., 1999). Unlike CD98LC, which remained intracellular when expressed alone, CD98HC reaches the plasma membrane in the absence of light chain
(Mastroberardino et al., 1998; Teixeira et al., 1987). However, it is still an open question as to whether the heavy chain has an impact on the amino acid transport specificity and/or kinetics of the hetero-oligomer transporters or whether it only fulfills a chaperone function (Tsurudome and Ito, 2000; Verrey et al., 1999).
The human heavy chain (hCD98HC) gene is located on the long arm of chromosome 11
(Francke et al., 1983), and is composed of nine exons and spans approximately 8 kb.
The gene also includes a housekeeping promoter which is G+C rich and four potential binding sites for the ubiquitous Spl transcription factor in 5’ end of the gene
(Gottesdiener et al., 1988). The regulation of CD98 gene expression is controlled by major regulatory elements, locating on exon 1-intron 1 region, and which act through blocking transcriptional elongation (Leiden et al., 1989). An active and powerful enhancer has been reported in the first intron from some cells including malignant T cells (Karpinski et al., 1989). This enhancement is regulated by binding with two novel nuclear proteins (NF-4FA and NF-4FB), which flank a consensus binding sites for the
AP-1 transcription factor (Karpinski et al., 1989).
1.3.2 Light chain (CD98LC)
Although the early biochemical studies had revealed that CD98 is a heterodimer containing two chains, there was little evidence on the nature of the light chain before
1997. One year later, however, Mastroberadino and coworkers (1998) found indirect evidence that the permease-related protein E l6, functioning in amino acid transport.
35 Introduction
was able to covalently associate with CD98HC when this chain was co-expressed with various amino acid transporters in Xenopus oocyts. Recently, biochemical approaches have directly verified that the CD98HC-associated light chain is a glycoprotein- associated amino acid transporter (Tsurudome et al 1999). To do this, CD98LC was co purified with CD98HC from HeLa S3 cells, and a partial N-terminal sequence was obtained. Using this sequence, a full length cDNA clone of CD98LC was cloned from a cDNA library, and shown to be identical to the amino acid transporter hLATl/E16.
The I1LATI/EI6 gene locates on the long arm of chromosome 16 (16q24.3) (Tsurudome et al., 1999). A related human LAT-2 gene was found at chromosome 14ql 1.2-13 (13 cR or approximately 286 kb from marker D14S1349) (Pineda M et al., 1999). The human y+LAT-1 gene localizes to chromosome 14qll.2 (17cR approximately 374 kb from D14S1350) (Torrents et al., 1998), within the lysinuric protein intolerance (LPI) locus (Lauteala, et al., 1997).
Since the first finding of CD98LC, 6 kinds of light chains (Table 1.2), called (i) CD98-
Icl (LATl: E16, TAl, AmAT-L-lc, ASUR4) (Kanai et al., 1998; Marmion et al., 1998;
Nakamura et al., 1999; Mastroberadino et al., 1998), (ii) CD98-lc2 (y+LATl) (Torrents, et al., 1998), (iii) CD98-lc3 (y+LAT2) (Pfeiffer et al., 1999), (iv) CD98-lc4 (xCT) (Sato et al., 1999), (v) CD98-lc5 (LAT2) (Pineda et al., 1999; Rossier et al., 1999; Segawa et al., 1999) and (vi) CD98-lc6 (Rajan et al., 1999) have been reported. According to Na"^ dependency and substrate specificity, these light chains are divided into 4 types (Verrey et al., 1999); (i) LATl and LAT2 (specific for system L), (ii) y+LATl and y+LAT2
(specific for system y+L), (iii) xCT (specific for system Xc-) and (iv) CD98-lc6
36 Table 1.2 Summary of light chain of CD98 (I).
Reference Mastroberadino et al., 1998 Torrents et al., 1998 Pfeiffer et al., 1999
Name LATl (CD98-lcl) y+LATl (CD98-lc2) y+LAT2 (CD98-lc3)
Physiological properties - Transporter type system L system y+L system y+L - AA transport ( Na+ dependency] 1 large,neutral (-) dibasic(-) ; some neutral(+) cationic ; large neutral(+) - Representative AA leucine arginine ; leucine arginine ; leucine Biochemical properties - Cloned sites activated lymphocytes(human) (human) - AA size 507 511 515 - Transmembrane domains 12 12 12 - cDNA size - M.W. (estimated) 55.5 - Tissue distribution (high level) Ovary; testis; placenta; brain kidney ; small intestine ubiquitous - Chromosomal location 16q24.3 14qll.2 - Cys for disulfide bond Cys 164 Cys 151 Structural features (potential) - Glycosylation sites 1 - Phosphorylation sites - for PKC-dependent 1 - for cAMP-, cGMP-dependent PK - for casein kinase Il-dependent 2 Pharmacological properties - Sensitivity to -BCH Yes NO -MeAIB NO NO
Pathophysiology a candidate for lysinuric protein intolerance
AA : amino acid, M.W. : molecular weight, Cys : cysteine. Table 1.2 Summary of light chain of CD98 (II).
Reference Sato et al., 1999 Pineda et al., 1999 Raj an et al., 1999
Name xCT (CD98-lc4) LAT2 (CD98-lc5) CD98-lc6
Physiological properties - Transporter type system X c system L system b®’"*’ - AA transport ( Na+ dependency) anionic small, large zwitterionic(-) cationic (-) ; neutral (-) ; cystine (-) - Representative AA cystine ; glutamate leucine; arginine ; proline arginine ; alanine
Biochemical properties - Cloned sites activated macrophage (mouse) opossum kidney cells rabbit small intestine - Number of AA 502 535 503 - Transmembrane domains 12 12 12 - cDNA size 1.858 kb - M.W. (estimated) 53.6 kD - Tissue distribution (high level) activated macrophage; brain kidney; small intestine kidney; small intestine - Chromosomal location 14ql 1.2-13 - Cys for disulfide bond Cys 158 Cys 154 Cys 137
Structural features (potential) - Glycosylation sites 1 1 - Phosphorylation sites - for PKC-dependent 3 - for cAMP-, cGMP-dependent PK 1 - for casein kinase Il-dependent
Pharmacological properties - Sensitivity to - BCH NO Yes Yes - MeAIB NO NO NO
Pathophysiology a possible candidate for cystinuria
AA ; amino acid, M.W. : molecular weight, Cys : cysteine. Introduction
(specific for b°'^-like amino acid transport system which is specific to rBAT). A defect in the y+LATl gene has recently been shown to be responsible for the genetic disorder lysinuric protein intolerance (Rajan et al, 1999) (see Section 1.7.1.1) (Gitomer and Pak,
1996; Pierides, 1997). Type 1 Cystinuria has been shown to be mediated by a mutation in the gene encoding rBAT (Calonge et al, 1994; Chairoungdua et al, 1999; Chillaron et al, 1997).
Table 1.2 shows that these light chains have several common characteristics. All are predicted on the basis of hydrophobicity plots to have 12 membrane-spanning regions
(unlike the heavy chain which has only one hydrophobic region) and a highly conserved
L-cysteine (164 Cys) which locates on the second extracellular loop between the putative third and fourth TM domains (Pfeiffer et al, 1998). The light chain is therefore a membrane-bound protein, and has structural similarity to other transport proteins. The conserved Cys 164 residue of CD98LC is believed to take part in disulfide bond formation with the heavy chain (Rajan et al, 1999; Mastroberardino et al, 1998; Sato et al, 1999; Kanai et al, 1998; Nakamura et al, 1999). In addition, CD98LCs possess a conserved central hydrophobic domain (418-432 amino acids without cytoplasmic tails) and variable NH 2- and COOH-terminal residues, respectively (Verrey et al, 2000). By pairwise comparison, the minimal level of identity between the different CD98LCs is more than 40%. 21.1% of amino acids are identical in all CD98LC sequences and
82.9% are similar (conservative substitutions), out of the 454 residues which are always present (Verrey et al, 2000).
A prediction from cDNA sequences is that CD98-lc6 has one potential site for N-linked
39 Introduction
glycosylation in the extracellular loop between transmembrane domains 7 and 8, three potential sites for PKC-dependent phosphorylation and one potential site for cAMP- and cGMP-dependent protein kinase phosphorylation in the intracellular domain (Rajan et al, 1999).
1.4 Regulation of CD98 expression
CD98 expression is potentiated by some mitogens such as lectin, alloantigens (Haynes et al, 1981; Yagita and Hashimoto, 1986), lipopolysaccharide (LPS) and phorbol 12- myristate 13-actate (PMA)/calcium ionophores (Tanaka et al, 1989) in T cells. A low level of CD98HC expression in resting T cells is markedly up-regulated upon activation, reaching a maximum level within 6 to 12 hours. The maximal expression of CD98HC is shown at the late G1 phase and the levels are maintained throughout the rest of cell cycle before the expression of interleukin (IL)-2 and transferrin receptor (Helmer et al,
1982; Haynes et al, 1981; Yagita et al, 1986). Mouse mRNA of CD98HC is also shown to be very low in resting mouse spleen cells. However, phorbol-12-myristate-13- acetate (PMA) strongly stimulates the transcription of the gene up to 60 fold (Teixeira and Kuhn, 1991). Furthermore, it has been reported that the expression of the fourth amino acid transporter of CD98, mouse xCT, is greatly enhanced when mouse macrophages are stimulated by LPS (Sato et al, 1999). This massive increase of CD98 mRNA by activation stimuli is mediated by a transcriptional enhancer (Karpinski et al,
1989).
1.5 Tissue distribution
The tissue distribution pattern is more or less consistent with an understanding of the
40 Introduction
functional role of CD98. Thus, CD98HC is highly expressed in tissues related to proliferation, peptide or protein secretion, and cell fusion such as hair follicle
epithelium, skeletal muscle sarcolemma, stomach surface and glandular epithelia, pancreatic islets, kidney proximal tubules, parathyroid gland, follicular epithelium, testicular seminiferous tubules and ova in the ovary (Tabata et al., 1995; Azzarone et al.,
1984) (Table 1.1 and 1.2). Other cells such as peripheral blood monocytes, alveolar macrophages and some bone marrow stem cells are also reported to exhibit higher
surface level of CD98HC (Azzarone et al., 1984; Woodhead et al., 1998).
The tissue distribution of murine CD98HC showed a different pattern from human. The
mCD98HC is highly expressed in brain, kidney, lung, spleen and testis, but a lower
level of CD98HC was found in heart, skeletal muscle, thymus, pancreas and liver. The
mCD98HC is also expressed on thymocytes, splenocytes, peripheral lymphocytes and
blood monocytes (Parmacek et al., 1989). Another report similarly demonstrated that
mCD98HC is highly expressed in adult testis, lung, brain, kidney, and spleen, and is
expressed at significantly lower levels in adult liver and cardiac and skeletal muscle
(Tsumura et al., 1999).
hCD98HC expression is linked to the maturation of dendritic cells (DC) derived from 2-
hr adherent peripheral blood monocytes, and cultured for 7 days with granulocyte-
macrophage colony-stimulating factor and IL-4. The kinetics of monocyte to DC
transition revealed a rapid activation phase within the first 24 hr, with a considerable
increase in expression of CD98; this was followed by a down-regulation of CD 14 and a
more gradual development of the other dendritic cell features over the remaining 6 days
41 Introduction
(Woodhead et al., 1999). Thus, the report suggests that CD98HC may regulate DC maturation or participate in DC function at an early transition stage.
Warren et al (1996) also showed that mCD98HC is expressed on haematopoietic stem cells, T- and B-cell precursors, and a subset of myeloid and erythoid-lineage cells. mCD98 is strongly detected in 70 to 90% of haematopoietic cells in murine yolk sac at day 8/8.5 gestation and 100% of mononuclear cells from fetal liver at day 12 of gestation. Furthermore, CD98 is also expressed in more than 90% of thymocytes at days
13, 14 and 15 of gestation.
The tissue distribution of human CD98LC has also been evaluated by Northern hybridization analysis. Although it is controversial whether or not normal tissues including brain, lung, liver, kidney, colon and stomach do express hCD98LC mRNA
(Gaugitsch et al., 1992; Wolf et al., 1996), all colorectal carcinoma and adenocarcinomas from breast, endometrium, salivary gland and esophagus have a strong detectable level of hCD98LC mRNA (Prasad et al., 1999). A weak but easily detectable signal was found in other normal tissues, such as heart, colon, thymus, spleen, kidney, liver, lung and leukocytes (Prasad et al., 1999). There was no detectable level in retina and thymus (Prasad et al., 1999). Leucocytes or quiescent peripheral blood lymphocytes displayed lower level of CD98LC transcripts or were negative, whereas stimulation with PMA/ionomycin clearly increased mRNA expression (Gaugitsch et al.,
1992).
Northern blot analysis of the distribution of rat and mouse CD98LC also suggests that
42 Introduction
neoplastic tissues and tissues which undergo extensive proliferation possess abundant expression of mCD98LC. Some other tissues, such as brain also express mCD98LC mRNA (Kanai et al., 1998; Sang et al., 1995). However, normal liver does not express mCD98LC, whereas fetal liver evidently expresses mCD98LC (Nakamura et al., 1994).
Abnormal level of CD98 in some diseases is consistent with this idea that CD98 could contribute to the functional impairment of lymphocytes. The diseases includes Wiskott-
Aldrich syndrome (Gerwin et al., 1996), ulcerative colitis (UC) (Yacyshyn, 1993), rheumatoid arthritis (Hoy et al., 1993), chronic infection with Leishmania. major (Rosat et al., 1994), chronic hepatitis type B (Garcia-Monzon et al., 1992) and active systemic lupus erythematosus (Alcocer-Varela et al., 1991). In these diseases, B or T lymphocytes from peripheral blood, UC lamina propria or synovial fluid showed relatively increased or significantly decreased expression level of CD98 as well as other activation markers.
1.6 Ligand for CD98HC
One valuable approach to understand the extraordinary diversity of functional roles of
CD98 is to identify possible ligands for CD98. To date, galectin-3 is regarded as the only identified CD98 ligand (Dong and Hughes 1996). Galectin-3 is a carbohydrate- binding protein of approximately 30 kD expressed on the surface of inflammatory macrophages and several macrophage cell lines. Addition of galectin-3 to Jurkat cells induced a CD98-mediated sustained influx of extracellular Ca^”^ by a voltage-gated Ca^^ channel-independent manner, suggesting that galectin-3 released by accessory cells such as macrophages may bind to CD98 and participate in Ca^^ signalling (Dong and Hughes,
43 Introduction
1996). However, it is not clear yet whether galectin-3 is able to induce other CD98- mediated biological activities. Several groups are now searching for other possible ligands for CD98.
1.7 The function of CD98 in physiological and pathological processes
Presently, the function of CD98 under normal physiological conditions remain largely unknown, but its surface abundance and the high degree of cross-species conservation in some domains of the protein (Parmacek et al., 1989) are suggestive of an important role. Experimentally, CD98 has been shown to regulate amino acid transport, T cell costimulation, cell aggregation and adhesion, and cell death, survival and differentiation
(Fig. 1.2), although the exact mechanisms remain unknown (see Tsurudome and Ito,
2000; Deves and Boyd, 2000; Diaz and Fox, 1998 for review).
1.7.1 Involvement in transport systems
Amino acid and metal ions play a major role in many aspects of cellular metabolism and metabolic communication, such as protein synthesis, ATP formation and activation of cell signalling, between cells and tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed in part by the recently identified family of CD98LC upon cooperation with CD98HC (Verrey et al., 1999). Although the understanding of the functional relationship between CD98HC and LC is still deficient, molecular cooperation between two chains seems to be necessary to carry out the functional role of CD98 in these cellular events.
44 CD98 activation Intracellular events > Cell Outcome
Proliferation/ Tumor cells ^ Transport Costimulation/ Regulation Activation CD98LC Amino acids of T lymphocytes" Na+/Ca2+ cellular Amino acid Others (?) Cardiac/skeletaJ/ function reabsorption muscle cells / Hematopoiesis Kidney cells Intracellular y signalling Hematopoietic Apoptosis Integrin progenitor , cells CD98HC regulation Aggregation Cytoskeleton Virus-infected, cells rearrangement Fusion pi integrins Monocytes ■ Osteoclasto- genesis
Fig. 1.2 CD98-mediated regulation of cellular function. Introduction
1.7.1.1 Amino acid transport
Since it was first reported that CD98HC has structural and functional similarities to rBAT, which is a surface membrane glycoprotein bound to a cysteine transporter
(Bertran et al., 1992; Wells and Hediger, 1992), a possible function of CD98 has been proposed to be amino acid transport. For example, CD98 and rBAT display 30% overall amino acid sequence identity with 52% sequence similarity, and the cysteine residue at position 109 is highly conserved in CD98HC as well as rBAT (Bertran et al., 1992;
Wells and Hediger, 1992). Direct evidence was obtained by showing that CD98HC cRNA injected into Xenopus oocytes stimulated transport of dibasic amino acids and neutral amino acids in a sodium-independent manner (Bertran et al., 1992; Wells and
Hediger, 1992; Kanai et al., 1992). However, it was unlikely that CD98HC itself had transporter properties, because of its structure, as compared to other known amino acid transporters. CD98HC is a simple type II membrane glycoprotein in which there is no evidence of multiple membrane-spanning regions which are necessary to carry out transport function (Verrey et al., 1999). This suggests that CD98HC regulates amino acid transport, indirectly. This prediction was later confirmed by the evidence that
CD98HC undergoes molecular association with one of six amino acid transporters, possessing multiple membrane-spanning regions (Verry et al., 1999).
As mentioned above, 6 different amino acid transporters have been reported to associate with the CD98HC (Kanai et al., 1998; Mannion et al., 1998; Nakamura et al., 1999;
Mastroberadino et al., 1998; Torrents, et al., 1998; Pfeiffer et al., 1999; Sato et al., 1999;
Pineda et al., 1999; Rossier et al., 1999; Segawa et al., 1999; Rajan et al., 1999).
Commonly, these light chains transport neutral amino acids such as leucine, but the
46 Introduction
properties are divided by their differing specificity for substrates and by difference in
their sodium dependency (Verrey et al., 1999; Verrey et al., 2000).
As indicated in Table 1.2, the first representative class of CD98LC is an L-type specific
amino acid transporter which exchanges large neutral amino acids, in particular those
with branched and aromatic side chains (Christensen, 1990). Two CD98HC-associated
light chains (LATl and LAT2) have been identified to be L-type specific (Kanai et al.,
1998; Mastroberardino et al., 1998; Pineda et al., 1999; Rossier et al., 1999; Segawa et
al., 1999). In spite of being of the same L type, these two light chains show striking
differences in tissue distribution and substrate specificity. LATl transports neutral
amino acids (leucine, histidine, isoleucine, phenylalanine, tyrosine, tryptophan, valine, methionine and glutamine) and is predominantly localised to the testis, ovary, placenta, brain, activated lymphocytes and some tumor cells (Kanai et al., 1998; Nakamura et al.,
1999; Rossier et al., 1999; Wolf et al., 1996). In contrast, LAT2 has a higher affinity for
small neutral amino acids (phenylalanine, tyrosine, tryptophan) and it is more
specifically found in kidney (in epithelial cells of the proximal tubules, mostly
localised to the basolateral membrane) and small intestine, suggesting that this
transporter contributes to the renal reabsorption of neutral amino acids by the epithelial
cells. The molecular mass (total aa : 534 or 531) of LAT2 is also larger than that of
LATl (total aa : 512) (Verrey et al., 1999).
Two y+L-type amino acid transporters (y+LATl and y+LAT2) have also been identified
as CD98HC-associated light chains (Pfeiffer et al., 1999; Torrents et al., 1998). Like
LAT2, y+LATl is highly expressed in small intestine and proximal kidney tubules.
47 Introduction
whereas y+LA.T2 is ubiquitously found in most tissues and cells (Pfeiffer et al., 1999;
Torrents et al., 1998). These light chains transport neutral amino acids more effectively than cationic amino acids in a Na^-dependent manner (Deves et al., 1992). Defective y+LATl function due to mutation of its gene (SLC7A7) is regarded as a highly probable cause of autosomal recessive disease lysinuric protein intolerance (see below) (Borsani et al,, 1999; Rajantie et al., 1981; Torrents et al., 1998, 1999). y+LAT2 shows very similar molecular characteristics to y+LATl.
The cysteine-glutamate exchanger xCT is also a significant CD98HC-associated light chain (Sato et al., 1999). This amino acid transporter is found in activated macrophages and some cultured cells. This transporter exchanges extracellular L-cystine in its anionic form for intracellular L-glutamate, in a Na^-independent manner. Thus, this light chain might regulate the immune response by controlling L-cystine/L-cysteine and intracellular glutathione levels in activated cells (Sato et al., 1998).
The last member of the CD98LC family (CD98-lc6) is a Na^-independent amino acid transporter, b^'^AT, which is also known to associate with rBAT (Well et al., 1992;
Bertran et al., 1992; Tate et al., 1992). CD98-lc6 mediates apical influx of L-cysteine and cationic amino acids in epithelial cells of the kidney proximal tubule and the small intestine. Because of this, it has been suggested that mutations of CD98-lc6 might be a cause of cystinuria (Rajan et al., 1999), a genetic disease in which cystine and other dibasic amino acids fail to be reabsorbed from the tubular fluid.
Impairment of amino acid transport has been linked to some genetic diseases, including
48 Introduction
lysinuric protein intolerance (L PI; OMIM 222700) (Myl^anen et al ^000: Torrents et ^1
1999; Torrents et al 1998; Gitome and Pak, 1996; Pierlides, 1997). LPI is a rare, autosomal recessive disorder characterised by defective transport of the cationic amino acids lysine, arginine and ornithine at the basolateral membrane of the polar epithelial cells in the intestine and renal tubules, and by hyperammonemia after high-protein meals (Navar, 1980). Symptoms include failure to thrive, growth retardation, muscle hypotonia and hepatosplenomegaly (Navar, 1980). LPI is caused by mutations, such as deletions, missense mutations, frameshift mutations, nonsense mutations, a splice site mutation and a tandem duplication, in the SLC7A7 (solute carrier family 7, member 7) gene encoding y(+)LAT-l (y(+)L amino acid transporter-1) (Torrents et al., 1999).
Functional genetic analysis using five mutations (L334R, G54V, 1291delCTTT,
1548delC and LPI(Fin)) also indicated that residues L334 and G54 play a crucial role in the function of the y(+)LAT-l transporter (Mykkanen et al., 2000).
Light chain may also be implicated in abnormal modulation of T4-T3/rT3 metabolism in the hypothyroid state (Ritchie et al., 2001). Although experimental hypothyroidism
(28-day propylthiouracil treatment) has no significant effect on System L-like transport of the amino acid tryptophan in adipocytes, uptake of T3 and especially T4 is substantially reduced in adipocytes from hypothyroid rats, partly due to reduction of the
BCH [2-(-)-endoamino-bicycloheptane-2-carboxylic acid] -sensitive transport component (L-type amino acid transporters) in liver cells. This may be due to down- regulation or dissociation of iodothyronine receptors from the System L transporter complex.
49 Introduction
1 ,1,12 Na^/Ca^^ transport
It has been suggested that the CD98HC participates in the regulation of intracellular
Ca^^ concentration via activation of a NaVCa^^exchanger (Michalak et al., 1986; Latarte et al., 1986; Wacholtz et al., 1992). This activity is specific to certain cell types; especially, cardiac and skeletal muscle sarcolemmal vesicles. An mAh to CD98HC, when added to rabbit skeletal muscle or bovine cardiac sarcolemmal vesicles, inhibited
Na^-dependent Ca^^ uptake by up to 90%. Neither Na"^-dependent release of accumulated Ca^^ nor Ca^^/calmodulin-dependent ATPase activity and ATP-dependent
Ca^^ uptake by sarcolemmal vesicles were inhibited (Wacholtz et al., 1992). Cardiac and skeletal muscle sarcolemmal vesicles are also reported to express higher levels of
CD98HC than skeletal muscle transverse tubule membrane and sarcoplasmic reticulum membranes (Michalak et ah, 1986). The role of CD98HC in regulation of Ca^^ uptake was also investigated with human parathyroid adenoma cells. Incubation with the anti-
CD98HC antibodies transiently increased intracellular Ca^^, resulting in decreasing basal level of parathyroid hormone secretion (Posillico et al., 1987). Therefore, these findings suggest that CD98 is able to regulate NaVCa^^ exchanger molecules in a specific manner.
1.7.2 CD98 and the control of cell survival, growth and differentiation
1.7.2.1 Regulation of cell proliferation
CD98 is reported to be abundant at the surface of endothelial cells in metaphase, anaphase, and cytokinesis but not the surrounding interphase cells (i.e.: mitotic cells are intensely stained by CD98 mAh whereas interphase cells are not) suggesting that cells
50 Introduction
with high mitotic index such as tumor cells would possess abundant cell surface CD98.
Indeed, the localisation and expression level of CD98 in tumor cells parallels that shown in mitotic primary cells (Papetti and Herman, 2001). CD98 mAh also block
DNA synthesis in activated peripheral blood mononuclear cells (Diaz et al., 1997a).
CD98 is also important in the regulation of tumor cell proliferation, because some anti-
CD98 mAbs inhibited cell growth in vitro (Azzarone et al., 1984; Yagita et al., 1986;
1986b; Papetti and Herman, 2001). Especially, CD98 mAbs strongly suppressed fibroblast sarcoma cells, colon carcinoma cells and prostate tumor cells, but it showed little inhibition in certain tumor cell line (HEPG2 hepatocarcinoma) and primary cells.
The inhibition did not due to apoptosis. The cells arrest at a particular phase of the cell cycle, and proliferation is restored if the mAh is removed from the culture.
Early reports have demonstrated that significant amino acid transport occurs in mitosis as well as in interphase (MacMillan and Wheatley, 1981). Therefore, the mAbs might be act by inhibiting amino acid transport during interphase and possibly mitosis.
1.7.2.2 Programmed cell death (apoptosis)
Like other surface molecules such as CD43 (Pace et al., 1999) and CD99 (Sohn et al.,
1998), it has been reported that CD98 might regulate cell survival and death via the induction of apoptosis. Warren et al (1996) demonstrated that CD98 ligation with a
CD98 mAh, Joro 177 induces programmed cell death (apoptosis). Joro 177 increased
DNA fragmentation in Lin' haematopoietic precursor (FL) cells, resulting in inhibition of differentiation and induction of apoptosis. However, conflicting observations report
51 Introduction
that CD98 may have oncogenic potential (Hara et al., 1999; Hara et al., 2000; Shishido
et al., 2000b), and that CD98 ligation did not affect cell viability of U937 cells (Cho et
al., 2001). CD98-induced apoptosis may, therefore, be restricted to certain cell types or
to specific differentiation steps.
1.7.2.3 Regulation of differentiation
Although the underlying mechanism is not completely understood, it is generally
accepted that CD98 plays an important role in the regulation of differentiation of Lin'
haematopoietic precursors and blood monocytes (Warren et al., 1996).
1.7.2.3.1 Differentiation of Lin haematopoietic precursors
Warren et al (1996) showed that CD98 participates in the differentiation of Lin'
haematopoietic precursors. A CD98 mAh inhibited the generation of lymphoid, myeloid,
and erythroid lineage cells in vitro from early Lin' haematopoietic precursors. This
inhibition may be due to suppression of cell survival and proliferation, because CD98
HC ligation also induced apoptosis (see above).
1.7.2.3.2 Induction of osteoclast-like cells
Long exposure of blood monocytes to CD98HC mAbs for 7 to 14 days induced the
formation of multinucleated giant cells (Higuchi et al., 1998; 1999; Tajima et al., 1999).
These cells were positive for the osteoclast markers (such as tartrate-resistant acid phosphatase (TRAP), actin ring and calcitonin receptors) (Suda et al., 1992) and gained bone resorption ability (Higuchi et al., 1998; 1999; Tajima et al., 1999). In agreement with this data, CD98 is expressed on both osteoclasts isolated from human bone and the
52 Introduction
osteoclast-like cells obtained from human giant cell tumors of bone (Higuchi et al.,
1999).
1.7.2.3.2.1 Molecular mechanism of CD98-osteoclastogenesis
To define the molecular mechanisms by which CD98 induces differentiation of monocyte to osteoclast-like cells, Higuchi et al (1999) analysed messenger (mRNA) expression of osteoclast markers, during differentiation of osteoclasts from monocytes.
Of selected markers (cathepsin-K, carbonic anhydrase II, vacuolar H^-ATPase, vitronectin receptor, TRAP, osteopontin, galectin-3, receptor activator of NF-KB and its ligand, c-src, c-fos, and c-frns), only c-src mRNA was selectively expressed from 3 h after CD98 ligation, suggesting that c-src may play an important role in the CD98- induced differentiation process. In turn, c-src induction requires tyrosine kinase activity,
Ras activation, mitogen-activated protein kinase (MAPK) activation and Spl, a ubiquitously expressed transcription factor (see Section 1.9.2.4) (Mori et al., 2001;
Higuchi et al., 1999; Tajima et al., 1999). Specific pharmacological inhibitors of these classes of enzymes could block c-src expression and osteoclastogenesis induced by
CD98 mAb.
1.7.2.3.2.2 Possible regulation of CD98-osteoclastogenesis
Importantly, it has been reported that some mAbs to CD98 can block osteoclast formation (Table 1.3). The CD98 mAh, HBJ 127, induced extensive cell aggregation, but not polykaryocyte formation (Tajima et al., 1999). Interestingly this mAh also blocked the ability of other anti-CD98 mAbs (6-1-13, 4-5-1, or 38-2-2) to induce osteoclast formation, and the induction of c-src message. CD98 may therefore play a
53 Table 1.3 Differential biological activities of anti-CD98 mAb(Tsurudome and Ito, 2000).
Effects on Effects on NDV- Effects on biological activity biological activity Cd+U2ME-7 infected Cd+JEM2 of 4-5-1 in ofHB127in mAb U937-2 (U937-2/gpl60) HeLa Monocytes (Jurkat/gpl60) Cd+U2ME-7 Cd+U2ME-7 Aggregation event
4-5-1 ++-H- ++-H- + + + NT NT 6-1-13 ++++/+ +++/- +-H- Inhibitory No effect 4F2 -- -H-+ Inhibitory Inhibitory HBJ127 +++ ++++ +++ NT NT H227 - - -H-+ No effect Inhibitory 38-2-2 -- + + + No effect No effect
Fusion event
4-5-1 - t - t - No effect NT NT 6-1-13 ++ No effect Inhibitory Inhibitory 4F2 + Inhibitory Inhibitory Inhibitory HBJ127 ++ Inhibitory NT NT H227 + Inhibitory No effect Inhibitory 38-2-2 + + Inhibitory Inhibitory Inhibitory Introduction
dual role in both inducing or inhibiting c-src expression, and hence osteoclast formation.
1.7.3 A potential role for CD98 in oncogenesis
A cancerous (transformed) state is due to abnormal control of cell proliferation. Many
studies have shown that CD98 expression is strongly increased during lymphocyte
proliferation, suggesting that CD98 is linked to the cell cycle. One possible role for
CD98 is to increase the intracellular pool of amino acids, including several essential
amino acids (notably large aliphatic ones), and hence allow increased protein synthesis.
This could be one explanation why actively proliferating cancer cells, including
osteosarcomas and rhabdomyosarcomas as well as simian virus-transformed cells
(Haynes et al., 1981; Azzarone et al., 1984; Dixon et al., 1990; Esteban et al, 1990)
display high levels of CD98.
The involvement of CD98 in tumorigenicity was firstly suggested by clinical findings
that CD98 was highly expressed on various cancer cells such as thyroid cancer cells,
cholesteatoma (Sudhoff et al, 1994; Holly et al, 1993), childhood acute lymphoblastic
leukemia (Taskov et al, 1996), squamous cell carcinomas (Esteban et al, 1990), plasmacytic neoplasms and B-cell non-Hodgkin's lymphomas (Lewandrowski et al,
1990). The level of CD98 expression was shown to be correlated with different tumor-
spreading, differentiation and metastatic behaviors in some cells (Esteban et al, 1990),
including childhood acute lymphoblastic leukemia (Taskov et al 1996), brain tumors
and non-Hodgkin's lymphomas (Holte et al 1989).
However, several experiments have demonstrated a more direct link between CD98 and
55 Introduction
tumorigenicity. Hara et al., (1999; 2000) and Shishido et al., (2000b) overexpressed
CD98 in BALB3T3 or N1H3T3 cells, and showed that these cells had increased
tumorigenicity. The cells grew to higher saturation density, had a greater efficiency in
colony formation in soft agar, and allowed development of tumors in athymic mice
(Hara et al., 1999).
In contrast, ligation of CD98HC inhibited tumor cell proliferation (Yagita et al., 1986)
as well as mitogen-induced DNA synthesis in T and B lymphocytes (Haynes et al.,
1981). Thus, although CD98 and cell cycle are linked in some way, the physiological
role of CD98 in the regulation of the cell cycle requires further investigation.
1.7.3.1 Mechanism of CD98-induced tumorigenicity
To dissect further the structural requirements for CD98-mediated tumorigenicity, the
effect of introducing a number of mutations was investigated. Truncated forms of CD98
in which extracellular domains are deleted (Hara et al., 2000) or mutants (C103S,
C325S and 103/325) in which Cys 103 and/or Cys 325 were replaced with serine
(Shishido et al., 2000) were tested. Truncation (up to 292 aa) of the extracellular domain
did not affect CD98-mediated tumorigenicity, and indeed further enhanced tumor
growth (Hara et al., 2000). However, disulfide linkage between CD98HC and CD98LC was found to be essential to induce malignant transformation (Shishido et al., 2000b).
These results suggest that the interaction of CD98HC with its ligand (if such exists) is not necessary for induction of cell transformation, but that the light chain(s) probably play an essential role.
56 Introduction
1.7.3.2 Other possible functions in tumor cells
The L-phenylalanine transporter, which is one of the CD98 light chains, mediates the uptake of melphalan, an anticancer drug susceptible to development of drug-resistance
(Harada et al., 2000). Down-regulation of CD98LC might be therefore responsible for drug resistance, by reducing melphalan uptake. Altered tumor associated gene-
1/CD98LC (LATl) is also hypothesized to be involved in an early event in hepatocarcinogenesis giving neoplastic cells a growth or survival advantage, particularly under conditions of limited amino acid availability (Campbell et al., 2000).
1.7.4 Role of CD98 as an adhesion molecule
In parallel to its role in transport, and regulation of cell growth/differentiation, CD98 has been implicated in the regulation of cellular adhesion and virus-induced cell fusion
(Suga et al., 2001; Cho et al., 2001 Fenczik et al., 1997; Ohta et al., 1994; Ohgimoto et al, 1995).
1.7.4.1 Involvement in cell adhesion
To carry out many biological activities, cells must dynamically interact with other cells or with the extracellular matrix. These activities include both physiological and pathological processes, such as development, wound healing, homeostasis, migration, tumor cell metastasis, immune activation, inflammation, and thrombosis (Springer,
1990; Albelda and Buck, 1990). The control of cell adhesion plays an especially important role in the regulation or generation of immunological and inflammatory responses (Patarroyo, 1991). Cell adhesion is initiated and maintained by a complex interaction and association between cell surface and intracellular molecules (Springer,
57 Introduction
1990; Helmer, 1990).
1.7.4.1.1 Cell-to-extracellular matrix adhesion
One example of an adhesion process is integrin-mediated adhesion to matrix components. Binding of many types of cells to extracellular matrices (e.g. fibronectin, collagen etc.) through integrin transmembrane receptors initiates the assembly of an actin cytoskeletal complex at the inner surface of the membrane, which in turn drives formation of filopodia, lamellipodia, focal adhesions, and stress fibers (Giancotti and
Ruslahti, 1999; Hubbard and Rothlein, 2000). Continuously, multiple intracellular signalling molecules are stimulated during this process, many of which are dependent on assembly of these cytoskeletal structures for their function.
CD98 has also been reported to regulate cell adhesion to extracellular matrix (Fenczik et al., 1997; Chandrasekaran et al., 1999). A CD98 mAh increased cell adhesion of the small cell lung-cancer line (SCLC) and breast carcinoma cell line (MDA-MB-435 cells) to laminin, collagen, fibronectin and other sulfate glycoproteins (Fenczik et al., 1997).
The adhesion of SCLC was abrogated by a blocking mAb to CD29 (pi integrin) and
EDTA treatment, suggesting that CD98-induced cell adhesion is dependent on pi integrin activation. The thrombospodin (TSF)-1 -mediated adhesion of MDA-MB-435 to fibronectin and collagen is also functionally regulated by CD98, because CD98 ligation increased cell adhesion of the cell line, via activation of pi integrins (Chandrasekaran et al., 1999).
1.7.4.1.2 Cell-to-cell adhesion
58 Introduction
In the immune system, cell-to-cell adhesion (homotypic and/or heterotypic aggregation) has been observed for neutrophils (Simon et al, 1993), lymphocytes (Andrew et al.,
1995), eosinophils (Teixeira et ah, 1995), platelets (Gordon, 1981), dendritic cells
(Delemarre et ah, 2001) and macrophage/monocytes (de Smet et ah, 1993) after activation. These aggregation events are believed to play important roles in many immunological processes, including antigen presentation, cell-mediated cytotoxicity, and cell extravasation into inflammation sites (Singer, 1992; O’Rourke, 1996; Gille and
Swerlick, 1996). Like adhesion events, cell aggregation is accomplished through a variety of cell surface receptors, including the p2 integrin / lymphocyte function- associated molecule-1 (LFA-1, CD18/CD11) (Zapata et ah, 1995), intercellular adhesion molecule-1 (ICAM-1, CD54), tetraspans (such as CD9, CD53, CD81 and CD82)
(Lagaudriere-Gesbert et ah, 1997), pi-integrins (CD29) (Nieswandt et ah, 2001), CD43
(de Smet et ah, 1993), CD54 (Smith et ah, 1989), CD99 (Hahn et ah, 2000), CD 147
(Kasinrerk et ah, 1999), CD151 (Fitter et ah, 1999) and T cell activation antigen
(TABS) (Andrew et ah, 1995).
1.7.4.1.3 Homotypic aggregation induced by CD98 ligation
CD98 mAb-induced homotypic aggregation has been reported in many experiments with various cells such as U937 (Cho et ah, 2001), FTH cells (lymphoid progenitor cells) (Warren et ah, 1996), BAF-3 (pro-B cells) and BAF3/hLAF-l (BAF3 cells transfected with human LFA-1 a (aL and p2) chain cDNAs) (Suga et ah, 2001), U2ME-
7C (CD4^ U937 cells transfected with the human immunodeficiency virus (HIV) gpl60 gene) (Ohgimoto et ah, 1996), U937-2 cells (CD4^ U937 cells) (Ohta et ah, 1994;
Ohgimoto et ah, 1995) and JME2 cells (Ohgimoto et ah, 1996). Although it is unclear
59 Introduction
what physiological purpose (if any) is served by this type of homotypic aggregation, it
was found to lead to cell fusion in certain cases, such as virus-infection or
osteoclastogenesis (Ohta et al., 1994; Ohgimoto et al., 1996).
The aggregation of JME2, U2ME-7C and U937-2 cells by CD98 mAh is strongly
induced within 1 to 2 hours, but these aggregates become loose after approximately 6
hours (Ohta et al., 1994; Ohgimoto et al., 1996). In contrast, U937 homotypic
aggregation is slowly induced, maximal at 5 to 6 hours, and clusters remain for up to 2
days. In comparison, CD29- and CD43-induced aggregation is much more rapid, and is
strongly induced within 1 hour (Cho et al., 2001). Since cytochalasin, azide,
deoxyglucose and low temperature (4°C) all strongly block the formation of aggregates,
CD98-induced homotypic aggregation requires both cytoskeletal changes and metabolic
energy ((Ohta et al., 1994; Ohgimoto et al, 1996; Tabata et al, 1997; Warren et al,
1996). In agreement with this, it has been reported that CD98 associates or co-localises
with cytoskeleton proteins such as heat shock protein (hsp) 70, actomyosin, vimentin
and various tropomyosin isoforms (Suga et al, 1995; Nakamura et al, 1999; Shishido et
al, 2000a). As will be discussed in Section 1.9, CD98-induced homotypic aggregation
requires a series of intracellular signalling steps, such as MAPKs and G-proteins (Ohta
et al, 1994; Ohgimoto et al, 1996; Warren et al, 1996; Suga et al, 2001; Tabata et al,
1997).
1.7.4.2 Adhésion molecule involvement
Although the mechanism whereby CD98 ligation induces adhesion is not fully understood, the involvement of adhesion molecules is well documented. Most studies
60 Introduction
report that CD98-induced adhesion is mediated by activation of pi and p2 integrins.
Both monocyte and BAF3/hLAF-l aggregation is suppressed by p2 integrin (LFA-1)
mAbs (Ohgimoto et al., 1995; Suga et al., 2001), providing evidence of p2 integrin
involvement. Similarity, blockade of pi integrins attenuated the aggregation of U2ME-
7C and U937-2 cells (Ohta et al., 1994; Ohgimoto et al., 1996).
However, mouse CD98 HC mAh Jorol77-induced aggregation of FTH5 Pro-T cells was
not blocked by several blocking mAbs against CD49d/CD29, CD49f/CD29,
CD 11 a/CD 18 and CD54, indicating that integrin-independent adhesion molecules might
be involved in mCD98-mediated homotypic aggregation (Warren et al., 1996). As
described in Chapter 3 (Cho et al., 2001), CD 147, which is a member of
immunoglobulin superfamily, also plays an important role in U937 homotypic
aggregation (Cho et al., 2001).
1.7.5 Regulation of cell fusion
Normal cells do not normally undergo cell fusion, with the exception of sperm fusion
with an egg, myogenesis, osteogenesis and placenta formation (White, 1990; Burger and
Verkleij, 1990). However, virus infection often induces cell fusion, and formation of
giant polykaryocytes and/or syncytia (White, 1990). Many enveloped viruses such as paramyxoviruses, HIV and herpes simplex virus can induce cell fusion (White, 1990).
Although the underlying mechanisms remain unknown, these viruses seem to be able to
overcome the inhibitory mechanisms by which cell fusion is normally prevented
(Tsurudome and Ito, 2000). Virus-induced cell fusion is started by the fusion between the virus envelope and the cell membrane, and usually results in cell death.
61 Introduction
In order to understand how virus-induced cell fusion is regulated, Ito and colleagues screened a number of mAbs for their ability to induce fusion of the U2ME-7 cell line, and identified two such mAbs (Tsurudome and Ito, 2000). One of these mAbs recognised a proteins which was shown to be identical to CD98/4F2, both by N- terminal amino acid sequence analysis and other biochemical characteristics, such as molecular mass and covalent association with a non-glycosylated light chain (Ohgimoto et al., 1995).
Studies on the role of CD98 in membrane fusion have been conducted mostly in two models: I) Virus-mediated cell fusion by Newcastle disease virus (NDV)-infected HeLa cells (Yamamoto et al., 1984; Ito et al., 1987) and 2) HIV gpl60-mediated cell fusion by
HIV gpl60-transfected U937-2 (Cd+U2ME-7) cells or CD4+ Jurkat T-cell (JME2) line
(Ohgimoto et al., 1995; Ohgimoto et al., 1996). In the first model, CD98 ligation using
CD98 mAh was shown to increase HeLa cell fusion in a coculture system in which dispersed BHK cells infected with NDV were seeded on monolayers of HeLa cells (Ito et al., 1992). In the second model, CD98 mAbs initiate the formation of syncytia and polykaryocytes of HIV gpI60-transfected U937-2 cells (Ito et al., 1992). The polykaryocytes begin to appear at about 10 h and increase in number for approximately
15 h (Ohta et al., 1994).
The role of CD98 was further confirmed using dominant negative mutants (Okamoto et al., 1997a). Two mutated CD98HC molecules, [CD98/HN, in which the cytoplasmic domain is replaced by human parainfluenza vims type 2 haemagglutinin neuraminidase
62 Introduction
(HN), and CD98/330 (serine), in which Cys 330 is replaced by serine], showed little or no effect on CD98 mAb-mediated enhancement of polykaryocyte formation in parent
HeLa cells induced by NDV, and rubulavirus-induced cell fusion (Okamoto et aL,
1997a).
The CD98-mediated cell fusion phenomenon is further complicated, because CD98 mAbs can differentially modulate the event (Table 1.3). For example, CD98 mAb
(HBJ 127) displayed two opposite effects on virus-induced fusion events; this mAb enhanced cell fusion in NDV-infected HeLa cells, but delayed human parainfluenza type 2 virus (hPIV2)-induced fusion without diminishing virus growth or viral protein synthesis (Okamoto et al., 1997a).
1.7.6 T cell co-stimulation
CD98 expression is strikingly regulated during T cell differentiation and activation, but the role of CD98 in T lymphocyte responses is not yet understood. Using ligation with mAbs, several functions have been proposed. Firstly, CD98 showed comitogenic properties when T lymphocytes were treated with CD98 mAbs and soluble or immobilized CD2 and CD3 mAbs (Freidman et al., 1994; Warrant et al., 2000), because these CD98 mAb strongly up-regulated T cell proliferation even in the absence of antigen presenting cells (APC). These results suggest that CD98 might act as a costimulatory molecule. Secondly, some different CD98HC mAbs inhibited lL-2 receptor expression and progression of T cells from G1 to S phase, suggesting that
CD98 might be involved in T cell activation (Diaz et aL, 1997b). Finally, the effects of
CD98 ligation may also be mediated via the regulation of APC function, since CD98HC
63 Introduction
is expressed at very high levels on human peripheral blood DC (Woodhead et al, 1998)
as well as monocyte and monocyte-derived lines (Stonehose et al., 1999). Diaz et al.,
(1997) demonstrated that the interaction between CD98 mAh and CD98 on monocytes,
acting as APC, was more important than a direct effect on the T cell in mediating
regulation of T cell activation. This model used a novel xenogeneic system, which took
advantage of the lack of xenoreactivity of purified human T cells against mouse
splenocytes (Diaz et al., 1997b). Mouse splenocytes, therefore, acted as “accessory
cells” for CD3-mediated activation of T cells. In this model, mAbs recognised mouse
but not human CD98. Two further studies (Stonehouse et al., 1999; Woodhead et al.,
2000) demonstrated a functional role for CD98 on APC. In these studies, CD98 mAbs
inhibited T cell proliferation induced by CD3 mAb in the presence of U937 or DC. This
suppressive effect was maintained, even when CD98 mAh was prepulsed on U937 cells
and DC, and was washed off before the U937 cells and DC were added to the T cells.
Taken together, these results implicate that CD98 is involved in a novel pathway for
APC regulation of T cell activation and proliferation.
1.8 CD98-associating proteins
CD98 ligation induces activation-dependent signalling pathways, particularly tyrosine
phosphorylation, despite the fact that CD98 contains no catalytic region within its
cytoplasmic domain (Warren et al., 1996). This suggests that phosphorylation might
result from association with membrane-associated tyrosine kinases, or with
serine/threonine-specific protein kinases to activate the tyrosine phosphorylation system.
More generally, CD98 function may be mediated by proteins that associate with CD98.
Recent data indeed support this hypothesis. Immunohistochemical, immunological and
64 Introduction
biochemical studies have shown that CD98 can associate with various cytoskeletal and structural proteins, such as heat shock protein 70 (hsp70), actomyosin, vimentin,
ADAM3 disintegrin, tropomyosin, (31 integrins and cadherin from various type of cells
(Suga et aL, 1995; Nakamura et aL, 1999; Shishido et aL, 2000a; Takahashi et aL, 2001).
These results further suggest that the function of CD98 may be linked to the regulation of cell cytoskeleton and integrin function.
1.8.1 CD98 as an integrin function regulator
1.8.1.1 What are integrins ?
Integrins are heterodimeric transmembrane proteins composed of a and (3 subunits that functionally mediate cell adhesion to extracellular matrix components or cell-to-cell interaction and serve as signalling receptors for cell migration, cell growth and differentiation, cell cycle and adhesion events (Kishimoto et aL, 1989; Larson and
Springer, 1990). The a and (3 chains each include an extracellular region, a transmembrane segment and a small cytoplasmic domain. These (3 chains have been identified as pi, P2, p3 and P7. The pi-integrins are mainly involved in binding of cells to extracellular matrix such as fibronectin, etc. (Wood and Shimizu, 2001), p2-integrins participate in leucocyte adhesion to endothelium and to other immune cells (Hubbard and Rothlein, 2000), and p3-integrins are involved in the interactions of platelets and neutrophils at inflammatory sites or sites of vascular damage (Seftor, 1998). Each p chain associates with one of a number of different a chains to make at least 22 different integrin receptors (Springer, 1990). The binding of the integrin complex to its ligand is accompanied by rearrangements in the actin cytoskeleton. This rearrangement is mediated by activation and nucléation of a complex of proteins inside the cell, including
65 Introduction
the cytoplasmic tail of the p subunit of the integrin receptor and a number of other signaling proteins. The p subunit is important in determining which proteins are nucleated (to form the focal adhesion) and which signals are generated, p and a chains also bind directly to structural proteins (such as talin, actin, vinculin and a-actinin) and signaling proteins (such as focal adhesion kinase (FAK), pl30CAS, Src, Csk and Crk) to regulate cytoskeleton rearrangement, formation of focal adhesion and signalling
(Giancotti and Ruoslahti, 1999; Boudrea and Jones, 2000; Schlaepfer et al., 1997).
Integrin-mediated signalling pathways are very complex, but include activation of
MAPK via RJio GTPases (especially Cdc42) (Boudreau and Jones, 1999; Hill and
Treisman, 1995).
1.8.1.2 Regulation of integrins is of therapeutic interest
Integrins have been implicated in the pathogenesis of many diseases, such as rheumatoid arthritis, inflammatory bowel disease, asthma, cancer and coronary heart disease (Kishimoto et al., 1989; Larson and Springer, 1990; Engers and Gabbert, 2000).
For this reason, it is of great interest to learn how to control and regulate the function of integrins, which are promising targets for the development of therapeutic agents. To do this, there are two main approaches at present. The first is to develop antagonists of the extracellular domain and the second is to identify new regulators of integrin extra- and intracellular domain function.
1.8.1.3 Direct inhibition of integrin function : receptor antagonism
Interest in the development of specific antagonists of integrin function has been principally driven by efforts to design more potent curative (e.g. anti-thrombotic) agents
66 Introduction
than conventional drugs (Scarborough, 1999). One example is the development of a
ROD tripeptide mimic as a functional blocker of P3 integrins. Several possible clinical settings for targeting the P3 integrins are currently being evaluated.
1.8.1.4 Indirect inhibition of integrin function
Recently a dominant and concentration-dependent suppression of integrin activation was reported, using a chimaeric integrin protein. Fenczik et al (1997) showed that adhesion, spreading or migration mediated by integrin function was strongly blocked by a chimaeric protein (Tac-pl), composed of the cytoplasmic region of the pi integrin chain and the extracellular domain of the Tac (CD25) subunit of the IL-2 receptor. The suppression was believed to be due to the binding of the free pi integrin cytoplasmic domains of the Tac-pl chimaeras to putative cellular factors. Identifying this factor, or factors, was clearly an important question (Lasky, 1997). Integrins were already known to associate physically with many cell-surface and intracellular proteins (so called integrin-associating proteins). Such proteins included CD47, glycosylphosphatidylinositol-liked receptor (CD87, CD 16b and CD 14), the tetraspan or transmembrane-4 superfamily (CD9, CD53, CD63, CD81, and CD82), CD98 and
CD 147 (Hemler, 1998; Berditchevski, 1997; Zhang et al., 2001). Any of these proteins might be involved in the regulation of integrin function. However, the clever genetic complementation strategy employed by Fenczik and collaborators (1997), specifically identified that CD98 is involved in Tac pi dominant suppression as the putative cellular factor. Blocking CD98 function may therefore be an alternative tool to suppress integrin function, and such a blockade may have potential therapeutic applications, for example anti-inflammatory or anti-metastatic (Lasky, 1997).
67 Introduction
1.8.1.5 Biochemical evidence : molecular mechanism
The hypothesis that CD98 regulates integrin function was therefore explored further.
The CD98HC gene was manipulated to make several deletion mutants and the function of these recombinant CD98 molecules was evaluated using immunoprécipitation and the capacity to reverse dominant negative suppression (DONS) of integrin activation
(Fenczik et al., 1997; Zent et al., 2000; Fenczik et al., 2001). Using this approach, CD98 was shown to specifically associate with the integrin pi A cytoplasmic domains
(Fenczik et al., 1997). There was no association with other domains, such as the muscle- specific splice variant, piD, or the leukocyte-specific p7 cytoplasmic domain (Zent et al., 2000). Both the cytoplasmic and transmembrane domains of CD98HC were found to be required for its interaction with integrins, because deletion or replacement of either domain completely blocked DONS capacity of CD98, and physical association with pi integrins (Fenczik et al., 2001).
1.8.1.6 Functional evidence that CD98 associates with integrins
Several further reports support the biochemical evidence for CD98/pl integrin interaction. Most studies have used cell adhesion models, and immunological blockade using blocking mAbs.
The first evidence was obtained in a model system in which CD98 ligation induces adhesion to collagen (see Section 1.7.4.1.1). In this model, the pi integrin blocking mAb P5D2 strongly diminished CD98-mediated cell adhesion to collagen, suggesting that antibody-mediated crosslinking of CD98 stimulated pi integrin-dependent cell
68 Introduction
adhesion (Fenczik et aL, 1997). The second example uses a model in which cell adhesion is mediated by TSP-1 (Chandrasekaran et aL, 1999; 2000), a matricellular protein that displays both pro- and anti-adhesive activities (Bomstein, 1995).
Attachment and spreading of MDA-MB-435 (breast carcinoma) cells on immobilized
TSP-1 are known to be primarily pi integrin-dependent. Under these conditions, CD98 ligation increased the activity of pi integrins. Conversely, serum stimulation, which leads to integrin activation, is accompanied by increased surface expression of CD98.
Thus, it was suggested that the pro-adhesive activity of TSPl for breast carcinoma cells was in part controlled by CD98 activation through regulation of the aSpi integrin.
Evidence for pi integrin regulation by CD98 was also shown in the case of virus- mediated cell fusion (Tabata et aL, 1994; Ohta et aL, 1994) (see Section 1.7.5). pi integrin mAh completely inhibited the enhancement of NDV-mediated cell fusion by
CD98 mAb, whereas mAbs to P3 and a4 integrins, did not affect the CD98-mediated potentiation of the cell fusion. Furthermore, pi integrin mAb also blocked syncytium formation in CD98 mAb-treated Cd^U2ME-7 cells, suggesting that pi integrins are necessary for CD98-induced fusion.
A role for pi integrins was also demonstrated in the co-stimulatory activity of CD98 for
T cells (Warren et aL, 2000) (see Section 1.7.6). As reported previously (Nakao et aL,
1993), some CD98 mAbs synergised with CD3 mAh in stimulating proliferation of peripheral blood T lymphocytes. However, a blocking pi integrin mAb suppressed the
CD98-mediated costimulatory activity. Therefore, it was proposed that interaction between CD98 and integrins is necessary for the T cell costimulatory role of CD98.
69 Introduction
Although pi integrins have been the main CD98-associated molecule studied, other
important proteins may exist. The evidence for one such molecule, CD 147, is presented
in Chapter 4.
1.9 Role of CD98 as a signal transduction molecule
Despite the multitude of studies addressing CD98 function, there is little information on
the intracellular signalling pathways which underlie the extraordinary diversity of
function. Tyrosine phosphorylation, G-protein involvement, and MAPK activation
following CD98 ligation with activating mAbs have all been documented as possible
signalling pathways in CD98 function (Fig. 1.3). Most of the experiments have been
carried out using U937 (Cho et al., 2001), BAF/hLFA-1 cell (Suga et al, 2001), FTH5 cells (Warren et al, 1996), Cd+U2ME-7 (Tabata et al, 1997) and blood monocytes
(Miyamoto et al, 2000; Tsurudome and Ito, 2000), under normal culture or virus-
infected conditions. A definite consensus pathway for CD98-mediated signalling caimot yet be clearly defined, however. This may be due to the variety of cell types examined
or the different outcomes evaluated with the frequent absence of quantitation. The data
do suggest that different CD98 functions may be mediated by different intracellular
signalling pathways.
70 CD98HC CD29
CD98LC
Plasma membrane
PTK-1 i i Signalling molecule complex
PTK-2 PI3-K MAPKKK 4 Homotypic f MAPKK aggregation Others MAPKsI (Erk,p38)
Nuclear membrane
Transcription factor activation New protein synthesis (Spl) (including c-src)
Fig. 1.3 The interaction of CD98 ligand with CD98 and the formation of molecular complex within the cell membrane initiates signal transduction. CD98 may transduce signals by association with pi integrins, cytoskeleton proteins (CP) and other signalling molecules such as PTK (protein tyrosine kinases), and PI3-K (phosphatidylinositol 3-kinase). The activation can lead to downstream activation of MAPKs. With the activation of MAPK, transcrip ion factor (e.g., Spl) activation directs new protein synthesis (eg., c-src) Introduction
1.9.1 Engagement or ligation of CD98
To stimulate activation / interaction of surface molecules with their ligands, cells are often incubated with crossing-linking antibodies, a process called engagement or ligation. To date, dissection of the signal transduction pathways induced by activated
CD98 has been mostly done by means of CD98 ligation. The CD98 activation by mAb treatment leads to activation of protein tyrosine phosphorylation, serine/threonine phosphorylation, G-protein/Ras cascade, and MAPK to mediate CD98-induced biological activities (see alsoChapter 4).
1.9.2 Intracellular changes upon CD98 ligation
1.9.2.1 Tyrosine phosphorylation
CD98 ligation rapidly induces or strongly potentiates tyrosine phosphorylation of many cellular proteins in U2ME-7 (U937-2 cells transfected with HIV gpl60 gene), U937 and
FTH5 (pro-T cell line) cells (Warren et al., 1996; Tabata et al., 1997; Miyamoto et al.,
2000; Cho et al., 2001). However, in contrast to integrin-mediated signalling, none of the CD98-induced tyrosine-phosphorylated proteins have been previously identified
(Warren et al., 1996; Tabata et al., 1997). Using U2ME-7 cells undergoing aggregation and cell fusion, it was demonstrated that CD98 ligation induces and enhances tyrosine phosphorylation of several cellular proteins; two proteins including ppl30 were newly phosphorylated, phosphorylation of ten proteins was increased, and the phosphorylation of two polypeptides was decreased (Tabata et aL, 1997). The CD98-induced phosphoprotein, ppl30, was clearly different from FAK (ppl25FAK), pi integrin, and vinculin which all have similar molecular weights, suggesting that CD98-induced tyrosine phosphorylation is distinct to the integrin-mediated phosphorylation cascade
72 Introduction
(Tabata et al., 1997).
In terms of biological function, CD98-induced tyrosine phosphorylation was shown to be an early obligatory signal to induce multinucleated cell formation (Tabata et al.,
1007), c-src expression (Miyamoto et al., 2000), and murine pro-T cell homotypic aggregation (Warren et al., 1996), but not U2ME-7 homotypic aggregation (Tsurudome and Ito, 2000). Phosphorylation was strongly linked to c-src expression, an essential step in osteoclast-like cell formation (Miyamoto et al., 2000; Higuchi et al., 1999), because PTK inhibitors strongly blocked both c-src mRNA expression and osteoclastogenesis. CD98-induced tyrosine phosphorylation was also shown to be necessary for FTH5 homotypic aggregation (Warren et al., 1996), whereas CD98- induced U2ME-7 cell aggregation was not affected by PTK inhibitors (Tabata et al.,
1997). Therefore, CD98-induced tyrosine phosphorylation seems to be specifically required for certain cellular roles of CD98, especially the initiation of cell fusion. Some discrepancies between murine and human CD98 in this regard remains unresolved.
Because CD98 contains no PTK catalytic region or consensus PTK substrate sequences within its cytoplasmic domain (Warren et aL, 1996), CD98-induced tyrosine phosphorylation is thought to reflect its association with membrane associated tyrosine kinases or with serine/threonine-specific protein kinases, which indirectly activate tyrosine phosphorylation. Clear identification of CD98-induced tyrosine phosphorylated proteins, elucidation of possible association with PTK and the relationship between tyrosine phosphorylation and other kinase activities will be an interesting task for further investigation (see Chapter 3).
73 Introduction
1.9.2.2 Serine/threonine phosphorylation
Serine/threonine phosphorylation is mediated by several enzymes such as protein kinase
A (PKA), PKC, phosphatidylinositol 3-kinase (P13-K) and AKT. Like tyrosine phosphorylation, these enzymes play important roles in many cellular processes such as cytokine production. The involvement of these enzymes can be pharmacologically evaluated by broad or more specific inhibitors. In several experiments, these pharmacological inhibitors have been employed to investigate whether these enzymes control CD98-induced signalling pathways.
Broad specificity serine/threonine phosphorylation inhibitors (H-7 and H-89) inhibited
CD98-induced cell aggregation and fusion of human monocyte cell lines (Tabata et al.,
1997), and a mouse pro-T cell line (Warren et al., 1996). However, other reports did not find any increase in either PKC or PKA activities of whole cells after CD98 ligation, and there was no evidence of membrane translocation of PKCs by CD98 ligation in
U2ME-7 (Tabata et al., 1997).
1.9.2.3 G protein or G-like protein involvement
The heterodimeric guanine nucleotide-binding regulatory proteins (G-proteins) mediate signalling from a large number of diverse seven transmembrane spanning cell surface receptors to a variety of intracellular effectors (Ross and Wilkie, 2000; Sprang, 1997).
These signal transduction pathways control numerous essential functions in all tissues and cells. Ras proteins also play an important role in relaying signals from receptor tyrosine kinases to the nucleus to stimulate cell proliferation and differentiation
74 Introduction
(Kaibuchi et al., 1999). They function as signal mediators for receptor tyrosine kinases and tyrosine-associated receptors. Activation of cell surface receptors by a signalling molecule converts Ras proteins from their inactive, GDP-bound state, to active, GTP- bound state. In particular, these signalling molecules are of interest because they are specifically linked to regulation of cytoskeleton rearrangement which is necessary to mediate signal transduction pathways and changing cell shape, required for cell aggregation or adhesion.
There is some evidence that CD98 function does indeed require activation or involvement of G-protein or Ras proteins, particularly in cell cluster formation and fusion. Using murine cell lines, BAF3 and BAF3/hLFA-l, Suga et al (2001) found that cross-linking CD98 with mAh induces activation of Rap 1 which is a Ras-related small
GTPase. This activation was found to be linked to induction of homotypic aggregation via activation of PI3-K (Suga et al., 2001), although it was required only for fusion, and not aggregation or s-src expression (Miyamoto et al., 2000). In contrast, however, in
Cd^2ME-7 and blood monocytes, the Ras pathway was apparently not involved in triggering homotypic aggregation (Miyamoto et al., 2000). A Ras inhibitor, manumycin
A, displayed inhibitory effects on c-src gene expression and cell fusion (Miyamoto et al.,
2000), suggesting that Ras-mediated pathways may play an important role in CD98- mediated cell fusion.
1.9.2.4 MAPK/ transcription factor activation
A few studies have evaluated the effects of CD98 engagement on MAPK signaling pathways and transcription factor activation. Ligation of CD98 on BAF3/hLFA-l cells
75 Introduction
activated extracellular signal-regulated kinases 1 and 2 (ERKl/2) (Suga et al., 2001).
MAPK involvement was also demonstrated pharmacologically in cell fusion events, although MAPK inhibitors did not affect cell aggregation (Miyamoto et al., 2000).
Specific MAPK inhibitors, PD98059 (a selective inhibitor of ERK 1/2 activator kinases) and SB20380 (an inhibitor of p38 MAPK) strongly suppressed fusion of Cd'^U2ME-7 cells or blood monocytes (Miyamoto et al., 2000).
CD98-induced MAPK activation was also linked to activation of transcription factors.
Two transcription factors, Spl and AP2, bind to the 5’ flanking region of the c-src gene
(Bonham and Fujita, 1983). PD98059 and SB203580 blocked CD98-dependent induction of Spl mRNA in blood monocytes (Miyamoto et al., 2000). CD98 ligation may therefore induce Spl transcription and hence c-src and monocyte differentiation / fusion via activation of MAPK.
1.9,3 Possible view of CD98 and signal transduction
An overall model can be proposed in which interaction of ligands (eg. galectin-3) with
CD98 leads to the formation of CD98 multimers, and this in turn triggers out-side-in signal transduction (Fig. 1.2). The model incorporates the information from amino acid sequence analysis, suggesting that CD98 has no intrinsic tyrosine kinase activity, nor any consensus tyrosine phosphorylation motifs, but that CD98 could possibly be regulated by serine/threonine phosphorylation. The model also incorporates the evidence that CD98 activation is able to activate different biological functions in a specific and cell-dependent manner.
76 Introduction
In human primary monocytes or monocytic cell lines, protein tyrosine kinases appear to be required for CD98 induction of cell fusion. Tyrosine phosphorylation is specifically required for induction of c-src expression, which is an essential step for osteoclast-like cell formation. Unidentified serine/threonine kinases also seem to be involved in both homotypic aggregation and cell fusion processes. Although the details of the signalling pathways related to G-protein or Ras proteins are still to be determined, these pathways may participate in CD98-induced signalling via PI3-K and MAPK. Clearly, the available data are not enough to define the full details of CD98-induced signalling, and further studies are needed to complete our understanding of CD98 regulation of cellular events.
1.10 Conclusion
Although CD98 was originally identified as a phenotypic marker indicating lymphocyte activation, this molecule has now been implicated in several fundamental cellular functions. These are not all mediated by amino acid transport function by CD98LC, and indeed the functions for amino acid transport and CD98HC-mediated pi integrin regulation can be separated by molecular manipulation. Presently, CD98LC-mediated amino acid transport might be involved in regulating proliferation, but CD98HC seems to play important roles in other cellular functions such as Ca^"^ transport, cell adhesion and fusion and T cell costimulation via association with cytoskeleton proteins and pl- integrins. The CD98 molecular complex mediates its multiple biological functions via activation of intracellular signalling pathways, including tyrosine phosphorylation and serine/threonine phosphorylation.
77 Introduction
1.11 Aims
CD98 is important in many aspects of cellular function, such as amino acid transport, cell aggregation and adhesion, virus-induced cell fusion, T cell costimulation, and oncogenic transformation, but an understanding of its molecular mode of action, which would link together its diverse functional roles, is still lacking.
The first aim of this project is to establish a suitable model for looking at CD98-induced signalling and its function in cell aggregation and costimulation of antigen presenting cells. Previous work in this laboratory has described a role for CD98 in the interaction between antigen presenting cells (either U937 as a model cell line, or human peripheral blood dendritic cells) and T cells. The molecular analysis of antigen presenting systems, however, are made much more difficult because the experiments involve co-culture of two cell types, both of which express CD98, and both of which can be the target for pharmacological intervention. As a first step in understanding the role of CD98 on these cells, therefore, we established a quantitative model system in which CD98 induced homotypic aggregation of U937 cells. The establishment and characterisation of this system is described in the first part (Chapter 3) of this thesis. In this context, we mainly focused on understanding the aggregation mechanism, and which surface adhesion molecules cooperated in mediating CD98-induced U937 homotypic aggregation.
The second aim of this thesis is to use the model described above to dissect the major signalling pathways activated in response to CD98 ligation (Chapter 4 and 5). As mentioned above, only a few papers address CD98-induced intracellular signaling.
Furthermore, the available data are not enough to define the full details of CD98-
78 Introduction
induced signalling, and further studies were needed to understand CD98 regulation of cellular events. Using pharmacological and biochemical approaches, therefore, more quantitative dissection of CD98-induced signalling was carried out and the results will be described in this thesis (Chapter 4 and 5).
79 Chapter 2
Materials and Methods.
80 Materials and Methods
2.1 Materials
Metabolic inhibitors (Table 2.1) and broad or more specific signaling enzyme inhibitors or activators (Table 2.2) were purchased from Sigma (Poole, UK), Calbiochem (La
Jolla, CA, USA) and Daewoong Pharm. (Sungnam, Korea), respectively.
Table 2.1 List of metabolic inhibitors.
Compound Specificity Solvent Company Azide ATP synthesis Medium Sigma Colchicine Microtubule formation Ethanol Sigma Cycloheximide Protein synthesis Ethanol Sigma Cytochalasin B Cytoskeleton arrangement Ethanol Sigma Deoxyglucose ATP synthesis Medium Sigma EDTA Ca^^ Medium Sigma
able 2.2 List of enzyme inhibitors.
Compound Specificity Solvent Company
Genistein PTK inhibitor Ethanol Calbiochem
Herbimycin A PTK inhibitor Enthanol Sigma
A77,1726 PTK inhibitor Medium Daewoong Pharm H-7 Broad specific serine/threonine Medium Calbiochem kinase inhibitor H-89 Broad specific serine/threonine Medium Calbiochem kinase inhibitor KT5720 Specific PKA inhibitor DMSO Calbiochem
Forskolin Adenylate cyclase activator DMSO Sigma
81 Materials and Methods
Rottlerin Specific PKCÔ inhibitor DMSO Calbiochem Myristoylated Specific PKCa/p inhibitor Medium Calbiochem PKC peptide Inhibitor 19-27 Calphostin C Specific diacylglycerol (DAG)- DMSO Calbiochem sensitive PKC inhibitor Go6976 Specific Ca^^-sensitive PKC DMSO Calbiochem Inhibitor Chelerythrine Broad specific PKC inhibitor DMSO Calbiochem Chloride Staurosporin Broad specific PKC inhibitor Ethanol Sigma
GF109203X Broad specific PKC inhibitor DMSO Calbiochem
Go6983 Broad specific PKC inhibitor DMSO Calbiochem
PMA PKC activator Ethanol Sigma
Dioctanoylglycerol DAG analogue Ethanol Calbiochem D609 Phosphatidylcholine-specific DMSO Calbiochem phospholipase C inhibitor Propranolol Phosphotidate Medium Calbiochem phosphohydrolase inhibitor inhibitor) PD98059 ERK 1/2 kinase inhibitor DMSO Calbiochem SB20380 p38 inhibitor DMSO Calbiochem LY294002 Phosphatidylinositol 3-kinase DMSO Calbiochem Inhibitor Okadaic acid Protein phosphatase 1/2A DMSO Calbiochem Inhibitor Cyclosporin A Protein phosphatase 2B Ethanol Sigma Inhibitor
A panel of mAbs to surface proteins (Table 2.3), direct-labelled mAbs (Table 2.4) and
82 Materials and Methods
mAbs to signaling enzymes (Table 2.5) were used. The mAb concentration was
determined by SDS PAGE with Coomassie Blue staining compared to standard mAb
(CD44, El/2). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and all other chemicals were purchased from Sigma Chemical Co. (Poole, UK). Protein
G-Sepharose was obtained from Calbiochem (La Jolla, CA, USA). Nitrocellulose
membrane and ECL reagent were from Amersham Life Science (Little Chalfont,
Buckinghamshire, UK). Bradford reagent was obtained from Bio-Rad
(HeidemannstraBe, München, Germany). Fetal calf serum (PCS) and RPMI1640 were
obtained from GIBCO (Grand Island, NY, USA). U937 cells were purchased from
ATCC (Rockville, MD, USA).
Table 2.3 List of unlabelled mAbs to surface molecules.
Specificity MAb name Isotype Species Originator / Company CD18 CLB-LFAl G1 Mouse CBL BU86 G1 Mouse D. Hardie CD29 MEMIOIA GI Mouse V. Horejsi P5D2 G1 Mouse N. Hogg MAR4 Gl Mouse S. Menard CD43 161-46 Gl Mouse R. Vilella HI161 Gl Mouse D. C. Shen CD44 El/2 Gl Mouse Leinco Tech P2A1 G2a Mouse Gene Tex CD98 BK19.9 Gl Mouse A. J. Van Agthoven MEM 108 Gl Mouse V. Horejsi CAF7 Gl Mouse H. Taskov AHN-18.1 Gl Mouse K. M. Skubitz
83 Materials and Methods
AHN-18 Gl Mouse K. M. Skubitz BU53 G2a Mouse D. L. Hardie BU59 Gl Mouse D. L. Hardie 4F2 G2a Mouse D. Fox CD147 MEM M6/1 Gl Mouse V. Horejsi H84 G2b Mouse K. Segawa
Table 2.4 List of direct labelled antibodies.
Ab name Specificity Fluorescence Company 7E4 CD18 FITC Immunotech 4B4-RD1 CD29 TRITC Coulter FlO-89-4 CD45 FITC Serotec 84H10 CD54 FITC Immunotech UM-8D6 CD147 FITC Ancell 9-49 HLA-DR FITC Immunotech
Table 2.5 List of antibodies to signaling enzymes.
Ab name or Specificity Dilution Species Originator / catalogue # Company 4G10 p-Tyr 0.25 Mouse Upstate Biotech pg/ml C-17-G JNK 1/2000 Rabbit Santa Cruz
G-7 phospho-JNK 1/2000 Mouse Santa Cruz
06-333 ERK 1/2000 Mouse Upstate Biotech
PTEpY phospho-ERK 1/4000 Rabbit Promega
9212 p38 1/1000 Rabbit New England Biolabs
9211S phospho-p38 1/1000 Rabbit Cell signaling
84 Materials and Methods
P16520 PKCa 1/1000 Mouse Transduction lab P17720 PKCp 1/250 Mouse Transduction lab
P20420 PKCy 1/5000 Mouse Transduction lab
P36520 PKCÔ 1/500 Mouse Transduction lab P14820 PKCe 1/1000 Mouse Transduction lab P15120 PKCe 1/250 Mouse Transduction lab P22529 PKCA. 1/250 Mouse Transduction lab C-20 PKCÇ 1/2000 Rabbit Santa Cruz T(P)505 phospho-PKCô 1/2000 Rabbit P. J. Parker T(P)560 phospho-PKCÇ 1/2000 Rabbit P. J. Parker
2.2 Cell culture
Promonocytic U937 cells, established from malignant cells from the pleural effusion of a 37 year-old Caucasian male with diffuse histiocytic lymphoma (Sundstrom and
Nilsson, 1976) were maintained in RPMI 1640 supplemented with 10% PCS. Cells were grown at 37°C and 5% CO 2 in humidified air.
2.3 Quantitative homotypic cell aggregation assay
Twenty pi of cells in RPMI 1640 medium supplemented with 10% PCS at 10^ cells/ml were placed in round bottom wells of a 96-microwell plate. An equal volume of medium, with or without appropriate antibody, was added, and the cells were incubated at 37°C for 4-7 h. The cells were resuspended gently, so as not to break up the clusters, and the number of unaggregated and total cells were counted in a haemocytometer and percentage of cells in aggregates was determined by the following equations:
Equation 1: % of cells in aggregates = [total cells-free cells/total cells] x 100.
85 Materials and Methods
Equation 2: % of control = [% of aggregation (drug or blocking mAb-treated group / % of aggregation (only aggregating mAb-treated group)] x 100.
2.4 Cytofluorometric analysis
Expression of CD98 on cell surface of U937 cells was determined by flow cytometry.
Cells (10^) were washed with phosphate-buffered saline (PBS) staining buffer
(containing 2% PCS and 0.1% sodium azide) and incubated in 50 pi of staining buffer containing 10% rabbit serum for 10 min on ice and then with the primary antibody for a further 45 minutes. After washing three times with staining buffer, cells were treated with 1/20 dilution of fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG secondary antibody (Dako, Dakopatts, High Wycombe, UK). Cells were then washed three times with staining buffer and analyzed on a FACScan (Becton Dickinson).
Expression of CD29, CD 147, CD 18, CD45 and HLA-DR was analysed by direct binding of fluoresceinated antibodies (Table 2.4) using flow cytometry as above.
2.5 Subcellular fractionation for PKC translocation assay
Four ml U937 cells (5 x 10^ cells/ml) were plated in 6 well plates under serum-free conditions. After 3 h recuperation, pharmacological inhibitors were added to the cells as appropriate, for a further 30 min. CD98 mAh (ANH-18) was then added for 20 min, and the cells were then transferred on to ice, and washed three times with ice-cold phosphate-buffered saline. 800 pi of homogenisation buffer A (20 mM Tris-HCl, pH 7.4,
2 mM EDTA, 2 mM EGTA, 50 mM p-glycerophosphate, 1 mM dithiothreitol (DTT), 1 mM Na3V0 4 , each 10 pg/ml of leupeptin, aprotinin and pepstatin, 0.4 mM phenylmethylsulfonyl floride (PMSF), 1 mM benzimide, 2 mM hydrogen peroxide,
86 Materials and Methods
0.25 M sucrose) were added and the cell lysate was sonicated for 10 s three times. The homogenate was centrifuged at 2000 x g for 5 min at 4°C. The supernatant was further centrifuged at 100,000 x g for 25 min at 4°C to obtain the cytosolic fraction. The pellet was resuspended in 300 pi of homogenisation buffer B (2% Triton X-100 in buffer A) and sonicated for 10 s twice. After incubation on ice for a further 25 min, the suspension was centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was collected and used as membrane fraction. Protein concentration of each sample was determined by the Bradford method and 2 x Laemmli sample buffer was added before boiling for 5 min.
For preparing whole cell lysates, cells (5x 10^ cells/ml) were washed three times in cold
PBS containing 1 mM sodium ortho vanadate, and lysed in lysis buffer (20 mM Tris-
HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 50 mM p-glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, 1% Triton X-100, 10 % glycerol, 10 pg/ml leupeptin, 10 pg/ml aprotinin, 10 pg/ml pepstatin, 1 mM benzimide and 2 mM hydrogen peroxide) for 30 min with rotation at 4°C. The lysates were clarified by centrifugation at 16,000 x g for 10 min at 4®C.
2.6 Western Blotting
Samples containing equal amoimt (40 or 80 pg/lane) of protein were loaded in each lane and separated on 10% SDS-polyacrylamide gel. The gel was transferred by electroblotting to nitrocellulose membrane. Membranes were blocked for 60 min in
TTBS (3% BSA in Tris-buffered saline containing 20 mM NaF, 2 mM EDTA, 0.2%
Tween 20) at room temperature. Immunoblots for phosphorylated or non-
87 Materials and Methods
phosphorylated proteins of eight PKC isoforms, MAPK/p38, MAPK/ERK, and
MAPK/JNK, and phosphotyrosine proteins were carried out using specific antibodies against phosphorylated or nonphosphorylated forms, and secondary horseradish peroxidase-conjugated rabbit anti-mouse or swine anti-rabbit immunoglobulins.
Peroxidase activity was visualised by chemiluminescence detection (ECL reagents,
Amersham, Little Chalfont, Buckinghamshire, UK). Intensity of bands on film was quantified using Synoptic image analysis system and software (Synoptic, Cambridge,
UK).
2.7 Immunoprécipitations
The cells were washed with ice-cold PBS and lysed in lysis buffer. After centrifugation for 5 min at 12,000 x g, the supernatant (whole cell extract (0.5 mg/ml)) were subjected to immunoprécipitation with both anti-PKCÔ and anti-phospho PKC5 antibodies overnight at 4°C. Then, protein G-Sepharose was added, and the mixture was incubated for 2 h. The immunoprecipitates were washed three times with washing buffer containing 50 mM Tris-HCl, 0.5% NP-40, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA,
1 mM Na3V0 4 , 1 mM DTT, 1 mM NaF, 1 mM PMSF and each 10 pg/ml pepstatin, aprotinin and leupeptin. The immunoprecipitates were boiled in the sample buffer of
SDS-PAGE. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The residual binding sites were blocked by incubating the membrane with
3% BSA or 5% non-fat milk protein in TTBS. The membranes were incubated with anti-phosphotyrosine antibody for 1 h. After washing, the membranes were incubated with anti-mouse IgG peroxidase conjugate and developed by ECL. Materials and Methods
2.8 Purification of CD98 mAb (AHN-89)
Protein A coupled to Sepharose CL-4B (Sigma, 1 ml containing 2 mg protein) was swollen in PBS containing 0.1% sodium azide for 24 hours. One ml of gel solution was packed into the column and washed with 10 ml 0.1 M phosphate buffer (pH 8.0), 4 ml citric acid solution and 10 ml 0.1 M phosphate buffer, respectively. On the other hands, mAb culture supernatant (total 2 1, a gift from Prof. K.M. Skubitz, Minnesota University,
USA) was precipitated with ammonium sulfate and dialysed with 0.1 M sodium phosphate buffer (pH 8.0, final 100 ml). Ten ml from the dialysate was applied to the
Protein A column at approximately 5 ml/h. After collecting a flow-through, the column was washed with 5 ml of 0.1 M phosphate buffer. The unretarded fraction containing non-immunoglobulin components was also collected. Elution to purify IgGl isotype was started by treatment with 15 ml 0.1 M sodium citrate buffer (pH 4.5). The fraction size was 1 ml per Eppendorf tube, containing 60 pi of 2 M Tris solution. The column was washed with 4 ml citric acid solution and then stored in 0.1 M phosphate buffer containing 0.2% sodium azide. The protein elution profile was monitored by measurement of the absorbance at 280 nm. The purity was checked by SDS-PAGE.
Three fractions with the highest OD values were pooled and dialysed two times in PBS.
2.9 Preparation of F(ab’)2 and Fab fragments from purified AHN-18
CD98 mAb (AHN-18) solution dialysed against 0.1 M citrate buffer, pH 3.5 were subjected to pepsin digestion to prepare F(ab’)2 fragments. Briefly, 3 ml of mAb solution was digested with 50 pg pepsin (EC 3.4.23.1) (Sigma) for F(ab’)2 or papain for
Fab. The mixture was incubated at 37°C for 10 h and then neutralized with 2 M Tris
(270 pi in 3 ml mixture) to stop the enzymatic reaction. Undigested antibody was
89 Materials and Methods
removed on a protein A-Sepharose column by allowing it to bind to protein A. Purity of
F(ab’)2 or Fab fragments (flow-through) was confirmed by SDS-PAGE.
2.10 Biotin conjugation of AHN-18
CD98 mAb (AHN-18) solution dialysed against 0.1 M sodium bicarbonate buffer was subjected to biotin conjugation to prepare labelled antibody. 5 ml of mAb solution was mixed with 150 pi of 2 mg/ml biotin (biotin N-hydroxysuccinimide, sigma) solution dissolved in DMSO. The mixture was incubated at room temperature for 4 h with stirring. Unreacted biotin was removed by dialysis against PBS overnight
2.11 Protein determination (Bradford method)
Protein determination was performed by using the Bradford reagent as described by the manufacturer (Bio-Rad). Eight hundred pi of diluted samples was mixed with 200 pi
Bradford reagent and incubated for 10 min at room temperature. Optical density (OD) values were determined at 595 nm. Bovine serum albumin was used as standard protein.
2.12 MTT assay (colorimetric assay) for measurement of cell viability
Cell viability was measured by standard MTT assay. 10 pi of MTT solution (10 mg/ml in phosphate buffered-saline) was added to each well of U937 cultures for 3 h before the end of the culture period. The cells were lysed by the addition of 15% SDS for solubilization of formazan and the OD at 570 nm (OD570-630) was measured using a
Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
90 Materials and Methods
2.13 Statistical analysis
All values expressed as mean ± SEM were obtained from 3 ~ 6 observations (for one experiment). The Student's ^-test for unpaired observations between control and experimental samples was carried out to evaluate statistical difference; p values of
=/<0,05 were regarded as statistically significant.
91 Chapter 3
The functional interactions between CD98, pi-integrins, and CD147 in the induction of U937 homotypic aggregation
92 Functional molecular complex including CD 147
Abstract
CD98 is expressed on both haematopoietic and non-haematopoietic cells and has been implicated in a variety of different aspects of cell physiology and immunobiology. This study examines the functional interactions between CD98 and other adhesion molecules on the surface of the promonocyte line U937, using a quantitative assay of cell aggregation. Several of the CD98 antibodies induced homotypic aggregation of these cells, without affecting cellular viability or growth. Aggregation induced by CD98 antibodies could be distinguished from that induced by pi-integrin (CD29) ligation by lack of sensitivity to EDTA, and by increased sensitivity to deoxyglucose. Aggregation induced via CD98 and CD29 could also be distinguished by the pattern of protein tyrosine phosphorylation induced. Some CD29 antibodies partially inhibited CD98- induced aggregation, and these antibodies were neither agonistic for aggregation, nor inhibitors of pi-integrin binding to substrates. Conversely, some CD98 antibodies were potent inhibitors of CD29-induced aggregation. Antibodies to p2-integrins also partially inhibited CD98-induced aggregation. Unexpectedly, two antibodies to CD 147, an immunoglobulin superfamily member whose function has remained unclear, were also potent inhibitors of both the aggregation and the protein tyrosine phosphorylation induced via CD98 ligation. The results of this study support a central role for CD98 within a multi-molecular unit which regulates cell aggregation.
93 Functional molecular complex including CD 147
3.1 Introduction
CD98 is the heavy chain (85 kD) of a cell-surface dimeric molecule found on the surface of many haematopoietic cells (Warrant et ah, 1996; Lumadue et ah, 1987). It is a type II integral membrane protein, with a long cytoplasmic portion of 81 amino acids.
Some studies have suggested that it may act as ligand for the cell surface lectin galectin-
3 (Dong and Hughes, 1997). CD98 is found covalently linked to one of several (six have been identified to date) alternative light chains, at least four of which have been identified as members of an amino acid transporter family (Verrey et ah, 1999). There is also good evidence that CD98 is functionally associated with pi integrin molecules on the cell membrane (Zent et ah, 2000; Fenczik et ah, 1997), although the consequences of this association remain unclear.
The most striking feature of CD98 is the extraordinary diversity of functions in which it has been implicated (Deves and Boyd, 2000). Antibodies against CD98 block the formation of cell syncitia by HIV and other viruses (Ohgimoto et ahm 1996; Okamoto et ah, 1997), and it appears also to play a more general role in regulation of integrin- mediated cell adhesion (Fenczik et ah, 1997; Tabata et ah, 1994; Chandrasekaran et ah,
1999). CD98 has been implicated in haematopoietic cell differentiation, growth, transformation and apoptosis (Warrant et ah, 1996; Hara et ah, 1999), and recently this role has been extended to the regulation of osteoblast differentiation (Higuchi et ah,
1999). CD98 also plays a role in the regulation of amino acid transport by virtue of its associated light chain, as discussed above. Most recently, the molecule has been implicated in the regulation of both antigen presenting cell function and T cell
94 Functional molecular complex including CD 147
activation (Diaz and Fox, 1998; Diaz et al., 1997). Our group's interest in CD98 arose first because we described its presence at very high levels on the surface of human dendritic cells Woodhead et al., 1998). Subsequently we showed that some antibodies to this molecule could block the ability of both the U937 promonocyte line (Stonehouse et al., 1999) and human peripheral blood derived dendritic cells (Woodhead et al., 2000) to deliver essential co-stimulatory signals to T cells.
One central question in the biology of CD98 has been how to understand the nature of its functional interactions with integrins and other adhesion molecules at the cell surface.
In this study we have developed and used a new quantitative assay of homotypic aggregation of U937 cells, and we have re-examined this question in detail. Our results confirm that there is an important interaction between CD98 and CD29 (pi-integrin), but demonstrate that the cellular events following activation via these two molecules can be clearly distinguished pharmacologically, biochemically and in terms of their differential sensitivity to antibody modulation. Thus the down stream functional effects of CD98 ligation are not mediated solely via pi-integrins. Furthermore, both the specificity profile and kinetic data suggest that pi integrins are involved in modulating
CD98 activity, but not in mediating the U937 homotypic adhesion. Finally, our study identifies a new member of the CD98 functional unit, the immunoglobulin superfamily member CD 147 , whose function has remained unclear hitherto. Taken together with the previously published data on CD98, the results presented below suggest that CD98 plays a central role within a multi-molecular unit which may serve to regulate cellular responses to changes in the tissue micro-environment.
95 Functional molecular complex including CD 147
3.2 Results.
3.2.1 Aggregation-inducing activity of CD98 antibodies is heterogeneous, and is not correlated with binding activity.
The binding of a panel of seven mAbs specific for CD98 to U937 cells is shown in Fig.
3.1A. All showed a unimodal binding profile by flow cytometry, and the level of binding was dependent on antibody concentration. The panel of mAbs was then tested for their ability to induce homotypic aggregation of U937 cells (Fig. 3.1B panels B-E, and Table 3.1). The mAh CD98-AHN-18 was the most potent inducer of aggregation; several others induced weaker aggregation (BK19.9, BU53, 4F2), while some mAbs did not induce any aggregation. An mAb known to activate signalling and homotypic aggregation through the CD29 pi-integrin chain (MEM 101 A), and an mAh to CD43
(161-46), which is believed to induce aggregation via a pi-integrin independent pathway (King and Katz, 1989) were also tested in the aggregation assay. Both these mAbs did induce aggregation of the U937 cells (panels F, G), although the morphology of the clusters was different from that induced by CD98-AHN-18, as the clusters tended to be tighter and more compact. Aggregation was not induced simply by the presence of mAh on the surface of U937, since mAbs to CD44, another molecule present on the surface of U937 (Freistadt and Eberle, 1997), did not induce aggregation (panel H).
The ability of the panel of CD98 mAbs to induce aggregation did not correlate with the level of binding to the U937 cell surface. To confirm this further, flow cytometry at a range of mAh concentrations was used to select a titre that gave a mean fluorescent
96 No AHN-18.1
\10* ni* T6 ^ To* 10* To* F L 1 -H
AHN-18
10"in* 10’’ i in* 10* in10* 10^ 10 * 10 ’ 10 * 10 * 10 *
BK19.9 BU53
°io" 10^ 1 0 " 10* 10’ 10 * 10 * 10 * FL 1-H LL
BU89 MEM 108
10* 10 ’ 10 * 10 * 10* 10* 10’ To*k To* io*
Fig. 3.1 A CD98 mAbs differ in their ability to bind to the surface of U937 cells, and their ability to induce homotypic aggregation.A panel of seven CD98 mAbs were tested for their ability to bind U937 eelis using flow cytometry. All mAbs were tested over a wide range of concentrations (1-30 pg/ml), and the histograms shown in the figure are those obtained at saturating doses of mAb (between 1 and 30 pg/ml). k' O
l ^ m
W-##È
Fig. 3.1 B CD98 mAbs differ in their ability to bind to the surface of U937 cells, and their ability to induce homotypic aggregation. U937 cells were incubated with mAbs (at dilutions which induced maximal aggregation in each case, between 0.3 and 1.5 pg/ml in each case) for 6 h, as described in methods. Images of cells in culture at this time point were obtained using an inverted phase contrast microscope, attached to a video camera, and captured using NIH image software. A. Medium alone. B. CD98-AHN-18 (CD98). C. BU53 (CD98). D. BU89 (CD98). E. 4F2 (CD98). F. MEM lOlA (CD29). G. 161-46 (CD43). H. El/2 (CD44). Functional molecular complex including CD 147
Table 3.1 Lack of correlation between cell surface binding and aggregation- inducing activity of CD98 mAbs.
MAb Cell surface binding Aggregation AHN-18.1 134 ±0.6 9 ± 2 AHN-18 159 ±19 54 ±9 BK19.9 152 ±10 17±3 BU53 200 ±4 32 ±6 BU89 184 ±5 10 ± 1 MEM 108 193 ± 1 11 ±6 4F2 108 ±2 20 ±2
U937 cells were incubated with the CD98 mAbs shown at a wide variety of concentrations (between 0.5 and 30 pgm/ml), and tested both for binding to U937 and for the ability to induce aggregation. The data shown in the Table 3.1 were selected so as to show aggregation at a concentration that gave comparable cell surface binding, with MFI between 100-200. Binding to the U937 cells was measured by indirect immunofluorescence and flow cytometry. MFI was calculated using WIN-MDI software on a minimum of 5000 cells. Aggregation at 6 h was measured using the quantitative assay described in Methods. The results are shown as mean for triplicate cultures ±
SEM.
99 Functional molecular complex including CD 147
channel number (MFI) of between 100 and 200, Aggregation at this titre was then measured using the quantitative assay. As shown in Table 3.1, aggregating activity is clearly independent of binding activity, and aggregation levels vary widely even when the concentrations of mAh used were chosen to give comparable levels of binding to the
U937 cells.
The characteristics of CD98-AHN-18 induced aggregation, as well as binding, were examined in more detail (Fig. 3.2). Binding increased in a linear fashion for six hours and then reached a plateau. In contrast, aggregation induced via CD29 (MEM 101 A) or
CD43 (161-46) was much more rapid (Fig. 3.2A). The quantitative level of CD98-
AHN-18 induced aggregation was dose dependent, but was maximal at sub-saturating doses of bound mAb. Further increase in mAh concentration above this optimal level resulted in a lesser degree of aggregation (Fig. 3.2B) F(ab’)2 fractions of CD98-AHN-
18 also induced aggregation (Fig. 3.2B) The presence of high levels of human or rabbit
Ig did not inhibit clustering (data not shown). In contrast. Fab fragments of CD98 failed to induce any clustering.
3.2.2 CD98-induced aggregation does not lead to cell death.
Since a previous study (Warrant et al., 1996) had suggested that CD98 triggering could induce cell death, we tested whether the CD98 mAh AHN-18 also caused killing of
U937. As shown in Fig. 3.3, CD98-AHN-18 did not induce decrease MTT reduction at
48 h (Fig. 3.3A). Furthermore, CD98-AHN-18 did not appear to cause growth arrest of the U937 cells, since cell numbers during 48 h in culture increased to the same amount in both treated and control groups (Fig. 3.3B).
100 (A) (B)
2000 -
O)
0)20 -
0 1 2 3 4 5 6 7 Time (hours) % MFI whole antibody -$ -C D 9 8 % aggregation whole antibody - * -C D 2 9 % aggregation (Fab)2 • A CD43 % aggregation Fab
Fig. 3.2 Time and dose dependency of aggregation induced by CD98- J3/AHN-18. A) U937 cells were incubated with CD98-AHN-18 (1 pg/ml), MEM lOlA (CD29, 0.3 pg/ml) or 161-46 (CD43, 0.3 pg/ml) for various times as shown. Aggregation was measured as described in Methods. The results show mean aggregation from triplicate cultures for one representative experiment out of 2. B) U937 cells were incubated with CD98-AHN-18 or fragments of CD98- AHN-18 at different concentrations for 6 h. Aggregation and binding were measured as for Fig. 3.1. The results show mean aggregation ± SEM for triplicate cultures for one representative experiment out of 2. (A) (B)
180 2.5 160 140 120 100 ■o 80 S 60 40 I 20 0 20 10 5 2.5 24 hours 48 hours Antibody concentration (fig/m!) ■ No antibody — isotype control □ CD98-AHN-18 -*-CD98-AHN-18 0 Isotype control
Fig. 3.3 CD98-AHN-18 mAb does not induce apoptosis of U937 cells, or inhibit cell division. A) U937 cells (5 x 10^ cells/ml) were incubated in the presence of different concentrations of CD98-AHN-18 mAh or isotype control in complete medium. MTT reduction was measured after 48 h. Results are expressed as % OD (mean ± SEM for triplicate cultures), relative to OD in the absence of mAb. The isotype control significantly stimulated MTT reduction at the highest concentration (p < 0.01), but all other values did not differ significantly from control. B) U937 cells (5 x 10^ cells/ml) were seeded into 96 well plates, and incubated for 24 or 48 h in the presence of CD98-AHN-18 mAh (1.5 pg/ml) or an isotype control. Cells were harvested and counted using trypan blue staining and light microscopy. The results show mean ± SEM for triplicate cultures. No value differed significantly from control. Functional molecular complex including CD 147
3.2.3 CD98-induced aggregation, but not CD29 induced aggregation is resistant to
EDTA but sensitive to deoxyglucose.
Since CD98 has been reported to associate with CD29 (pl-integrin) in the cell surface
(Fenczik et al., 1997), we compared the ability of a number of inhibitors of cell function to block the aggregation induced by mAbs to these two molecules, as well as aggregation induced by CD43. As shown in Fig. 3.4A, aggregation by mAbs to CD29 and CD98 share the properties of being sensitive to colchicine, cytochalasin B, and low temperature, but insensitive to cycloheximide. EDTA blocks the ability of the activating pl-integrin mAh (MEM 101 A) to induce aggregation; however, aggregation induced by the CD98 mAh AHN-18 is insensitive to the presence of EDTA. Aggregation induced by CD43 mAb, in contrast, was sensitive only to low temperature, suggesting a completely different mechanism of action for this molecule.
CD98-dependent aggregation was also much more sensitive than CD29 or CD43- dependent aggregation to inhibition by the metabolic inhibitor deoxyglucose (Fig. 3.4A).
However, sensitivity to deoxyglucose was lost soon after addition of aggregating m (Fig.
3.4B), suggesting that ATP-dependence was an early step in the CD98-induced signalling pathway.
3.2.4 CD98*induced aggregation and CD29-induced aggregation are associated with distinct patterns of tyrosine phosphorylation.
Previous studies have identified tyrosine phosphorylation as a down-stream event in signalling by both pi-integrins (Meng et al., 1998) and CD98 (Tabata et al., 1997). As
103 (A)
EDTA (2 mM)
Deoxyglucose (10 mM)
Cytochalasin (luM)
Colchicine (10 uM)
Cycloheximide (10 ugm/ml)
No antibody
0 20 40 80 100 120 Aggregation (% of control) CD98 mcm9 HCD43 (B) 120 -
80
O)
0 2 4 6■2
Time of deoxyglucose addition (hours) Fig. 3.4 Metabolic inhibitor profile for CD98-, CD29- and CD43- induced aggregation. A) Aggregation was measured under standard conditions as described in Methods, in the presence of CD98-AHN-18 (1.5 pg/ml), MEM lOlA (CD29, 0.3 pg/ml) and 161-46 (CD43, 0.3 pg/ml). The inhibitors were added 1 h prior to the mAh and remained in the culture. Results are expressed as aggregation relative to control cultures in the presence of each aggregating antibody, but absence of inhibitor (mean ± SEM, triplicate cultures). All values which differ significantly from controls (p <0.01) are indicated by an asterisk. B) As for A) but the deoxyglucose (10 mM) was added either before, simultaneously or after CD98-AHN-18. Aggregation in all eases was measured at 6 h. Results are expressed as aggregation relative to control cultures in the presence of each aggregating antibody, but absence of inhibitor (mean ± SEM, triplicate cultures). Functional molecular complex including CD 147
shown in Fig. 3.5, a comparison of the pattern of phosphotyrosine proteins induced following activation of the U937 cells with CD98-AHN-18 is similar, but distinct to that induced by the activating CD29 mAh MEM 101 A. In particular, CD98-AHN-18, but not MEM 101 A, induces a rapid strong phosphorylation of a 72 kD band. Both mAbs induced bands at 114 and 155 kD.
3.2.5 Reciprocal cross-inhibition by antibodies to CD98 and CD29.
In order to probe further the interaction between CD29 and CD98 in the induction of
U937 aggregation, the abilities of blocking mAbs to each of these two molecules to inhibit aggregation induced by the other was tested. As shown in Fig. 3.6A two non aggregating CD98 mAbs, MEM 108 and BU89 strongly inhibited CD98-AHN-18 induced aggregation. At the same concentration, CD98-MEM108 showed no inhibition of aggregation induced by CD29 agonist mAb MEM 101 A, while CD98-BU89 showed a significant, but partial inhibition (panel B). In the reciprocal experiment, two inhibitory mAbs to CD29, P5D2 and MAR4 showed strong inhibition of CD29-induced
(MEM 101 A) aggregation (panel E). P5D2, which almost totally blocked the agonist action CD29-MEM 101 A, and is known to block binding of pl-integrin dimers to fibronectin (Fenczik et al., 1997; Wayner et al., 1993) did not have any inhibitory activity against CD98-AHN-18 induced aggregation, and indeed reproducibly enhanced the aggregation observed (panel D). The other CD29 blocking mAb, MAR4 (which does not block binding of pl-integrins to fibronectin (Tanaka et al., 1997)) showed a small but reproducible inhibition of CD98-AHN-18 induced aggregation (Panel D).
None of the blocking mAbs to either CD98 or CD29 tested had any inhibitory effect on
CD43-induced aggregation (Panels C and F).
105 V\) 13)
Time (min) 0 1 5 10 20 40 60 0 0.5 1 10 20 40
p p ii4 ^ i 00 §0 00 #0 ^ ^ pplOT PP72
Fig. 3.5 Ligation of CD98 or CD29 induces rapid and distinct patterns of tyrosine phosphorylation. U937 cells (5 x 10^ cells/ml) were incubated in the presence of CD98-AHN-18 (1.5 ug/ml, panel A) or MEM 101A (CD29, 0.5 pg/ml, panel B) for various time periods. Cells were lysed and analysed for phosphotyrosine proteins as described in Methods. Molecular weights of the major phosphorylated species are shown on the left, and were calculated by image analysis of the Western blot, using molecular weight standards to calibrate the software. CD98 CD29 CD43 150i 120 100 80 CD98 60 I I 1 60 BU89 I I 40 MEM1 20 0 0 1 2 4 8 0 1 2 4
140i
CD29 120 120 — — - ICO ICO 80 60 40 20 0 0 1 2 4 0 1 2 4 8 0 1 2 4
Fig. 3.6 Cross-inhibition of U937 homotypic aggregation between CD98 and CD29 mAbs. Aggregation was measured under standard conditions in the presence of CD98-AHN-18 (1.5 pg/ml, left panels), MEM lOlA (CD29, 0.3 pg/ml., middle panels) or 161-46 (CD43, 0.3 pg/ml, right panels). Blocking mAbs were added to U937 cultures 1 h prior to the aggregating mAbs. Blocking mAbs used were to CD98 (panels A, B and C) or to CD29 (panels D, E and F). Results are expressed as % aggregation relative to the aggregation in the absence of blocking amAb (column labelled 0 in each panel). Means which differ significantly from control (absence of inhibitory mAb, p < 0.05) are shown with an asterisk. Antibodies were tested at a series of dilutions, starting with the most dilute and increasing in two fold steps along the x-axis. To obtain the actual concentration of mAh at each point (in pg/ml), the x-axis value should be multiplied by 0.6 for BU89 (CD98, empty bars), 0.3 for MEM 108 (CD98, solid bars), 1.25 for P5D2 (CD29, empty bars) and 0.5 for MAR4 (CD29, solid bars). Functional molecular complex including CD 147
3.2.6 Inhibition of CD98-induced homotypic aggregation by antibodies to p2 integrins.
Previous studies have implicated p2, as well as pi integrins, in mediating CD98- induced adhesion. As predicted, blocking mAbs to CD 18, the p2-integrin chain, partially blocked aggregation induced via CD98 (Fig. 3.7 top panel ), but not via CD29
(middle panel B) or via CD43 (bottom panel). Both antibodies, at the same concentrations, showed strong inhibition of aggregation induced by PMA (not shown).
A panel of ICAM-1 and -2 and -3 (CD54, CD 102, CD50,) mAbs were also tested, but none inhibited aggregation induced by CD98 (not shown).
U937 cells express relatively little p2-integrin at their cell surface, and the majority is retained within intracellular vesicles. One mechanism of action for CD98 might therefore be to cause a redistribution of CD 18 to the cell surface. The cell surface levels of both pi and p2 integrin chains were therefore measured following CD98 ligation, using directly fluoresceinated anti-CD 18 or CD29 mAbs, and flow cytometry. The levels of expression of CD29 and CD 18 were unchanged, however, following CD98-
AHN-18 induced aggregation (Fig. 3.7B).
3.2.7 Inhibition of CD98-induced homotypic aggregation by antibodies to CD147.
Since inhibition by integrin mAbs was only partial, we tested other mAbs previously shown to be expressed on U937 cells (Stonehouse et al., 1999) for the ability to block
CD98-induced aggregation. Unexpectedly, the only significant inhibition was by two mAbs to CD 147 (Fig. 3.8A left panel, and Fig. 3.8B). The CD 147 mAbs also significantly inhibited aggregation induced via CD29 (Fig. 3.8A middle panel). Neither
108 Functional molecular complex including CD 147
CD 147 mAbs inhibited aggregation induced via CD43 (Fig. 3.8A left panel). Addition of CD 147 mAb together with CD 18 mAh, CD29 mAb or all three together did not result in further inhibition over the level observed using each antibody independently
(data not shown).
In order to establish whether CD 147 was involved in the induction (signalling) phase of
CD98-induced aggregation, the influence of CD 147 mAh on CD98-AHN-18 induced tyrosine phosphorylation was examined (Fig. 3.9). The presence of CD 147 antibody, as well as the blocking CD98 mAb BU89, almost completely blocked phosphorylation of the 72 and 155 kD band induced by CD98-AHN-18 ligation (lanes 3 and 5).
Phosphorylation of the band (s) at around 114 kD was less strongly inhibited. CD 147 also partially inhibited CD29-induced tyrosine phosphorylation (lanes 4 and 6). CD 147 ligation alone did not induce any changes in protein tyrosine phosphorylation (Fig. 3.9 lane 2). CD 147 mAbs did not alter the level of CD98 expression when added either 4, 9 or 24 h before staining (data not shown).
Finally, to try to distinguish further between induction and effector (cell-cell binding) phases of the CD98-AHN-18 induced homotypic aggregation, the ability of the various mAbs to block aggregation was tested when added either prior to, simultaneously or after addition of the agonist antibody CD98-AHN-18 (Fig. 3.10). CD29 mAb MAR4 blocked CD98-AHN-18 induced aggregation when added 1.5 h before addition of
CD98-AHN-18, but not when added simultaneously or 1.5 h afterwards. In contrast, mAbs to CD98 itself, to CD 18 and to CD 147 inhibited to the same extent whether added before CD98-AHN-18 or up to 1.5 h afterwards.
109 1 2 0 -T------
CD98
4 8
CD29
4 8
140 n- 120 100 CD43 80 60 40 20 0 0
CD18/CLB-LFA1
CD18/BU86
Fig. 3.7A Functional interactions between CD98 and (32 integrins (CD 18). Aggregation was measured under standard conditions in the presence of CD98-AHN-18 (1.5 pg/ml, top panel), MEM 101A (CD29, 0.3 pg/ml, middle panel) or 161-46 (CD43, 0.3 pg/ml, bottom panel). Blocking mAbs to CD 18 were added to U937 cultures 1 h prior to the aggregating mAbs. Results are expressed as % aggregation relative to the aggregation in the absence of blocking mAb (column labelled 0 in each panel). Means which differ significantly from control (absence of inhibitory mAb, p < 0.05) are shown with an asterisk. mAbs were tested at a series of dilutions, starting with the most dilute and increasing in two fold steps along the x-axis. To obtain the actual concentration of mAb at each point (in pg/ml), the x-axis value should be multiplied by 0.5 for both CLB-LFAl (CD 18, empty bars) and BU86 (CD 18, filled bars). A) 24 h .ULl 10“ 10^ 10* 10* 10*
B) 48 h UJ
10" 10’ 10* 10' 10' FL1-H ai-H CD18 CD29
Fig. 3.7B CD98 ligation does not alter cell surface levels of CD18 or CD29. U937 cells were incubated in the presence of CD98-AHN-18 (1.5 |ag/ml) or control for 24 (top panel) or 48 (bottom panel) hours. Levels of cell surface CD 18 or CD29 were measured by flow cytometry as described in Methods. Note that the two fluorescence histograms obtained in the presence and absence of CD98 are almost coincident. Histograms marked C are the fluorescence profile of CD98-treated cells stained with iso type control. (A)
CD98 CD29 CD43
CD147/IVB/1 I I
CD147/H84AF ■
Fig. 3.8A CD147 mAbs block aggregation induced via CD98. Aggregation was measured under standard conditions in the presence of CD98-AHN-18 (1.5 pg/ml, left panel), MEM lOlA (CD29, 0.3 pg/ml., middle panel) or 161-46 (CD43, 0.3 pg/ml, right panel). Blocking mAbs to CD 147 were added to U937 cultures 1 h prior to the aggregating mAbs. Results are expressed as % aggregation relative to the aggregation in the absence of blocking mAb (column labelled 0 in each panel). Means (mean ± SEM, triplicate cultures) which differ significantly from control (absence of inhibitory mAb, p < 0.05) are shown with an asterisk. mAbs were tested at a series of dilutions, starting with the most dilute and increasing in two fold steps along the x-axis. To obtain the actual concentration of mAb at each point (in pg/ml), the x-axis value should be multiplied by 1.2 for MEM M6/1 (CD147, empty bars) and 1.1 for H84AF (CD 147, filled bars). (B)
Â| ^ i n m 'm#% B / ‘T LX-
^ " ,
Fig. 3.8B CD147 mAbs block aggregation induced via CD98. U937 cells were incubated in medium alone (A), CD98-AHN-18 (1.5 ug/ml) (B), or CD98-AHN-18 and MEM M6/1 (CD147, 1.2 pg/ml) (C) for 6 h, as described in Methods. Images of cells in culture at this time point were obtained using an inverted phase contrast microscope, attached to a video camera, and captured using NIH image software. 1 2 3 4 5 6 7 8
p p l 5 5 ^ ^ m p p l l 4 ^ m m A) p p l 0 7 ^ pp72 —►
B)
Lane No
Fig. 3.9 CD147 regulates tyrosine phosphorylation induced via CD98 or CD29 ligation. A) U937 cells (5 x 10^ cells/ml) were incubated in the presence of CD98-AHN-18 (1.5 pg/ml), MEM 101A (CD29, 0.5 pg/ml), MEM M6/1 (CD147, 2.5 pg/ml), BU89 (CD98, 2.3 pg/ml) or combinations of these mAbs for 20 min. Cells were lysed and analysed for phosphotyrosine proteins as described in Methods. Molecular weights of the major phosphorylated species are shown on the left, and were calculated by image analysis of the Western blot, using molecular weight standards to calibrate the software. Lane 1 - isotype control; lane 2 - MEM M6/1 (CD 147); lane 3 - CD98-AHN-18; lane 4 - MEM lOlA (CD29); lane 5 - CD98-AHN-18 + CD147; lane 6 - MEMIOIA (CD29) + CD 147; lane 7 - BU89 (CD98); lane 8, CD98-AHN-18 + BU89 (CD98). B) The relative intensities of the major phosphotyrosine species in bands 3, 5 and 8, calculated using gel image analysis software. 120 1
100 ■ -
2 c 80 • uo
c 60 • o O)ro 2 40 - * Ui <
20 -
CD29 CD98 CD147 CD18 Blocking Antibody
0-1.5 hours El 0 hours □ +1.5 hours
Fig. 3.10 Sensitivity of CD98 induced aggregation to blocking antibodies added at different times. U937 cells aggregation was measured under standard conditions in the presence of CD98-AHN-18 (1.5 pg/ml). mAbs to CD29 (MAR4, 0.5 pg/ml), CD98 (BU89, 1.2 pg/ml), CD 147 (MEM M6/1, 1.6 pg/ml) and CD18 (CLB-LFAl, 1.5 pg/ml) were added to the culture before, together with or after the aggregating mAb as shown. Results are expressed as % aggregation relative to the aggregation in the absence of blocking mAb (column labelled 0 in each panel). Means which differ significantly from control (absence of inhibitory mAb, p < 0.05) are shown with an asterisk. Functional molecular complex including CD 147
3.3 Discussion
The diversity of responses in which the 85 kD type II membrane protein, CD98, has been implicated supports the notion that the molecule probably plays a key role in cell cell interaction and signalling, but (perhaps because of CD98 diversity and ubiquity) this role remains poorly understood. Our recent studies identified CD98 as a major component of the human dendritic cell surface (Woodhead et al., 1998), and confirmed earlier reports (Diaz et al., 1997) that it is involved in T cell co-stimulation (Stonehouse et al., 1999; Woodhead et al., 2000). During the course of these studies, however, we also confirmed previous reports (Tabata et al., 1994) that some CD98 mAbs induced homotypic aggregation of the U937 cell line, and we focused on this model system to dissect the molecular basis of CD98 function, because of the advantages of working with a uniform cell line rather than a mixed population of T cells and antigen presenting cells.
A reproducible quantitative assay of U937 homotypic aggregation was developed as a prelude to pharmacological and molecular dissection of CD98 function, and the results from this assay form the basis for the present study. Using this assay, it was clear that
CD98 mAbs are highly heterogenous both in function and ability to bind to CD98 on the U937 cell surface (Fig. 3.1). It seems unlikely that this heterogeneity reflects simply concentration or affinity of the mAbs used, since each mAh was tested over a wide range of concentrations. The heterogeneity is very reminiscent of aggregation induced by anti-pi integrin mAbs (Tanaka, 1997), and presumably reflects the presence of specific conformational epitopes on the CD98 molecule, only some of which are
116 Functional molecular complex including CD 147
involved in stimulating the down-stream signalling cascade which leads to aggregation.
A cross-linking event, rather than a direct physical effect of antibody-binding to CD98 is also suggested by the requirement for divalent F(ab’)2 fragments, and by the lack of correlation between mAb binding levels and aggregation. Indeed very high levels of
CD98 occupancy, by either intact mAb or F(ab’)2 fragments, result in lower levels of aggregation, perhaps because cross-linking by mAb becomes less efficient under conditions of mAb excess.
The next question we addressed, using the same assay, was the relationship between
CD98 and other cell surface molecules thought to have a role in cell-cell interaction.
The functional association between CD98 and CD29 (pi integrin) in the cell membrane
(Fenczik et al., 1997; Warren et al., 2000) is well-documented, and has led to the suggestion that CD98 signalling into the cell is in fact mediated via integrin activation.
No clear biochemical data showing interaction between CD98 and CD29 in the cell membrane has been published, although a recent study has shown that CD98 can associate with isolated cytoplasmic portions of some pi integrin isoforms (Zent et al,m
2000). The results from the present study support the general hypothesis that there is a close link between pi integrin and CD98 function, but not the suggestion that CD98 functions simply by cross-linking pi integrins. Specifically, although aggregation by both CD29 mAh and CD98 mAh share many properties, such as a requirement for an intact cytoskeleton, and although there is some cross-inhibition by mAbs to the two molecules, significant differences between the two pathways exist. Thus pl-integrin mediated aggregation is completely abrogated by EDTA, reflecting the fact that both integrin chains require divalent cations for activity. In contrast, CD98-induced
117 Functional molecular complex including CD 147
aggregation is EDTA insensitive. An intact integrin heterodimer cannot therefore be an essential intermediate for CD98-induced signalling in these cells. Furthermore, the initial phase of signalling via CD98 appears to be much more sensitive to the metabolic inhibitor deoxyglucose, again suggesting a pathway distinct from that induced via CD29.
Finally, the pattern of protein tyrosine phosphorylation induced via CD98 appears to be quite distinct from that induced via CD29, though some substrates of phosphorylation appear to be shared between the two pathways. The identity of the phosphorylated proteins remains unknown, although some likely candidates, including FAK, vinculin and CD29 itself have been ruled out (Tabata et al, 1997). CD98 itself is also not tyrosine phosphorylated ((Tabata et al, 1997) and own unpublished data), and indeed its sequence does not contain any known tyrosine phosphorylation motif.
Several features of the CD29/CD98 results presented in this paper suggest that CD29 molecules play a role in the inductive phase of the response, rather than mediating the actual cell-cell adhesion. Firstly, the CD29 mAh which inhibited aggregation induced via CD98 is not the same as that which blocks the ability of pl-integrins to bind their ligand (e.g. fibronectin) in other systems. For example, the mAh P5D2 blocks aggregation induced by the agonist CD29-directed mAh MEM 101 A, and also blocks the ability of pl-integrins to bind extracellular substrates (Fenczik et al, 1997; Wayner et al, 1993), but does not influence CD98-induced aggregation at all Conversely,
MAR4 which does not block binding of pi integrin dimers to extracellular substrates
(Tanake, 1997) does inhibit CD98-induced aggregation. Secondly, MAR4, the CD29 mAb which does block CD98-induced aggregation does so when added prior to CD98, but not after CD98, even though significant aggregation does not occur until 2-3 h post-
118 Functional molecular complex including CD 147
activation. These results suggest that mAh blocking may reflect interference in
CD98/CD29 interaction in the membrane, rather than reflecting a block of CD29/ligand
interaction in mediating the cell-cell binding event itself.
In contrast, mAbs to p2-integrins block aggregation even when added after CD98,
consistent with a role in the actual cell-cell binding step. A role for p2-integrins in
mediating both homotypic (Hewison et al., 1994) and heterotypic (King and Katz, 1989)
aggregation is now well-documented. However, in comparison to other systems, the role
of the P2 integrins here seems rather minor, since aggregation is blocked only by
approximately 20-30%. Furthermore, no significant inhibition of aggregation was
observed by ICAM-1, 2, or 3 mAbs, perhaps because of significant overlap in the
function of these molecules as p2 integrin ligands .
The blocking of CD98-induced aggregation by CD 147 was an unexpected finding.
CD 147 is a member of the immunogloubulin superfamily, originally believed to be
involved in blood-brain barrier function (Schlosshauer, 1993). However, CD 147 knockouts did not reveal any defect in this function, but showed some behavioural and
immunological abnormalities (Igakura et al., 1996). The ligand (if such a molecule
exists) for CD 147 has not been described, and hence the partner for its role in mediating
cellular aggregation remains unknown. Most intriguingly, however, recent reports have
identified some striking parallels between CD 147 and CD98. CD 147 associates physically with pl-integrins in the membrane (Berditchevski et al., 1997), as does
CD98 with isolated cytoplasmic pi domains (Zent et al., 2000). Antibodies to both molecules can induce aggregation of U937 cells, and the aggregation appears to be
119 Functional molecular complex including CD 147
mediated in part by p2-integrin activation (Kasinrerk et al., 1999). Levels of CD98 and
CD 147 correlate on T cells, with high levels in the thymus, low in resting mature T cells,
and higher levels on activated mature T cells (Kirsch et al., 1997). Finally, and most
intriguingly it appears that CD 147, like CD98 is acting as a chaperone for multi
membrane spanning transporter molecules. In the case of CD98 these are amino acid
transporters (Pfeiffer et al., 1999); in the case of CD 147 they are the MCT family of
proton-linked monocarboxylic acid transporters (Halestrap and Price, 1999).
In CD98 - mediated U937 aggregation it is unclear whether CD 147 is acting as an
ancillary adhesion molecule, or, alternatively mediates the aggregation event itself. The
inhibitory action of CD 147 is manifest even when antibody is added 1.5 h after CD98-
AHN-18, consistent with a possible role for CD 147 in mediating the cell-cell binding.
However, an alternative hypothesis for this data is that continuous signalling via CD98
is required to produce aggregation, and that CD 147 interferes with this signalling event,
or even sends negative signals that oppose CD98-induced changes. This model is
consistent with the observation that CD 147 ligation profoundly inhibits the tyrosine
phosphorylation induced by CD98-AHN-18 ligation.
The data presented above, taken together with previous data on this pleiotropic
molecule, suggest that CD98 is a central component within a multi-molecular complex
which can regulate outcomes as diverse as adhesion, growth, differentiation and antigen
presentation. This data prompts us to suggest a speculative model which links CD98
induced aggregation, the central role of pi integrins in CD98 function, the involvement
of CD 147, and the curious structural parallels between this molecule and CD98 (Fig.
120 Functional molecular complex including CD 147
3.11). This envisages that CD98 forms one component of a “sensory complex”, containing pi integrins, CD98 and CD 147, together with all their associated transporter molecules. The complex induces signals, perhaps via the transporter molecules themselves, to regulate multiple aspects of cell physiology. Parameters which would regulate the function of such a complex may include extracellular matrix, levels of amino acids and levels of carboxylic acids (e.g. lactic acid) which are found in their environment. Work is therefore in progress to dissect how this complex might function at a molecular level.
121 Pyruvic acid Amino acids Extra cellular matrix Lactic acid
membrane
CD147 CD98 (31 integrins
signalling
Fig. 3.11 Model of possible functional complex containing CD98 (heavy and light chains, pl-integrins (a/p chains) and CD147 (heavy and light chains).The figure shows the 3 components of the CD98 signalling complex and their topology within the plasma membrane. Chapter 4
CD98 regulates mitogen activated protein kinases and adhesion via the selective activation of protein kinase C6
123 Positive signalling pathway
Abstract
CD98 is a protein found on the surface of many activated cell types, and is implicated in
the regulation of cellular differentiation, adhesion, growth and apoptosis. Despite many
studies addressing CD98 function, there is little information on the intracellular
signalling pathways that mediate its activity. In this study, we examine the major
signalling pathways which are activated following ligation by the CD98 antibody AHN-
18, an antibody which induces U937 homotypic aggregation, and inhibits antigen presenting activity and T cell activation. Pharmacological and biochemical analysis,
using a quantitative assay of U937 aggregation, demonstrate that CD98 ligation induces
a selective membrane translocation of the novel PKCô isoform, as an essential step in
mediating U937 homotypic aggregation. PKCô activation in turn mediates activation of
ERKl/2 and p38, as well as tyrosine phosphorylation of multiple proteins, but only
MAPK activation is essential for cellular aggregation. One of the targets of CD98
induced tyrosine phosphorylation is itself PKCÔ, suggesting that this phosphorylation
may act as a negative feedback to limit the overall activation of the CD98 pathway.
124 Positive signalling pathway
4.1 Introduction
CD98 is the heavy chain (85 kD) of a dimeric molecule found on the surface of many haematopoietic cells (Warren et al., 1998; Lumadue et al., 1987). It is a type II integral membrane protein, with a long cytoplasmic portion of 81 amino acids. Some studies have suggested that it may act as ligand for the cell surface lectin galectin-3 (Dong and
Hughes, 1997). CD98 is found covalently linked to one of several alternative light chains. Thus far six have been identified, of which at least four are members of an amino acid transporter family (Verry et al., 1999). CD98 is functionally associated with both pi integrin molecules (Zent et al., 2000), and the immunoglobulin superfamily member CD 147 on the cell membrane (Cho et al., 2001; Fenczik et al., 1997). The most striking feature of CD98 is the extraordinary diversity of functions in which it has been implicated (Deves and Boyd, 2000). Antibodies against CD98 block the formation of cell syncitia by HIV and other viruses (Ohgimoto et al., 1996; Okamoto et al., 1978), and it appears also to play a more general role in regulation of integrin-mediated cell adhesion (Fenczik et al., 1997; Tabata et al., 1994; Chandrasekaran et al., 1999). CD98 has been implicated in haematopoietic cell differentiation, growth, transformation and apoptosis (Warren et al., 1996; Hara et al., 1999), and this role has been extended to the regulation of osteoblast differentiation (Higuchi et al., 1999). CD98 also plays a role in the regulation of amino acid transport by virtue of its associated light chain, although this function, in contrast to its association with integrins in the membrane, is mediated principally via the extracellular portion of the molecule. Most recently, the molecule has been implicated in the regulation of both antigen presenting cell function and T cell activation (Diaz et al., 1998; Diaz et al., 1997). CD98 is present at very high levels on
125 Positive signalling pathway
the surface of human dendritic cells (Woodhead et al., 1998), and our group showed that some antibodies to this molecule could block the ability of both the U937 promonocyte line (Stonehouse et al., 1999) and human peripheral blood derived dendritic cells
(Woodhead et al., 2000) to deliver essential co-stimulatory signals to T cells.
Despite the multitude of studies addressing CD98 function, there is little information on the intracellular signalling pathways which underlie the extraordinary diversity of function of this molecule. A few studies have documented tyrosine protein phosphorylation following CD98 ligation (Warren et al., 1996; Tabata et al., 1997), but there is limited evidence linking this to downstream functional outcomes. In this study, we examine the major signalling pathways which are activated following ligation of
CD98 by the CD98 mAh AHN-18, an antibody which we previously selected as the most potent activator of U937 homotypic aggregation (Cho et al., 2001), as well as being the most potent inhibitor of antigen presenting activity and T cell activation.
Pharmacological and biochemical analysis, using a quantitative assay of U937 homotypic aggregation which we described previously (Cho et al., 2001), demonstrate that CD98 ligation induces a very selective membrane translocation of the novel PKCô isoform, as an essential step in mediating U937 homotypic aggregation. PKCô activation in turn mediates activation of ERKl/2 and p38, as well as tyrosine phosphorylation of multiple proteins, but only MAPK activation is essential for cellular aggregation. Interestingly, one of the major downstream targets of CD98 induced tyrosine phosphorylation is itself PKCô, suggesting that this phosphorylation may be part of a negative feedback loop which limits the overall activation of the CD98 pathway. This study suggests that selective regulation of PKCÔ is a key intracellular
126 Positive signalling pathway
target of the CD98 signalling complex.
127 Positive signalling pathway
4.2 Results.
4.2.1 Protein tyrosine phosphorylation induced by CD98 ligation is not essential for U937 homotypic aggregation.
The addition of CD98-AHN-18 mAh (or F(ab’)2 fragment, not shown) to U937 cells induced strong homotypic clustering (Fig. 4.1A) as described previously (Cho et al.,
2001). The extent of clustering was dependent on the concentration of mAh, and on time, with maximum clustering observed after 3-6 h (Cho et al., 2001). In parallel,
CD98-AHN-18 induced rapid tyrosine phosphorylation of multiple proteins. The addition of genistein (Fig. 4.1B) almost completely blocked tyrosine phosphorylation, in a dose dependent maimer. However, neither genistein (Fig. 4.1C, left panel) nor herbimycin A (Fig. 4.1C, right panel), nor A77,1726 (not shown), all inhibitors of tyrosine phosphorylation , blocked CD98 dependent clustering. Both genistein and herbimycin A enhanced U937 clustering at lower concentrations.
4.2.2 Protein kinase C activity is necessary for CD98-induced aggregation.
Since CD98 mediated aggregation did not require protein tyrosine phosphorylation, a number of inhibitors of serine/threonine kinase activity were tested in this model. Two broad specificity serine/threonine kinase inhibitors, H-89 and H-7, both blocked aggregation at concentrations of 10 (H-89) or 50 (H-7) pM respectively (Fig. 4.2A).
Two major classes of protein serine/threonine kinases are the PKA and PKC families.
The selective PKA inhibitor KT5720, however, did not block CD98-induced aggregation, and indeed enhanced aggregation (Fig. 4.2B). Furthermore, forskolin, a potent inducer of cAMP, which itself activates PKA, inhibited CD98-induced
128 Positive signalling pathway
aggregation. The pharmacological data therefore suggest that PKA plays a negative, rather than positive role in CD98 signal transduction.
In contrast, the broad specificity PKC inhibitors, chelerythrine chloride and
GF109203X both completely inhibited CD98-mediated aggregation (Fig. 4.2C).
Chelerythrine chloride was a potent inhibitor, showing maximal inhibition at concentrations of 3 pM and above. In contrast, GF109203X showed a biphasic dose response, inducing enhanced aggregation at concentrations of 10 pM or below, but inhibiting aggregation at concentrations of 80 pM.
The involvement of PKC family members was dissected further in Fig. 4.3. Two selective inhibitors of the classical Ca^^ dependent PKC isoforms (a,p and y), Go6976 and 19-27, showed no inhibitory activity. 19-27, a pseudosubstrate peptide which very specifically blocks the a and P isoforms, enhanced aggregation at concentrations of 25 pM or above. In contrast, aggregation was inhibited by low concentrations of rottlerin, an inhibitor which has been reported to show 10 fold or more selectivity in vitro for the
PKCÔ isoform over other PKC isoforms (although recent studies have suggested that other serine/threonine kinases in the cell may also be inhibited (Davies et al., 2000))
(Fig. 4.3B, C and D). Rottlerin blocked aggregation when added prior to, or together with CD98-AHN-18, but did not block aggregation when added 1.5 h after addition of
CD98-AHN-18 (but before aggregation occurred) (Fig. 4.3C). The rottlerin-sensitive step was thus an early signalling event in the aggregation process. Rottlerin also strongly inhibited CD98-induced protein tyrosine phosphorylation (Fig. 4.3E), suggesting that the rottlerin-sensitive step was up-stream of tyrosine kinase activation.
129 B) PY
a o O M + + + + i f f Cmstdn(plV^ - - 50 100 200
p l 5 5 P107/114 Nii p 7 2 » «•
—. - d
Q Genistein Itrbin-^n * 200 X 200 *
150 ■ 150
■~ 100 -c 100 X I I §g 50 §g 50 <
12 25 50 100 0.3 0.6 1.2 pM pM Fig. 4.1 CD98-induced aggregation is independent of tyrosine phosphorylation. A) U937 cells were incubated for six hours in the presence of CD98-AHN-18 (1 pg/ml). Images of cells in culture at this time point were obtained using an inverted phase contrast microscope, attached to a video camera, and captured using NIH image software. A representative field is illustrated, showing strong U937 homotypic aggregation.B) U937 cells were incubated for 20 min in the presence or absence of CD98-AHN-18 (1 pg/ml), with or without the prior addition of genistein as shown. Cells were washed, lysed and analysed for protein tyrosine phosphorylation as described in Methods. C) U937 cells were incubated for six hours in the presence of CD98-AHN-18 (1 pg/ml), with or without the prior addition of genistein or herbimycin A as shown. Aggregation was measured as described in Methods. The results [aggregation % (% of control)] show mean aggregation ± SEM for triplicate cultures. * p < 0.01. A) H-89 H-7 — 120 1 ^ 140 - ^ 100 X % 120 ■ ,2 80 ^ .2 100 & 80 a 8° 1 X ! * 2 60 - 40 - 50 50 4 0 20 20 0 CL A 1 5 10 5 10 50
pM |iM KT5720 forskolin
B) 2 5 0 -1 * * 1 4 0 - 1 20 - 200 - .1 1 00 ■ 1 5 0 - « 80 - 00 10 0 - K 60 ■ 4 0 - 50 - < 20 - 0 . a x 125 250 500 1000 25 n M HM
C) Chelerythrine chloride GF109203X
3 00 - 1 60 * * 140 2 50 1 20 1 2 0 0 - 1 00 80 1 50
60 - 1 00 40 50 20 - 0 0 0.7 1.5 3 1 0 20 40 80 pM jiM
Fig. 4.2 CD98-induced U937 aggregation requires protein kinase C activity. U937 cells were incubated for 4 h in the presence of CD98-AHN-18 (1 pg/ml), with or without the prior addition of inhibitors as shown. A) Broad spectrum threonine/serine kinase inhibitors H-89 and H-7. B) An inhibitor of PKA, KT5720, and the cAMP agonist forskolin. C) Broad spectrum PKC inhibitors chelerythrine chloride, and GF109203X. Aggregation was measured as described in Methods. The results [aggregation % (% of control)] show mean aggregation ± SEM for triplicate cultures. * p < 0.05. ** p < 0.01. Go6976
H M R ottlerin R o ttlerin
hours
aCD98 R ottlerin
,155 P I 07/1 1 4
U937 alette + CD98-AHN-18 + rottlerin (10|iM) + C D 9 8 -A H N -1 8 + rottlerin (I0|4M) Fig. 4.3 Rottlerin, a selective inhibitor of novel PKCô, blocks CD98- induced aggregation and protein tyrosine phosphorylation. A) - C) U937 cells were incubated for 6 h in the presence of CD98-AHN-18 (1 pg/ml), with or without the prior addition of inhibitors as shown. Aggregation was measured as described in Methods. The results [aggregation % (% of control)] show mean aggregation ± SEM for triplicate cultures. * p < 0.01. A) Selective inhibitors of conventional Ca^^-dependent PKC isoforms, Go6976 and peptide 19-27 (a peptide substrate analogue of PKCa/p. B) - D) Selective inhibitor of the novel (Ca^^-independent) PKCÔ isoform, rottlerin. In panel C), the inhibitor was added either 1.5 h before, or together with, or 1.5 h after CD98- AHN-18. Aggregation was measured after 4 h. In panel D) images of cells in culture at 4 h were obtained using an inverted phase contrast microscope, attached to a video camera, and captured using NIH image software. E) U937 cells were incubated for 20 min in the presence or absence of CD98-AHN-18 (1 pgm/ml), with or without the prior addition of rottlerin as shown. Cells were washed, lysed and analysed for protein tyrosine phosphorylation as described in Methods. Positive signalling pathway
Rottlerin did not block U937 aggregation induced by mAbs to CD43 (Cho et al, 2001), suggesting the inhibitory effect was not a generalised effect on U937 viability, or aggregation (data not shown), but was dependent on CD98 ligation.
4.2.3 CD98 ligation selectively induces PKCô membrane translocation.
The activation of PKC enzymes is frequently associated with translocation of the enzyme from a soluble to a membrane bound form. The relative distribution of eight
PKC isoforms between cytoplasm and membrane following CD98 ligation is shown in
Fig. 4.4A. A proportion of several PKC isoforms was found in the membrane fraction even before stimulation, perhaps reflecting the transformed phenotype of the U937 cell line. However, CD98 ligation induced a very selective additional translocation of the nPKCÔ isoform, while the distribution of all other isoforms tested remained unchanged.
In contrast, PMA, which is a potent agonist of many cPKC and nPKC isoforms, induced translocation of PKCa, p, and y (all the conventional isoforms) but not ô (not shown).
PKCÔ translocation was not a general effect of U937 aggregation, since antibodies to
CD43, another cell surface molecule which induces U937 aggregation via different signalling pathways (Cho et al., 2001), does not affect PKCô translocation (not shown).
Rottlerin, which inhibited aggregation as shown in Fig. 4.3C above, also abolished the translocation of PKC Ô (Fig. 4.4B), consistent with its known inhibitory activity against
PKCÔ.
4.2.4 CD98 induced aggregation requires downstream activation of £RK and p38
MAPkinases.
CD98 ligation induced rapid phosphorylation of both ERKl and ERK2, and a slower
133 A) Control aCD98 Fractions : W M C W M C Membrane fraction
Conventional PKCP m-m— mmm
PKCe _ . ___ Novel
aCD98 - + + PKC(
PKCÀ/1 Atypical
Membrane fraction B) 2.0 PKCÔ 8 15
Fractions W M c 10 aCD98 f + 0.5 Rottlerin + 0.0 CCD98 - Rottlerin -
Fig. 4.4 CD98-ligation induces a selective membrane translocation of PKCÔ. A) U937 cells were ineubated for 20 min in the presence (right hand lanes) or absence (left hand lanes) of CD98-AHN-18 (1 pg/ml). Cells were washed, lysed and fractionated into membrane and cytoplasmic fractions, as described in Methods. Whole lysate (W), membrane (M), and cytoplasmic (C) fractions were analysed for the presence of specific PKC isoforms by Western blot as described in Methods. The right hand panel shows the the relative intensities of membrane associated PKCô bands in the presenee or absence of CD98-AHN-18, calculated using Syngene gel image analysis software. The results from three separate experiments are shown. B) As for A) above, but rottlerin (10 pM) was added to some cultures 30 min prior to CD98-AHN-18, as shown. Positive signalling pathway
phosphorylation of p38, but no change in levels of phosphorylated JNK (Fig. 4.5A).
ERK phosphorylation was blocked by rottlerin (Fig. 4.5B), confirming that MAPKinase activation is downstream of PKCô activation. In addition, selective inhibitors of MEK
(PD98059), which is required for ERK activation, and of p38 (SB203580) inhibited
CD98-induced aggregation (Fig. 4.5C).
4.2.5 CD98 induced activation of PKCô is independent of diacylglycerol, and leads to PKCÔ phosphorylation.
PKCÔ can be regulated by binding of DAG. However, a number of pieces of evidence suggested that CD98 activation (translocation) of PKCô is independent of DAG formation. Firstly, calphostin C, a competitive inhibitor of the DAG/PKC interaction, did not block CD98-induced aggregation (Fig. 4.6). Secondly, inhibition of phosphatidyl choline-phospholipase C (D609 inhibitor), which catalyses the conversion of phosphatidyl choline into DAG, also did not inhibit CD98-induced aggregation (Fig.
4.6B). DAG can also be produced as a result of the cleavage of phosphatidylcholine by phospholipase D (PLD). Propranolol, which is often used as a selective blocker of phosphatidic acid phosphohydrolase, and hence an inhibitor of PLD-dependent DAG formation, did block CD98-induced aggregation (Fig. 4.6B). However, the activity of
PLD appeared to be downstream of PKCô activation, since propranolol did not block
PKCÔ translocation (Fig. 4.6C). In addition, propranolol blocked aggregation when added up to 1.5 h after CD98-AHN-18, consistent with a role in the late (effector) stages of aggregation. Alternative pathways of PKCô activation involve phosphorylation of
PKC, on either serine/threonine or on tyrosine. CD98 ligation appeared to induce a small increase in total levels of phosphorylated T505 PKCô (Fig. 4.7A). The
135 Positive signalling pathway
phosphorylated form is predominantly cytoplasmically localised in resting U937 cells
(Fig. 4.7B), but is strongly translocated to the cell membrane following CD98 ligation.
CD98 induced ligation also caused selective tyrosine phosphorylation of PKCÔ, as
shown by immunoprécipitation of PKCô, followed by blotting with anti- phosphotyrosine antibody (Fig. 4.7C). The tyrosine phosphorylation of PKCô was
strongly inhibited by genistein (data not shown), suggesting that this phosphorylation was not necessary for the induction of aggregation by PKCô.
136 A)
ERK p88 JNC
TmeCnir^O 1 25 5 10 D 0 10 2) 40 60 0 10 30 60
flrçh> ^
Tint(rrir^ 0 10 30 60 0 30 60 0 10 30 60
Tctal
B) Q ™ 8Q99pi^ æ3GS580(p38) Fhïçh>Bk
d J M - Rt ID æ
|JV1
Fig. 4.5 CD98-induced aggregation requires PKCô-dependent ERK and p38 activation. A) U937 cells were incubated for various times in the presence of CD98-AHN-18 (1 pg/ml). Cells were washed, lysed and analysed for ERK, p38 and JNK expression by Western blot as described in methods. The upper blots show gels probed with antibodies specific for phosphorylated forms of the kinases, while the lower blots show gels probed with antibodies which recognise both phosphorylated and non- phosphorylated forms of the kinases. B) U937 cells were incubated for 30 min in the presence or absence of CD98-AHN-18 (1 pg/ml), and in the presence or absence of rottlerin (10 pM), PD598059 (50 pM) or SB203580 (20 pM) as shown. Cells were washed, lysed and analysed for phospho- ERK, by Western blot as described in methods. C) U937 cells were incubated for 6 h in the presence of CD98-AHN-18 (1 pg/ml), with or without the prior addition of inhibitors as shown. Aggregation was measured as described in Methods. The results [aggregation % (% of control)] show mean aggregation ± SEM for triplicate cultures. ** p < 0.01. A)
160 1 Calphostin C 1 4 0 - 1 2 0 - 1 X 100 - X 1 80 -
60 -
40 -
20 - 0 50 100 200 400 nM
B) D609 (PLC) Propranolol (PLD) 160 - 140 - 120 1 2 0 - 100 100 - 80 80 - 60 * * 60 - 40 40 - * * 20 - 20 0 0 £L 75 pM pM C) PKCÔ aCD98 + Propranolol p72
Fig. 4.6 CD98-induced aggregation and PKCô translocation is independent of diacylglycerol formation. A) and B) U937 cells were incubated for 6 h in the presence of CD98-AHN-18 (1 pg/ml), with or without the prior addition of inhibitors as shown. Aggregation was measured as described in Methods. The results [aggregation % (% of control)] show mean aggregation ± SEM for triplicate cultures. * p < 0.01. A) Calphostin C is a PKC inhibitor, which acts by binding to the regulatory subunit at the same site as diacylglycerol. B) D609 is a seleetive inhibitor of phosphoryl choline phospholipase C. Propranolol is a selective inhibitor of phosphatidic acid phosphohydrolase, and hence of phospholipase D-dependent DAG formation. C) U937 cells were incubated for 20 min in the presence or absenee of CD98- AHN-18 (1 pg/ml), and the presence or absence of propranolol (50 pM). Cells were washed, lysed and fractionated into membrane and cytoplasmie fractions, as described in Methods. Membrane fractions were analysed for the presence of PKCô isoform by Western blot as described in Methods. (A) PKCÔ PKCC
Time(min) 0 5 10 20 40 60 0 5 10 20 40 60
Phospho - ** ^ ^ #
(B) PKCÔ
Control aCD98
Fractions W M C W M C
Phospho- %m
(C) IP: PKC5 aC D 98 - + Blot: PY
Fig. 4.7 CD98 regulation of PKCÔ phosphorylation. A) U937 cells were incubated for various times in the presence of CD98-AHN-18 (1 pg/ml). Cells were washed, lysed and analysed by Western blot, using an antibody specific for phospho PKCô (T(P)505) and phospho PKC(!^ (T(P)560) as described in Methods. B) U937 cells were incubated for 20 min in the presence (right hand lanes) or absence (left hand lanes) of CD98-AHN-18 (1 pg/ml). Cells were washed, lysed and fractionated into membrane and cytoplasmic fractions, as described in Methods. Whole lysate (W), membrane (M), and cytoplasmic (C) fractions were analysed for the presence of phospho (T505)-PKCô by Western blot as described in Methods. C) U937 eells were incubated for 20 min in the presence or absenee of CD98. Cells were washed, lysed and immuno- precipitated using an antibody specific for PKCô. The immuno-precipitate was solubilised and analysed by Western blot using an anti-phosphotyrosine antibody. Positive signalling pathway
4.3 Discussion
CD98 forms part of a multimolecular complex on the cell surface of many activated, or transformed cell types, and has been implicated in the regulation of amino acid transport, cell growth, differentiation, death, adhesion, fusion and immunological function. The intracellular events which transmit the necessary signals from the CD98 cell surface complex to the effector molecules that regulate these various functions remain very poorly understood. In this study, we have used a model system in which CD98 ligation by certain antibodies leads to homotypic aggregation of the U937 cell line, mediated by pi and P2 integrins. The functional properties of this model have been investigated in some detail both by our laboratory (Cho et al., 2001) and others (Tabata et al., 1997;
Ohgimoto et al., 1995). An important advantage of the model is that it allows a detailed pharmacological and biochemical analysis of the underlying molecular changes which follow CD98 ligation.
Previous studies on CD98 signalling have focused almost entirely on protein tyrosine phosphorylation, which is a rapid event following CD98 ligation in a variety of systems
(Cho et al., 2001 Tabata et al., 1997; Warren et al., 1996). Therefore we focused initially on the role of these signals in mediating aggregation of U937. Pharmacological data, however, clearly demonstrate that tyrosine protein phosphorylation is not necessary for this aspect of CD98 signalling. Neither genistein nor herbimycin A, both potent inhibitors of PTKs, block cell aggregation in this model. Indeed both inhibitors significantly enhanced CD98-dependent aggregation, suggesting that tyrosine phosphorylation may act as negative regulator of this signalling pathway (see below). In
140 Positive signalling pathway
contrast U937 cell fusion, which can be induced by CD98 ligation in the presence of
HIV gpl60, does require protein tyrosine phosphorylation (Tabata et al., 1997).
Our attention turned, therefore, to the role of serine/threonine kinases in mediating
CD98 signalling. Both the pharmacological and biochemical data from these experiments suggest that activation of the “novel” isoform PKCô is a key central step in orchestrating CD98 function. Pharmacological data include the inhibition of CD98- induced clustering by rottlerin. This inhibitor has been shown in vitro to have at least a ten-fold or greater selectivity for the PKCÔ isoform over others (Keenan et al., 1997), although may also inhibit other unrelated serine/threonine kinases (Davies et al., 2000).
The inhibition by rottlerin was not complete, and some homotypic aggregation occurred even in the presence of rottlerin, suggesting that additional pathways may exist. The dose response curve of GF109203X, which has a high affinity for “conventional” PKC isoforms, but also inhibits “novel” PKC s at higher concentrations (Chen et al., 1999), is also consistent with this model. A parallel biochemical approach used the membrane translocation of PKCô as an indication of PKC activation. Using this method, we showed that PKCô is the only PKC isoform tested which was translocated following
CD98 ligation in the U937 cells. The increase in membrane associated PKCÔ was even more marked when an antibody recognising pThr505 PKCô was used, suggesting a selective recruitment of the phosphorylated (activated) form to the membrane or selective phosphorylation of the membrane-bound form (see below). Finally, the pharmacological and biochemical data could be linked, since rottlerin inhibited not only
U937 aggregation, but also PKCô translocation. CD98 ligation is thus a highly selective way of regulating the activity of PKCô, and this activation is likely to be critical not
141 Positive signalling pathway
only for the induction of U937 homotypic aggregation but for many of the other documented downstream effects of CD98 ligation. In contrast, activation of conventional PKC isotypes may be involved in negative regulation of this pathway, since the selective inhibitor of PKCa/p, as well as low concentrations of GF109203X enhanced aggregation, while PMA inhibited aggregation and PKCô translocation
(Chapter 5).
Having established that regulation of PKCô is a pivotal step in CD98 signal transduction, we have examined both upstream and downstream steps in this pathway.
Activation of MAPkinases, especially ERK and p38 isoforms, has been previously implicated both in the regulation of cellular adhesion/migration (Lu et al., 1998; Sano et al., 2001; Rousseau et al., 1997) and as a downstream event following PKCô activation
(ERK) via integrin activation (Miranti et al., 1999). A similar signalling connection is apparently involved in the CD98 response, since CD98 ligation induces a PKCÔ- dependent activation of both ERK, and p38 kinases, and inhibition of either of these two
MAPkinases by selective inhibitors blocks the CD98-induced aggregation. In contrast to integrin mediated signalling (Miranti et al., 1999), however, the pathway from PKCÔ to
ERK activation does not appear to involve tyrosine kinase activity, since the pathway is resistant to genistein. The specific downstream targets of ERK and p38 which mediate aggregation in this model have not yet been investigated in detail. However, a number of likely candidates, which regulate cytoskeletal rearrangements and integrin activity have previously been identified, including paxillin (Ku et al., 2000) and pleckstrin
(Brumell et al., 1997). ERK-mediated PLD phosphorylation has also been implicated in the regulation of cellular adhesion (Sano et al., 2001). The relationship between PLD
142 Positive signalling pathway
and PKCÔ is complex, since PLD can be both an upstream activator of PKCs, via the release of DAG from phosphoryl choline, and a downstream target of PKCô (Lee et al.,
2000; Siddigi et al., 2000). Our data is more consistent with a downstream role for PLD, since aggregation is blocked by the propranolol, but PKCô translocation is not affected by propranolol. However, propranolol is likely to have other pharmacological effects, and further work is required to dissect the role of PLD in this system further.
The activation and regulation of PKCô is extremely complex, and can involve binding of DAG to the regulatory subunit (Gschwendt, 1999), serine/threonine phosphorylation
(Parekh et al., 2000) (although the significance of this phosphorylation has been questioned (Stempka et al., 1999)), and multiple tyrosine phosphorylation sites (Li et al.,
1994). The activity of the enzyme is also probably modulated by binding to specific sets of receptors, or linker molecules within the cell, which may act as scaffolds for promoting enzyme/substrate interaction (Mochly-Rosen and Gorden, 1998). In the present study, CD98 regulation of PKCô appears to be largely independent of DAG generation, since it is resistant to the DAG binding site inhibitor calphostin C, and also to phosphatidyl choline-PLC inhibition. Furthermore, PMA which is a potent DAG activator, and the DAG analogue dioctanoylglycerol, inhibits CD98 induced aggregation, and blocks PKCÔ translocation (Chapter 5). Increased phosphorylation at Thr505 is also unlikely to be the major regulatory step in PKCô regulation in this model, since resting U937 cells already contain high levels of this phosphorylated form (although there may be a small increase in the total levels of this phosphorylated form following
CD98 ligation). Finally, it is interesting that PKCô is itself tyrosine phosphorylated following CD98 ligation (this is the first identification of a CD98-induced
143 Positive signalling pathway
phosphotyrosine species). It is likely that this phosphorylation is itself a regulating step, since phosphorylation of PKCô has previously been shown to regulate function both positively and negatively Haleem-Smith et al., 1995; Deszo et al., 2001; Kronfeld et al.,
2000). In the case of CD98-mediated aggregation, this phosphorylation may be part of a negative feed-back loop, since genistein totally abolished PKCÔ tyrosine phosphorylation, and simultaneously enhanced aggregation.
The major steps in CD98 signalling identified in this study are depicted in Fig. 4.8.
Ligation of CD98 (by antibody in this study, but by interaction with appropriate counter-receptor in vivo), leads to a rapid recruitment of PKCô to the membrane. An analogous model has been proposed for the events which follow cross-linking of the
IgEy chain in mast cells (Haleen-Smith et al., 1995). Recruitment may require some post-translational modification of PKCô itself, or alternatively modification of a PKCô receptor associated with the CD98/integrin eomplex at the cell surface. Activated PKCô then activates a signalling caseade which involves both phosphorylation/activation of
ERK/p38, and downstream activation of unidentified proteins which initiate the changes in cell adhesion whieh result in U937 aggregation. In parallel, PKCô activation activates, either directly or indirectly, tyrosine kinase activity (both lyn and src have previously been shown to be regulated by PKCô (Song et al., 1998)). One target of this tyrosine kinase aetivity is PKCô itself, which may negatively regulate PKCô (Denning et al.,
1993), or alter the specificity of PKCÔ so as to phosphorylate different intracellular target proteins (Haleem-Smith et al., 1995; Deszo et al., 2001), and hence generate other functional outcomes. Other targets of the PKCô-activated tyrosine kinases remain undefined, but may be downstream pathways which regulate other CD98-dependent
144 Positive signalling pathway
functions, including growth and differentiation. CD98 forms part of multi-molecular complex, which includes integrins and CD 147 (Cho et al., 2001). This complex has been suggested to act as a sensory unit, which can be regulated by extracellular amino acid levels, pH, and matrix. CD98 is itself a key element in transducing signals from this complex to within the cell (Cho et al., 2001). The observation documented in this study that PKCÔ is a pivotal element in this pathway provides further support for this enzyme’s role as a key component of the intracellular signalling pathways which regulate a cell’s interaction with its complex microenvironment.
145 Ligation of CD98/integrin/CD147 complex
W PKCL
ERK/p38
Tyrosine phosphorylation Cytoskeleton Integrins PLD
Other functions Aggregation
Fig. 4.8 Model of CD98-induced signalling. Signal transduction steps which are essential for CD98-induced U937 aggregation: PKC translocation : 1 = ^ Regulatory signal pathways : ► Chapter 5
Conventional PKC plays a critical role in negative regulation of CD98-induced homotypic aggregation
147 Negative signalling pathway
Abstract
CD98, a heterodimeric type II transmembrane protein, is involved in many different cellular events, ranging from amino acid transport to cell-cell adhesion. Little is known about the signalling pathways involved in these responses. Therefore, we examined the role of conventional PKC isoforms during CD98-induced U937 homotypic aggregation.
The CD98-induced aggregation was blocked by a protein kinase C (PKC) activator
PMA, and enhanced by broad specific protein kinase inhibitors, GF109203X and staurosporin, and by specific PKCa/p peptide inhibitor 19-27. PMA inhibition was not affected by specific enzyme inhibitors of PKA), PTK, protein phosphatase (PPTase), and PI3-K. PMA inhibition was diminished by treatment with PKC inhibitors recognising the ATP-binding site in PKC (e.g. staurosporin, GF109203X and Go6983).
Inhibitors which bind to DAG or Ca^^-binding sites of PKC (calphostin C and Go6967) had no effect. In agreement with these observations, conventional PKC (cPKC) isozymes (a, p and y) were translocated during PMA treatment. PMA treatment did not decrease expression of CD 18, CD29 and CD 147 which have been implicated in CD98- induced cell adhesion, suggesting that PMA inhibition is not mediated by surface adhesion molecule. These data provide evidence that PMA-responsive conventional protein kinase C isoforms (a, p and y) play a key role in negative regulation of CD98 signalling and homotypic aggregation.
148 Negative signalling pathway
5.1 Introduction
Cell-cell adhesion interactions are known to play an important role in many immune functions, such as cell migration, effector target recognition and activation (Martz,
1987; Belitsos et al., 1990). Examples of such cell-to-cell interactions include those between T cell and APC, follicular dendritic cells and B cells, and tumor cells and cytotoxic T cells. Cell adhesion is strictly regulated by multiple molecules with different modes of action (Springer and Anderson, 1986; Pardi et al., 1992; Petty and Todd, 1996;
Hubbard and Rothlein, 2000), including integrins, selectins and intercellular adhesion molecules (ICAMs).
CD98 is a type II integral membrane glycoprotein, composed of a 80 kDa glycosylated heavy chain and a 45 kDa nonglycosylated light chain (Tsurudome and Ito, 2001). The molecule is highly expressed on both haematopoietic and non-haematopoietic cells.
CD98 expression is known to be tightly regulated in the process of differentiation. It has been implicated in haematopoietic cell differentiation, growth, transformation and apoptosis, and osteoclastogenesis (Warren et al., 1996; Hara et al., 1999; Higuchi et al.,
1999; Hara et al., 2000; Shishido et al., 2000). There is also tight regulation of CD98 expression during differentiation from monocytes to dendritic cells (Wooodhead et al,
1999). CD98 has been reported to be a regulator of cell-cell adhesion (Warren et al.,
1996; Ohgimoto et al., 1995; Ohgimoto et al., 1996; Ohgimoto et al., 1997; Fenczik et al., 1997; Chandraseran et al., 1999, Chapter 3), of pi integrin function (Fenczik et al.,
1997; Chandraseran et al., 1999), of T cell co-stimulation (Diaz et al., 1997; Stonehouse et al., 1999; Woodhead et al., 2000), and of virus-induced cell fusion (Ohgimoto et al..
149 Negative signalling pathway
1995; Ohgimoto et ai., 1996; Okamoto et al, 1997). The light chain functions as an amino acid transporter and is transported to the cell membrane by association with the heavy chain (Verrey et al 1999).
A key question in understanding the biology of CD98 is to resolve how the molecule can participate in such an extraordinary diversity of functions. Two approaches which could be used to explain this problem are (i) identification of CD98 ligand(s) and (ii) investigation of the intracellular signalling pathways that are triggered following CD98 ligation. At present, galectin-3 is the only identified ligand of CD98 (Dong and Hughes,
1997). There is very little information, however, on the intracellular signalling pathways which may mediate CD98-induced changes in cell function. Previous studies have identified tyrosine phosphorylation (Warren et al, 1996; Tabata et al, 1997; Miyamoto et al, 2000; Chapter 3), ras involvement (Miyamoto et al, 2000; Suga at al, 2001), activation of MAPK (Miyamoto et al, 2000; Suga et al, 2001) and PI3-K (Suga et al,
2001) as possible intracellular signals mediating CD98 ligation.
In a previous study, we reported that CD98 interacts functionally with both pi integrins and CD 147 to induce homotypic aggregation of U937 cells (Chapter 3), and we suggested that CD98 plays a central role within a multi-molecular unit which could serve to regulate cellular responses to changes in the tissue micro-environment. Further studies from our laboratory have focused on the role of the novel PKC (nPKC) isoform,
PKCÔ, in mediating CD98-induced homotypic aggregation of U937 cells (Cho et al, manuscript submitted). This work has now been extended to look at the role of other
PKC isoforms in this model system. We provide evidence that CD98-induced cell-cell
150 Negative signalling pathway
adhesion is suppressed by the activity of cPKC isoforms. The antagonism was not mediated by changes in surface adhesion molecule expression, but seemed to act downstream of the MAPK/ERK pathway. cPKCs may form part of a physiological regulatory system, which acts to limit cell-cell adhesion/aggregation mediated by ligation of CD98.
151 Negative signalling pathway
5.2 Results
5.2.1 Comparative kinetic analysis of CD98-induced homotypic aggregation.
The initial studies were based upon our observations that CD98 mAh (both intact and
F(ab’)2 fragments) not only abrogated U937-triggered T lymphocyte proliferation in the presence of soluble CD3 (Stonehouse et al., 1999), but also elicited homotypic aggregation of the U937 cells themselves under standard culture conditions (Chapter 3).
U937 homotypic aggregation was not induced by mAbs of the same isotype directed against several other major cell-surface molecules known to be expressed on U937 cells
(e.g. CD44, CD45 and CD 147). However, other mAbs to adhesion molecules, such as
CD29 and CD43, did cause strong homotypic aggregation of the cell line (Chapter 3).
In this study, CD98-induced aggregation was carefully compared to aggregation mediated by CD29 and CD43, because the shapes of the aggregates induced by these molecules were strikingly distinct. The kinetics of a representative assay are shown in
Fig. 5.1 A and B Aggregation induced by CD29 and CD43 was more rapid than the
CD98 response, reaching 30% (CD29) and 50% (CD43) at 30 min, and increasing further at one hour incubation, whereas CD98 induced cluster formation slowly, with only approximately 15% of the cells within clusters at one hour (Fig. 5.1 A). The maximum level of CD98-induced aggregation was also significantly less than that seen with CD29 and CD43 (Fig. 5.1C). These data raise the possibility that CD98-induced aggregation may be negatively regulated in some way to restrict cluster formation.
152 (A) (B) (C)
75 100 - # - CD98 CD29 60 - * - CEM3 » 45
% 15 ;C- C ‘CiC te 0 0 ^- 0 0.5 1 CD98 CD29 CD43 Time (hour) Treatment
Fig. 5.1 D98-induced aggregation shows a kinetically different pattern from CD29- and CD43-induced aggregation. A) U937 cells were incubated for 1 h in the presence of mAbs to CD98 (AHN-18, 1 pgm/ml), CD29 ( MEM 101 A, 1 pg/ml) and CD43 (161-46, 1 pg/ml). Images of cells in culture at this time point were obtained using an inverted phase contrast microscope, attached to a video camera, and captured using NIH image software. A representative field is illustrated, showing strong U937 homotypic aggregation. (1) Medium alone. (2) CD98 (AHN-18) (3) CD43 (161-46). (4) CD29 (MEMIOIA). B) U937 cells were incubated for indicated times in the presence of mAbs to CD98, CD29 and CD43 and aggregation was measured under standard conditions as described in Materials and Methods. The results show mean aggregation ± SEM for triplicate cultures for 1 representative experiment of 2. C) U937 cells were incubated for 6 h in the presence of mAbs to CD98, CD29 and CD43 and the results show mean aggregation ± SEM for triplicate cultures for 1 representative experiment of 2. Negative signalling pathway
5.2.2 PKC inhibitors enhance CD98-mediated homotypic aggregation
GFX109203X is a broad-specificity PKC inhibitor, which has been shown to inhibit both classical (a, P, and y) and novel (5 , e , r| and 0 ) forms of the enzyme family
(Martiny-Baron et al, 1993; Wilkinson et al, 1993). Fig. 5.2A shows that GF109203X increased CD98-induced aggregation significantly. This effect was dose-dependent, between 1 and 10 pM of the inhibitor. Furthermore, GF109203X (Fig. 5.2B) increased the rate of CD98-induced aggregation two or three fold, to rates comparable to that seen in the presence of CD29 and CD43. GF109203X alone did not induce any clustering
(Fig. 5.2A and B). The same enhancement was seen with another structurally related
PKC inhibitor, staurosporin (Courage et al, 1995) (Fig. 5.2C) and structurally unrelated
PKC inhibitors, polymixin B sulfate and D-sphingosine (data not shown). Isoform specific cPKC inhibitor, myristoylated PKC peptide inhibitor (19-27), which is a selective inhibitor of PKCa/p, also strongly up-regulated CD98-induced aggregation
(Chapter 4). Collectively, these data suggest that CD98-induced aggregation is under negative regulation by cPKC.
5.2.3 PKC activators inhibit CD98-induced homotypic aggregation.
Because PKC inhibitors potentiated CD98-induced aggregation, the next experiments examined the effect of PKC activators. PMA (a direct activator of DAG-dependent PKC isozymes), and dioctanolyglycerol (DOG) (a DAG analog), both induced a time- and concentration-dependent inhibition of homotypic aggregation of up to 90% (Fig. 5.3 A,
B, C and D). PMA inhibited aggregation when added either before or up to 1 h after
CD98 mAb (Fig. 5.3B), suggesting that PMA may inhibit the middle or late phase of
CD98-induced aggregation. These results suggest that PMA (or DOG)-responsive PKCs
154 (A) (B) (C)
100 n 350-1 300 1 CD98 GFXCD98 GFX 250-
1 0 0 - 1 0 0 - 2 0 - 50-
0 0.1 0.6 1.3 2.5 5 10 0 0.5 1 2 5 10 50 100 Time (hour) nM
GF109203X Staurosporin
Fig. 5.2 Conventional and novel PKC inhibitors potentiate CD98-induced intercellular adhesion. A) and C) U937 cells were ineubated for 4 h in the presenee or absence of AHN-18 and aggregation was measured under standard conditions as described in Materials and Methods. GF109203X and staurosporin were added 30 min prior to the antibody and remained in the eulture. B) U937 eells were incubated for indicated times in the presence or absence of AHN-18 and GF109203X (5 pM), and aggregation was measured under standard eonditions as deseribed in Materials and Methods. The results show mean aggregation ± SEM for triplieate cultures for 1 representative experiment of 2. (A) (B) (C) 120 ^ 120 OlOO I o 100 100 o OP 80 I (4-, O 80 60 i 60 - 60 a T 03 40 T SP 40
< 20 20 < 20
0 n
0 0.5 6 12 25 50100 200 -1 0 1 0 25 50 100 200 ng/ml Treatment time (hour) )liM
PMA DOG
(D) c
Fig. 5.3 PKC activators down-regulate CD98-mediated aggregation. A) and C) U937 ells were incubated for 4 h in the presence or absence of AHN-18 (1 pg/ml) and aggregation was measured under standard conditions as described in Materials and Methods. PMA (A and B) and dioctanolyglycerol (DOG (€)) were added 30 min prior to the antibody and remained in the culture. B) U937 cells were incubated for indicated times in the presence or absence of AHN-18 and PMA, and aggregation was measured under standard conditions as described in Materials and Methods. The results show mean aggregation ± SEM for triplicate cultures for 1 representative experiment of 2. D) U937 cells were incubated for 4 h in the presence of AHN-18 (1 pg/ml) and PMA (50 ng/ml). Images of cells in culture at this time point were obtained using an inverted phase contrast microscope, attached to a video camera, and captured using NIH image software. A representative field is illustrated, showing strong U937 homotypic aggregation. (1) AHN-18. (2) AHN-18 + PMA. (3) PMA alone. Negative signalling pathway
have a negative regulatory role in CD98-mediated cell-cell adhesion.
5.2.4 PMA-mediated inhibition is abrogated by conventional PKC inhibitors
To characterise the inhibitory mechanism of PMA, further pharmacological dissection was carried out using a panel of PKC inhibitors. These included three groups, competitive inhibitors for the ATP-binding (GF109203X, staurosporin and Go6983),
Ca^^-binding (Go6976) and DAG-binding (calphostin C) sites of conventional and novel PKC isoforms. Fig. 5.4 shows that ATP-binding site inhibitors abrogated the
PMA effect (Fig. 5.4A), but neither calphostin C (DAG-binding site inhibitor)
(Kobayashi et al, 1989), nor Go6976, (Ca^^ binding site inhibitor) (Martiny-Baron, et al.,
1993), diminished PMA inhibition (Fig. 5.4B and C).
5.2.5 PMA induces translocation of conventional PKC isoforms to the plasma membrane.
To identify which PKCs were responding to PMA, the membrane translocation of a panel of PKC isoforms was examined. U937 cells express all eight isoforms of PKC tested, as demonstrated by Western blot (Fig. 5.5A), although PKCe expression was relatively low. Only a very small proportion of the conventional isozymes was associated with the membrane fraction in unstimulated cells, but a significant proportion of PKC Ô, Ç, and X was membrane bound (Fig. 5.5B). CD98 ligation induced an increase in membrane associated PKCô (Chapter 4), but no change in membrane association of any other isotypes (Fig. 5.5B). In contrast, PMA treatment, both in the presence and absence (data not shown) of CD98, induced strong membrane translocation of PKC a, p, and y, but not atypical forms (Ç and X), as reported
157 (A)
GF109203X Staurosporin Go6983 120 1 180 1 P 200 -1
C 100 - 1 150 - 8 160 4 4-4 I 80 X 120 - X O I 1 _ 120 - T X, 60 - 90 - c 80 - o 40 - 60 - X X ts bX) 40 - 20 30 - X (U 5) Ü , 0 1 0 bX) 0 n < CD98 0 0.3 0.6 1.2 2.5 5 CD98 0 0.05 0.1 0.2 0.4 0.8 CD98 0 0.2 0.4 0.8 1.6 3.2 p M pM pM
+PMA
Fig. 5.4A Conventional and novel PKC inhibitors toward ATP-binding site abolish PMA-mediated inhibition of CD98-induced homotypic aggregation. U937 cells were incubated for 3 or 4 h in the presence or absence of AHN-18 (1 pg/ml) and aggregation was measured under standard conditions as described in Materials and Methods. All compounds were added 30 min prior to the mAb and remained in the culture. The results show mean aggregation ± SEM for triplicate cultures for 1 representative experiment of 2. (B) (C)
120 1 Calphostin C 120 - Go6976 g 100 -j 100 - c o o 80 80 - O ^ 60 60 - C o 40 - 40 - a g) 20 20 - bb bû 0 0 < CD98 0 30 60 125 250 500 CD98 0 0.1 0.2 0.4 0.8
nM pM
+ P M A
Fig. 5.4B and 5.4C Conventional and novel PKC inhibitors toward ATP-binding site abolish PMA-mediated inhibition of CD98-induced homotypic aggregation.U937 cells were incubated for 3 or 4 h in the presence or absenee of AHN-18 (1 pg/ml) and aggregation was measured under standard eonditions as described in Materials and Methods. All compounds were added 30 min prior to the antibody and remained in the culture. Aggregation was measured under standard conditions as described in Materials and Methods. The results show mean aggregation ± SEM for triplieate cultures for 1 representative experiment of 2. (A) (B) Total Membrane 1 2 3 PKCa
Conventional PKCp
PKCy
PKC8
Novel PKCe
PKC0
PKCÇ Atypical PKCX
Fig. 5.5 PMA induces translocation of conventional PKC isoforms to the plasma membrane. A) and B) U937 cells were incubated for 20 min in the presence or absence of CD98-AHN-18 (1 pg/ml) and PMA (50 ng/ml). Cells were washed, lysed and fractionated into membrane and cytoplasmic fractions, as described in Methods. Total (whole lysate (A)) and membrane (B) [lane 1: normal, 2: CD98 mAb (AHN-18), 3: CD98 mAb + PMA] were analysed for the presence of specific antibodies to PKC isoforms by Western blot as described in Methods. The results from three separate experiments are shown. Negative signalling pathway
previously (Zheng et al., 2000).
5.2.6 PMA does not inhibit CD98-induced ERK activation.
PKC activation has been linked previously to up-regulation of MAPK activity (Monick et al., 2001; Yoon et al., 2000; Chen et al., 1999), and PMA treatment increases phosphorylation of MAPK, an indicator of MAPK activation. Previous studies
(manuscript submitted) showed that CD98 induced activation of MAPK/ERK and
MAPK/p38, and that this activation was essential for CD98-induced homotypic aggregation. To identify possible target substrates of PMA-dependent inhibition, we first examined whether PMA inhibits CD98-induced MAPK activation. Fig. 5.6 shows that PMA enhanced CD98-induced ERK activation, but the total MAPK/ERK was not changed by PMA treatment (data not shown), suggesting that the inhibitory target of
PMA-responsive PKC is downstream of MAPK. PMA-induced ERK phosphorylation, like the inhibition of aggregation, was also blocked by GF109203X, but not calphostin
C or Go6983.
5.2.7 PMA does not block adhesion molecule expression during CD98 - induced aggregation
CD98-mediated homotypic aggregation has been reported to be regulated by pi and p2 integrins and CD 147. The expression level of these adhesion molecules following PMA treatment was therefore determined. The time period selected (three hours) is that required for maximum inhibition. As shown in Fig. 5.7 and Table 5.1, there was no marked down-regulation of any marker by PMA treatment. However, the surface
161 aCD98 PMA No - PMA GF Go Cal p-Erk
Fig. 5.6 PMA up-regulates CD98-iTiediated phospho- MAPK/ERK expression. U937 cells were incubated for various times in the presenee or absence of CD98-AHN-18 (1 pg/ml) and PMA (50 ng/ml). Cells were washed, lysed and analysed for phospho-MAPK/ERK expression by Western blot as deseribed in methods. The blots show gels probed with antibody specific for phosphorylated forms of the kinase. GF109203X (10 pM), Go6976 (1 pM), and ealphostin C (1 pM) were added 30 min prior to the antibody and remained in the eulture. The result shows a representative data of 3 separate experiments. CD18 CD29
10' 10 10* 10' FL 1-H FL1 -H CD43 CD44
i
1 “io“ o'* 10* 10* 10' 10* FL1 -H 10' FL 1-H
CD98 CD147
A .. 1 o' 10 1 0 “ 10 " ' 10 * 10 " 10 "
Fig. 5.7A Effect of PMA on the surface level of U937 adhesion molecule. U937 cells were incubated for 4 hours in the presence or absence of PMA (50 ng/ml or indicated concentrations). Surface adhesion molecule levels were tested with a panel of U937 adhesion molecule mAbs by means of flow cytometry. The histograms only are representative of the obtained data. Histograms marked C are the fluorescence profile of CD98-treated cells stained with isotype control (Black colour : Normal, Green colour: PMA). (B) (C)
120 120
110 110
^ 100 100
60 50 100 200 0 1.5 3 6 Concentration (ng/ml) Time (hour)
Fig. 5.7B and C PMA down-regulates the surface level of U937 CD98. U937 cells were incubated for 4 h (B) or indicated times (C) in the presence or absence of PMA (50 ng/ml or indicated concentrations). Surface adhesion molecule levels were tested with a panel of U937 adhesion molecule antibodies by means of flow cytometry, MFI values were calculated by means of WIN-MDI software on a minimum of 5000 cells. The results are shown as mean ± SEM (n=4) (* : p<0.05, ** :p<0.01). Negative signalling pathway
Table 5.1 Effect of PMA on surface level of U937 adhesion molecules.
MFI values Group Normal PMA No Ab 10.2 ±0.7 11.2 ±0.7 CD98 355.7 ± 12.1 288.6 ± 14.2 * CD147 2181.4 ±57.3 2130.9 ±83.7 CD44 1403.4 ± 18.6 1549.2 ± 11.4* CD29 539.3 ±7.1 584.8 ± 11.3 CD43 429.6 ± 67.2 398.2 ± 24.9 CD18 72.4 ± 1.8 89.0 ±2.5 ** CD45 66.1 ± 0.4 72.2 ± 2.2
U937 cells were incubated with PMA (50 ng/ml) for 4 h and tested its effect on surface level of adhesion molecule. MFI values were calculated by means of WIN-MDI software on a minimum of 5000 cells. The results are shown as mean ± SEM for triplicate for 1 representative experiment of 2 (* : p<0.05 and ** : p <0.01).
165 Negative signalling pathway
expression of CD98 itself was significantly decreased, and the surface levels of CD 18 and CD44 were significantly increased, in agreement with previous reports (Nueda et al.,
1995). No significant changes were seen in levels of CD29, CD43 and CD147. The down-regulation of CD98 occurred at inhibitory doses of PMA and began after 1 hour incubation (Fig. 5.7B and C).
5.2.8 The relationship between PMA-responsive PKC and other signalling pathways
A previous study (manuscript submitted) demonstrated that CD98-induced aggregation is strikingly potentiated by a selective PKA inhibitor, KT5720 and inhibited by PKA activator, forskolin. Protein tyrosine kinase (PTK) inhibitors also strongly augmented
CD98-induced cell adhesion, indicating that both PKA and PTK might be involved as negative regulator of CD98-induced signalling pathways.
The relationship between PKC, PTK and PKA pathways was therefore explored. As reported previously, GF109203X, KT5720 and genistein potentiated CD98-induced aggregation, whereas PKC activators such as forskolin and PMA had a strong inhibitory effect (Fig. 5.8A and B). The potentiation by genistein was not seen in the presence of
PMA (Fig. 5.8A), suggesting that PTK activity may lie upstream of PKC in this negative regulatory loop. In contrast, KT5720 potentiated clustering even in the presence of PMA, while GF109203X potentiated clustering in the presence of foskolin
(Fig. 5.8B). The PKA inhibitory pathway is therefore parallel to PKC.
166 (A) (B)
160 140 f 120 § 100
ol—L4--V- W--M-W--*-r- aCD98 + + 4- + aCD 98 + + + + + + +
Genisterin - - + - + GFX + - -- + -
PMA - + PMA - + --- KT5720 - - - + - - +
Forskolin - - _ - + + -
Fig. 5.8A abd B Relationship between PMA-inhibition and other enzyme activation in U937 homotypic aggregation. A) and B) U937 cells were incubated for 3 or 4 h in the presence or absence of AHN-18 (1 pg/ml) and PMA (50 ng/ml), and aggregation was measured under standard conditions as described in Materials and Methods. All compounds including genistein (50 pM), GFX (10 pM), KT5720 (IpM) and forskolin (50 pM) were added 30 min prior to the antibody and remained in the culture. Aggregation was measured under standard conditions as described in Materials and Methods. The results show mean aggregation ± SEM for triplicate cultures for 1 representative experiment of2. Negative signalling pathway
5.2.9 The effect of other intracellular signalling enzyme inhibitors on PMA inhibition
Previously, PMA has been shown to activate a variety of intracellular signalling enzymes (PMA-inducible enzymes) such as phosphatidylinositol 3-kinase (PI3-K) and serine/threonine phosphatase (STPPase) (Tardif et al., 1998; Djerdjouri et al., 1995).
Therefore, to address whether or not PMA inhibition is mediated by down-stream activation of these PMA-inducible enzymes, a panel of enzyme inhibitors were tested.
Fig. 5.8C illustrates that the inhibitory effect of PMA does not appear to be related to any of these pathways. Thus, a PI3-K inhibitor [LY294002 (LY)], STPPase inhibitors
[okadaic acid (OKA) and cyclosporin (Cys)], and a Ca^^ chelator (EDTA) did not interfere with the PMA inhibition of CD98-induced aggregation, suggesting that these
PMA-inducible enzymes were not involved in PMA inhibition.
168 120 -
2 i Ü 100 - O o o 80 60 -
c 40 - • o1-H 4—» cd I X 20 - i T (U i bX) bX) 0 1 I I I I I I I I I I I I < 50 100 0.05 0.1 0.5 1 1000 5000 CD98 PMA LY OKA Cys EDTA
+ PMA Concentration (|tM)
Fig. 5.8C Relationship between PMA-inhibition and other enzyme activation in U937 homotypic aggregation. U937 cells were incubated for 3 or 4 h in the presence or absence of AHN-18(1 pg/ml) and PMA (50 ng/ml), and aggregation was measured under standard conditions as described in Materials and Methods. All compounds including LY294002 (LY), okadaic acid (OKA), cyclosporin (Cys) and EDTA were added 30 min prior to the antibody and remained in the culture. Negative signalling pathway
5.3 Discussion
CD98 is now recognised as a cell surface molecule that is important in a range of cellular processes, but very little is known about the CD98 induced signal transduction pathways, and about bow these might affect the many proposed functions of the molecule. In this study, therefore, we have continued our exploration of the CD98 signalling mechanism, using a quantitative assay of CD98-induced homotypic aggregation of U937.
Initial experiments showed that the kinetics of cluster formation induced by CD98 was slower than the cluster formation seen with other adhesion molecule (CD29 and CD43)- mediated homotypic aggregation events, indicating that a more complex process than rapid receptor-ligand surface interactions is probably involved. The PKC activators
PMA and DOG inhibited the aggregation in a dose- and time-dependent manner, while cPKC inhibitors (GF109203X, staurosporin, and a selective cell-permeahle, myristoylated protein kinase Ca/p inhibitor 19-27) enhanced the response. The PKC inhibitors (GF 109203, staurosporin and Go6983) also reversed the PMA-induced inhibition, thus confirming that PMA itself inhibits via PKC. cPKC isoforms, and not novel or atypical PKCs, were translocated to the cell membrane during PMA-inhihition.
CD98 ligation did not induce cPKC translocation. These results therefore suggest that constitutive activity of cPKC isoforms limit CD98-induced aggregation, and that this activity is amplified by pharmacological activation of these enzymes. The results extend our previous findings that PKCô is a key enzyme in initiating CD98-induced aggregation, by demonstrating that cPKCs play an opposing negative regulatory role on
170 Negative signalling pathway
this process.
PKC activity is regulated by DAG, phospholipids, Ca^^ and ATP, each of which bind to distinct sites on the enzyme. Aggregation was enhanced by GF109203X, staurosporin, and Go6983, all competitive inhibitors of the PKC ATP-binding site (Toullec et al.,
1991; Martiny-Baron et al., 1993), whereas the other inhibitors were ineffective.
Furthermore, PMA inhibition was not affected by Ca^^ depletion conditions
(EDTA/EGTA) and by DAG-binding inhibitor (calphostin C). The binding of ATP to the N-terminal pseudosubstrate binding domain therefore seems to be the key regulatory step in mediating the negative regulation of CD98-induced U937 aggregation.
An important question raised by these studies is what are the target molecules of activated PKC which inhibit the CD98-induced aggregation ? cPKC activation could result either in direct phosphorylation of CD98, or indirect regulation of other enzymes which are able to phosphorylate CD98, and this phosphorylation could negatively regulate the function of the molecule. Similar regulatory loops, including the regulation of serine/threonine kinase Akt and Btk by PKC (Zheng et al., 2000; Yao et al., 1994) and serine/threonine phosphorylation of CD43, have been reported for other signalling pathways. Furthermore, CD98 has eight predicted potential PKC phosphorylation (S/T-
Xa-R/K) sites and two potential cAMP/cGMP-kinase phosphorylation (R/K-Xaa-S/T) sites (Warren et al., 1996). However, our data suggest that the target of cPKC is downstream of the initial signalling events since PMA enhanced, rather than inhibited
CD98-induced MAPK/ERK phosphorylation. Furthermore, PMA inhibited aggregation even if added one hour after CD98 ligation.
171 Negative signalling pathway
An alternative possibility is that inhibition of homotypic adhesion is mediated by ERK itself. A similar negative regulation of IGF-1-induced Akt activation and insulin receptor signalling has been reported to be mediated by PMA-mediated MAPK activation (Zheng et al, 2000). However, this pathway is unlikely to operate in our model, since the selective MAPK/ERK inhibitor, PD98059 strongly blocked CD98- induced aggregation, (Chapter 3; Lu et al., 1998) and did not reverse the PMA inhibitory effect. Furthermore, many actin-binding proteins regulated by conventional
PKC more usually enhance cell movement, spreading and cytoskeletal organisation, and adhesion (Ono et al., 1997; Adams et al., 1999; Rojnunckarin and Kaushansky, 2001).
Identification of the targets of cPKC in this system therefore requires further investigation.
The experiments also investigated the relationship between the inhibitory cPKC activity, and that of two other families of enzyme also apparently involved in the negative regulation of CD98-induced homotypic adhesion, PKA and PTK. PMA completely inhibited the enhancement of CD98-mediated aggregation induced by genistein (a PTK inhibitor), but not by KT5720 (a PKA inhibitor). One model to explain these findings is that a PTK lies up-stream of cPKC, and hence can be by-passed by the addition of exogenous PMA (see Fig. 5.9). In an analogous pathway, we recently reported that
PKCÔ is one of the targets of CD98-induced PTK activation (manuscript submitted). In contrast, PKA mediated inhibition may be independent of the cPKC pathway. Further biochemical studies will be required to resolve these issues.
172 Negative signalling pathway
In conclusion, this study defines a cPKC mediated regulatory pathway which limits both the speed and the maximum extent of CD98-induced aggregation. The pathway appears to operate constitutively, but can be enhanced by PKC activators such as PMA and
DOG. cPKC regulation is mediated predominantly by alterations in binding of ATP to the regulatory site on the cPKC. The target(s) of cPKC remain undefined, but lie downstream to the activation of MAPK/ERK. These results need to be seen in the context of our previous studies, which identified PKCô as a key enzyme involved in triggering of homotypic aggregation in response to CD98 ligation. The balance between cPKC activity and PKCô activity therefore determine the extent of CD98-triggered homotypic aggregation. Further studies will be required to determine whether a similar regulatory circuit operates in mediating other CD98-dependent functions.
173 CD98 ligation
membrane CD98LC CD98HC
ài ài Homotypic aggregation
PTK ^— PKCÔ
hypo-phosphorylation Erk cPKC Unidentified Substrate(s)
hyper-phosphorylation
Homotypic aggregation 1 1
Fig. 5.9 Model of PKC-mediated negative regulation of CD98-induced cell adhesion (CD98HC : heavy chain of CD98. CD98LC : light chain of CD98. PKC: protein kinase C. PTK: protein tyrosine kinase. cPKC : conventional PKC. PKA : protein kinase A. - : negative feedback loop). Chapter 6
Conclusions
175 Conclusions
6.1 Role of CD98 in the regulation of integrin function
Recently molecular genetic analysis has demonstrated that CD98 is able to regulate integrin functions such as adhesion, spreading and migration. It has been suggested that the mode of action for this modulation is regulated very specifically by means of structural interaction with pi integrins, but not with other integrin families (Fenczik et ah, 1997; 2001; Zent et al., 2000). The cytoplasmic and transmembrane domains of
CD98HC are absolutely required for its interaction with integrins (Fenczik et al., 2001).
The significance of CD98-mediated control of integrin function is supported by a wide variety of in vivo studies, including those dealing with cell aggregation, adhesion, fusion and APC-derived T cell costimulation (Fenczik et al., 1997;. Chandrasekaran et al., 1999; 2000; Tabata et al., 1994; Ohta et al., 1994; Stonehouse et al., 1999; Warren et al., 2000; Woodhead et al., 2000).
Chapter 3 of this thesis provides new support for a functional association between
CD98 and integrins, since blocking antibodies (MAR4 and CLB-LFAl) to CD29 and
CD 18 significantly inhibited CD98-induced aggregation, although the inhibitory activities are weaker than for CD29- or PMA-induced aggregation (data not shown). An interesting observation was that P5D2, a mAh to CD29 which strongly inhibits integrin- mediated adhesion (Fenczik et al., 1997), did not inhibit CD98-induced homotypic aggregation, but rather enhanced cluster formation. This discrepancy in inhibitory action of P5D2 on aggregation and adhesion events is still not fully explained. One explanation could be that pi integrins are not critically involved in mediating CD98- induced cell-cell adhesion, and that some other adhesion molecule is involved.
176 Conclusions
Alternatively, different integrin epitopes are involved in mediating cell-cell aggregation and cell adhesion to ECM, respectively.
On the basis of the data in Chapter 3, we favor the former hypothesis for several reasons. Firstly, all three blocking mAbs (P5D2, MAR-4 and Lia 1/2 (not shown)) either enhanced or showed small inhibitory effects on CD98-induced intercellular aggregation.
Secondly, the inhibitory effect of the CD29 mAh (MAR4) is abrogated if the mAh is added after CD98 ligation, suggesting that pi integrin might be involved in the induction phase of CD98-induced homotypic aggregation, rather than mediating cell cell adhesion. Thirdly, we did identify another molecule, CD 147 which may mediate
CD98-induced aggregation, because two mAbs (MEM M6/1 and H84AF) to CD 147 strongly diminished CD98-mediated cluster formation. Interestingly CD29-induced aggregation was also inhibited by the mAbs to CD 147, as much as by some mAbs to
CD98 (BU89 and MEM 108), suggesting that these three molecules may be functionally associated to make an adhesion complex. The blocking effect of CD 147 mAbs was seen even when these mAbs were added subsequent to CD98 ligation, suggesting that CD 147 may be directly involved in mediating cell-cell adhesion. This is further supported by confocal analysis (work in progress) showing that both CD 147 and CD98 are concentrated at the sites of cell contact, suggesting that CD 147 may also physically associate with CD98 at the contact area. Taken in the context of previous work showing that CD 147 is a pi integrin-associated protein (Berditchevski et al., 1997), our results suggest that the three molecules CD98, pi integrins and CD 147 can form a molecular cluster. Further studies, using biochemical (such as co-immunoprecipitation) and molecular biological (such as transfection of wild type and mutant genes) methods are
177 Conclusions
required to probe the nature of this complex.
The physiologic regulation of CD98-induced aggregation, and other CD98 functions remains unclear. However, it is intriguing that CD 147, like CD98, acts as a chaperon for multimembrane spanning transporters. While CD98 associates with amino acid transporters, CD 147 chaperons members of the monocarboxylate transporter family
(Halestrap and Price, 1999). The multimolecular complex of CD98, CD 147, their associated transporters, and the pi integrins might act as a sensory unit, which regulates outcomes as diverse as adhesion, growth, differentiation and antigen presentation. These functions would then be regulated by multiple parameters, including ECM (integrins), levels of amino acids (CD98), and levels of carboxylic acids (eg, lactic acid) (CD 147).
Our preliminary results that nutrient-deprived culture conditions suppressed CD98- induced aggregation, but not CD43-induced aggregation (Table 6.1) appear to support such a hypothesis.
6.2 CD98 induces intracellular signalling
CD98 participates in an extraordinary diversity of cell functions. How this molecule can be implicated in such a wide range of activities is still not fully explained. One possible approach to this question is to study how activation of CD98 via extracellular ligation affects the intracellular signalling pathways and hence provides an ‘inside-out’ signal as well as ‘out-side-in’.
178 Conclusions
Table 6.1 Effect of nutrient-deprived culture conditions on CD98-induced aggregation.
Aggregation (% of aggregation) Recuneration CD98 CD43 time (h) RPMI1640 HESS RPMI1640 HESS 1 27.5 ± 5.0 22.9 ± 2.5 77.8 ± 1.7 75.7 ± 3.3 2 26.5 ± 4.5 13.3 ± 3.7 48.9 ± 8.6 71.8 ± 1.2 3 33.0 ± 3.3 9.3 ± 4.6 70.4 ± 1.9 85.3 ± 0.9
U937 cells washed 3 times with PBS were cultured in RPMI1640 or HESS solution with Ca^^/Mg^"^ for indicated recuperation times and were stimulated with aggregating mAh solution [CD98; AHN-18 ( 1 pg/ml) and CD43; 161-46 (0.5 pg/ml)] with 10 % dialysed PCS for 4 h. Aggregation was measured as described in Materials and Methods.
The results are expressed as mean aggregation ± SEM (n=3).
179 Conclusions
It is perhaps surprising that despite the multitude of studies that have addressed CD98 function, there is little information on these pathways. Possible signaling events, which have been suggested as transducing CD98-mediated function post-ligation, include tyrosine phosphorylation, G-protein activity, and MAPK activation. Many of these experiments have been carried out using BAF/hLFA-1 cell (Suga et al., 2001), FTH5 cells (Warren et al., 1996), Cd+U2ME-7 (Tabata et al., 1997) and blood monocytes
(Miyamoto et al., 2000; Tsurudome and Ito, 2000), have also been used, sometimes in the presence of virus.
A definitive pathway for CD98-mediated signalling has not been defined, however, this may be due to the variety of different cell types examined, the different functional outcomes, and the frequent absence of quantitation. The data only suggest that CD98- induced function is mediated by a complex interaction of intracellular signalling pathways.
In this thesis, therefore, using a reproducible and quantitative aggregation assay, intracellular signalling events have been dissected, using a combination of pharmacological and biochemical approaches. One of the major findings in this thesis is that members of the PKC family may play a central role in induction of CD98-mediated homotypic aggregation, thus extending the known role of PKCs in cell migration, adhesion and aggregation via the regulation of cytoskeleton and activation of adhesion molecules (Yue et al., 1999; Masur et al., 2001; Kermorgant et al., 2001).
6.2.1 Positive signalling
180 Conclusions
The first part of the thesis dealing with signalling (Chapter 4) presents evidence that
PKCÔ is selectively activated by CD98 ligation and in turn activates MAPK (ERK and p38). Thus, the partially selective PKCÔ inhibitor, rottlerin, and selective MAPK inhibitors, PD98059 (ERK) and SB20380 (p38) showed strong inhibitory effects on
CD98-induced U937 aggregation. Biochemical studies confirmed both PKCô membrane translocation, regarded as an indicator of activation of PKCô, and a time- dependent increase in expression level of phospho-ERK and phospho-p38. Both these events were inhibited by rottlerin treatment. Both biochemical and pharmacological data suggest, therefore, that PKCô may be involved in mediating CD98 signals. Further molecular analysis is required to confirm this model. Our results conflict with a previous report that CD98 ligation did not induce PKC activation in mediating gpl20- mediated cell fusion (Tabata et al., 1997). The discrepancy may be due either to the use of a different cell line, or to using different CD98 mAbs, or because cell fusion and cell aggregation depend on different signalling pathways. There are no reports connecting
PKC, MAPK activation and CD98, although it has been reported that MAPK activation accompanies CD98 ligation (Suga et al., 2001;Miyamoto et al., 2000).
A second interesting finding in Chapter 4 is that CD98 is capable of regulating tyrosine phosphorylation of PKCô itself. Because the tyrosine phosphorylation itself appears to be a negative regulator of CD98-induced intercellular adhesion (see below), tyrosine phosphorylation of PKCô may act as a negative feedback on PKCô activity (Haleem-
Smith et al., 1995; Deszo et al., 2001; Kronfeld et al., 2000). In summary, the data suggest a model in which CD98 selectively activates PKCô, and MAPKs leading to induction of homotypic aggregation. This is the first report that PKCô is a key regulator
181 Conclusions
of cell-cell aggregation.
6.2.2 Negative signalling
In the second part of the thesis (Chapter 5), a key finding is that CD98-induced aggregation is negatively regulated by cPKC isoforms (a, p, and y). Thus, there was no spontaneous aggregation-inducing effect of the cPKC inhibitor, GF109203X, but it strongly augmented CD98-induced aggregation. In parallel, a strong PKC activator,
PMA, suppressed CD98-mediated aggregation. This suppression was antagonised by conventional PKC inhibitors, suggesting that PMA-inhibition is due to activation of conventional PKC, but not nPKC. The suppression appears to occur downstream of
MAPK, since PMA enhances, rather than inhibits, CD98-induced MAPK phosphorylation. This data is the first evidence that cPKC can negatively antagonise nPKC (PKCô)-induced aggregation, stimulated by CD98 ligation. Although the exact mechanism by which cPKC interferes with PKCÔ effect is still not known, cPKC presumably regulate the phosphorylation state of downstream protein substrate(s) following MAPK activation, so that this hyper- or hypo-phosphorylated condition may play an important role in the regulation of CD98-mediated homotypic aggregation.
6.3 Proposed signaling pathway in U937 cells
On the basis of the data presented in Chapter 4 and 5 and the information available in the literature (reviewed in Chapter 1), we have proposed a model for the intracellular signalling pathway which connects CD98 ligation to U937 homotypic aggregation. The major features of the model are depicted in Fig. 6.1. Ligation of CD98 (by mAh in this study, but by interaction with appropriate counter-receptor in vivo) leads to a rapid
182 Conclusions
recruitment of PKCô to the membrane. An analogous model has been proposed for the events which follow cross-linking of the IgEy chain in mast cells (Haleen-Smith et al.,
1995). Recruitment may require some post-translational modification of PKCô itself, or alternatively modification of a PKCô receptor associated with the CD98/integrin/CD147 complex at the cell surface. Activated PKCô then activates a signalling cascade which involves both phosphorylation/activation of ERK/p38, and downstream activation of unidentified proteins (including cytoskeleton and PLD) which initiate the changes in cell adhesion which result in U937 aggregation. In parallel, PKCô activation activates, either directly or indirectly, tyrosine kinase activity [both lyn and src have previously been shown to be regulated by PKCÔ (Song et al., 1998)]. One target of this tyrosine kinase activity is PKCô itself, which may negatively regulate PKCô (Denning et al.,
1993), or alter the specificity of PKCÔ so as to phosphorylate different intracellular target proteins (Haleem-Smith et al., 1995; Deszo et al., 2001), and hence generate other functional outcomes. Other targets of the PKCô-activated tyrosine kinases remain undefined, but may be downstream pathways which regulate different CD98-dependent functions, such as growth and differentiation. Simultaneously, the molecular complex activates negative regulatory systems (including cPKC and PKA) which are able to limit cell aggregation. These enzymes may catalyse serine/threonine phosphorylation of target substrate protein(s) to allow hyper- or hypo-phosphorylation states, which regulate CD98-induced homotypic aggregation.
183 Ligation of CD98/integrins/CD147 complex
Membrane CD98LC
PKC5 ERK/p38
Tyrosine Negative regulators phosphorylation Cytoskeleton cPKC integrins
~Pl ' Down-regulation^O Q Other functions ^ Hyper-phosphorylation ^ Unidentified Aggregation substrates I' Hypo-phosphorylation Up-regulation
Fig. 6.1 Signal transduction steps which regulate CD98-induced U937 aggregation.Ligation of CD98 leads to a rapid recruitment of PKCÔ to the membrane. Activated PKCô then activates a signalling cascade which involves both phosphorylation/activation of ERK/p38, and downstream activation of unidentified proteins, including cytoskeleton and PLD (phospholipase D). Simultaneously, the molecular complex activates negative regulatory systems [including cPKC (conventional PKC) and PKA (protein kinase A)] which are able to limit cell aggregation. These enzymes may catalyse serine/threonine phosphorylation of target protein(s) to allow hyper phosphorylation state, resulting in down-regulation of cluster formation. Conclusions
6.4 CD98-induced signaling in mammalian cells
A broad view of how CD98 might regulate other signalling molecules in mammalian
cells is depicted in Fig. 6.2. The interaction of CD98 ligand with CD98, and the formation of a molecular complex within the cell membrane, initiates CD98-mediated
signal transduction. The complex may then incorporate further surface molecules
(including pi integrins, CD 147), cytoskeletal proteins (CP), and other signalling molecules (such as PTK, PI3-K, ras protein and PKCÔ). Subsequently, activation leads to downstream activation of the MAPK signalling cascade. With the activation of
MAPK, there is continuous activation of either transcription factors (e.g. Spl) to direct new protein synthesis (e.g. c-src in fusion events, or other substrates, including PLD, resulting in cell adhesion events.
6.5 Functional role of CD98
In addition to its original description as a phenotypic marker for activated lymphocytes,
CD98 is now known to be a multifunctional molecule involved in many cellular events
(Fig. 6.3). The function of CD98 appears to depend on making molecular complexes with other surface and intracellular molecules. The ability to form complexes with a variety of molecules may be a possible way to determine differential CD98 function for each cell or tissue. For example, CD98HC association with one of 6 light chains, with each of different specificity (Deves and Boyd, 2000; Verrey et al., 2000) may help to determined the nature of the response. At the same time as diversity, these complexes also perform one of the most basic cellular functions, i.e. transport of amino acids and
NaVCa^"^ into and out of the cytoplasm. The transport function of the CD98 complex could augment and control both protein biosynthesis and/or other intracellular signalling
185 Conclusions
pathways. The molecular complex can be regarded as a focus for recruitment and regulation of integrand functions, once the signalling complex has been formed, and then this can participate in regulating an extraordinary diversity of cellular functions, such as proliferation, costimulation, haematopoiesis, apoptosis, cell aggregation, fusion, and osteoclastogenesis.
6.6 Future direction
On the basis of the data presented in this thesis, several future avenues of research can be proposed. These summarised below focus on understanding CD98 function in cell adhesion and T cell costimulation, the major immunobiological roles of CD98.
6.6.1 CD98 ligation and the reorganization of cell surface molecules such as integrins and CD147
Antibodies to several cell surface molecules have been shown to have a potential blocking effect on CD98-induced homotypic aggregation. As expected, these include partial block by pi integrin, and p2 integrin antibodies. Unexpectedly, our results have suggested that aggregation is also reduced by antibodies to CD 147. Since the avidity of
pi and pi integrins and perhaps other adhesion molecules such as CD 147 is known to be strongly regulated by their distribution within the membrane (Jun et al., 2001; Bleijs et al., 2001), we propose to measure changes in this parameter following CD98 ligation in two ways:
186 CD98HC CD29 D147 CD98LC
Plasma membrane
PTK-1 Signalling molecule complex
Homotypic PTK-2 Q PI3-K PKCÔ MAPKKK aggregation Q CP-2 i r Unknown Others Ras MAPKK substrates i PLD MAPKs (ErL p38) Nuclear membrane
Transcription factor activation New protein synthesis Fusion (Spl) (including c-src) Fig. 6.2 The interaction of CD98 ligand with CD98 and the formation of molecular complex within the cell membrane initiates signal transduction. CD98 may transduce signals by association with several proteins, including (31 integrins, CD 147, cytoskeleton proteins (CP) and other signalling molecules such as PTK (protein tyrosine kinases), PI3-K (phosphatidylinositol 3-kinase), PKCô (protein kinase CÔ). The activation can lead to downstream activation of MAPKs. With the activation of MAPK, transcripion factor (e.g., Spl) activation directs new protein synthesis (eg., c-src) CD98 activation "►Intracellular events ► Cell ► Outcome
Proliferation/ Tumor cells ^ Transport Costimulation/ Regulation Activation CD98LC Amino acids of T lymphocytes Na+/Ca2+ cellular Amino acid Others (?) Cardiac/skeletap / function reabsorption muscle cells / Hematopoiesis Kidney cells Intracellular y signalling Hematopoietic Apoptosis Integrin progenitor cells CD98HC regulation Aggregation Cytoskeleton CD 147 Virus-infected^ y rearrangement cells pi integrins Fusion Monocytes Osteoclasto genesis Fig. 6.3 CD98-mediated regulation of cellular function. Conclusions
6.6.1.1 Lateral mobility of pi and P2 integrins and CD147 in the membrane following CD98 ligation
Cells will be treated with CD98 mAh, (or as controls, CD29 mAh or CD43 mAh, both of which induce homotypic aggregation) and then capping and patching of adhesion molecules (both processes known to be tightly controlled by cytoskeletal interactions) will be followed using fluorescently-labelled antibodies, with or without addition of a second layer to promote capping. This approach is now a well-established way of looking at changes in integrin/cytoskeleton interaction. If interesting changes are observed in the distribution of cell surface molecules using this approach, this could be
followed up by more quantitative and sophisticated assay of lateral mobility using photo-bleaching, and fluorescence recovery.
6.6.1.2 Molecular complex of CD98 and adhesion molecules in lipid rafts
Recently, it has become clear that the phenomena described in section 6.6.1.1 above, have a biochemical correlation, in the re-assortments of proteins within lipid rafts within the cell membrane (Peyron et al., 2000; Claas et al., 2001). Thus U937 cells will be treated with CD98 mAh or controls, and the lipid rafts will be isolated using gradient centrifugation as described. The rafts will be then assayed for the presence of pi and p2 integrins or CD 147 (or other adhesion molecules) by Western blot.
6.6.2 Signalling pathways
Having identified the PKCs and MAPKs principally activated following CD98 ligation, the identification of the down-stream protein targets of this phosphorylation is an obvious next step. For this purpose, ^^P-labeling will allow detection of the major
189 Conclusions
protein species which become phosphorylated following CD98 ligation. Molecular weights alone of these species may provide some clues as to their identity, but attempts to scale up and obtain identifying sequence data by mass spectroscopy from gels will also be explored.
6.6.3 New cellular models with which to analyse CD98 structure and functions in cell aggregation and T cell costimulation
The objective would be to set up a system where 1) mutant variants of CD98 can be introduced, and ligated independently of or in the absence of endogenous CD98 and 2)
CD98 can be ligated on the surface of antigen presenting or U937 cells, without at the same time ligating CD98 on the surface of the responding T cells.
6.6.3.1 myc-tagged variants of CD98
U937 cells will be transfected with CD98 constructs with a C-terminal (i.e. extracellular) myc-tag added to the CD98-encoding sequence. CD98 ligation will be then be accomplished independently of endogenously CD98 by use of an anti-myc antibody.
6.6.3.2 CD98-negative variants of U937 cells
U937 cells will be mutagenised using UV or chemical mutagenesis, and then CD98- negative variants will be isolated by repeated rounds of cell sorting. This is potentially the most strait forward approach, and has been used previously to identify class IIMHC negative variants of a B cell line (Poirier et al., 1993) and this would allow for the identification of CD98-dependent versus independent events within the multimolecular
190 Conclusions
complexes postulated above.
6.6.3.3 Human/mouse chimaeric model
Antibodies to human CD98 do not cross-react with murine CD98. Therefore, human
CD98 cDNA will be transfected into mouse antigen presenting cells or into related cell lines, and the effects of CD98 ligation on homotypic aggregation and also on antigen presentation by the transfected cells to murine T cells will be measured. This approach would also address questions rasied in sections 6.6.3.1 and 6.6.3.2 above, allowing ligation independent of both endogenous CD98 (mouse) and T cell CD98 (mouse).
191 Chapter 7
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