Regulation of Folate Receptor Raft Recycling
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Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences Regulation of Folate Receptor Raft Recycling Submitted by: Hala Elnakat In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: Manohar Ratnam, Ph.D. Academic Advisory Committee: Han-Fei Ding, Ph.D. Sonia Najjar, Ph.D. Kandace Williams, Ph.D. Robert Trumbly, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: January 4, 2007 Regulation of Folate Receptor Raft Recycling Hala Elnakat The University of Toledo 2007 DEDICATION A mes parents, que j’aime énormement. ii ACKNOWLEDGMENTS I would like to thank my major advisor, Dr. Manohar Ratnam, for his guidance, his patience and his optimism when all else fails. I also would like to thank my advisory committee: Dr. Sonia Najjar, Dr. Robert Trumbly, Dr. Han-Fei Ding, and Dr. Kandace Williams for their support and suggestions. I would like to thank Dr. William Gunning for the many hours he spent helping me use the electron microscope. I would like to thank Dr. David Giovannucci for his time and help with the calcium ionophore experiment. I would like to thank Dr. Venkatesha Basrur for performing and analyzing the mass spectrometry data for us. I would like to thank Dr. Khew-Voon Chin for the replication-deficient adenoviruses expressing Ad5-type adenovirus vectors containing the cDNA for wild type PKCs (α, βII, δ and ε) and kinase negative mutants (DN-PKCα and DN-PKCβII). I would like to thank Dr. Kevin Pan for the PKCβI wild type construct. I would like to thank Dr. Darlene Dartt (Harvard Medical School) for the constitutively active myristoylated PKCα adenovirus. I would like to thank Dr. Hongjuan Cui and Sandra Beach for their help and guidance with the retroviral and lentiviral infection systems. I would like to thank all my colleagues George, Huiling, Ayman, Marcella, Remi, Karen, Thuyet, Hong and Juan for being great people to work with. iii Last but not least, I would like to thank Mariana Stoeva and Jenny Zak for always being there for me. iv TABLE OF CONTENTS DEDICATION.................................................................................................................... ii ACKNOWLEDGEMENTS............................................................................................... iii TABLE OF CONTENTS.....................................................................................................v INTRODUCTION ...............................................................................................................1 LITERATURE.....................................................................................................................4 MATERIALS AND METHODS.......................................................................................29 RESULTS ..........................................................................................................................45 DISCUSSION....................................................................................................................75 SUMMARY.......................................................................................................................80 BIBLIOGRAPHY..............................................................................................................81 ABSTRACT.....................................................................................................................112 v INTRODUCTION The two human folate receptor (FR) isoforms, FRα and FRβ share 71% amino acid sequence homology (Lacey et al., 1989; Ratnam et al., 1989) and have a glycosyl- phosphatidylinositol (GPI) anchor attached to the carboxyl-terminal end of the proteins at serine 234 and asparagine 230, respectively (Yan and Ratnam, 1995). The third isoform, FRγ lacks a GPI anchor due to the lack of an efficient signal for GPI modification (Shen et al., 1994, 1995). Like most GPI-anchored proteins, FRα and FRβ are located in special membrane microdomains referred to as rafts (Mayor et al., 1994; Wu et al., 1997) that are insoluble in cold non-ionic detergents and are additionally characterized by a concentration of cholesterol, glycosphingolipids and other signaling proteins (Edidin, 2003; Rajendran and Simons, 2005; Vereb et al., 2003). Caveolae are also detergent resistant membrane complexes that could be distinguished from rafts mainly by the presence of a coat of caveolin on the cytosolic surface of the membrane (Liu et al., 1997a; Rothberg et al., 1992) and mostly the absence of GPI-anchored proteins. Quantitative fluorescence microscopy data and kinetic studies in monkey kidney epithelial cells (MA104) have shown that FR quantitatively recycles, within minutes, between the cell surface and endocytic compartments via a Cdc42-regulated endocytic pathway (Kamen et al., 1988, 1989; Rothberg et al., 1990; Sabharanjak et al., 2002). Furthermore, FR recycling in these cells is modulated by phorbol-12-myristate-13-acetate (PMA) or other protein kinase C (PKC) activators which inhibit the internalization step of FR, thereby resulting in an increase in cell surface pool of FR (Smart et al., 1994). 1 This increase is abolished by incubating the cells with inhibitors specific for classical PKCs prior to the addition of PMA (Kamen and Smith, 2004). Diacylglycerol and PMA both activate classical and novel PKCs by binding to a highly conserved cysteine-rich regulatory motif located in the amino-terminal end of the kinase. As a result of this activation, different PKC isoforms have been visualized in live cells to rapidly translocate from the cytoplasm to the plasma membrane (Almholt et al., 1999; Ng et al., 1999; Ohmori et al., 1998; Sakai et al., 1997). Different activated PKC isoforms can bind to specific receptors for activated C kinase (RACKs) which act as shuttling proteins to the intracellular target sites of the kinase. Prolonged treatment with PMA results in the ubiquitination of the protein which targets it for degradation by the proteasomes (Lee et al., 1997; Leontieva and Black, 2004). Since rafts play a major role in cell signaling, understanding how raft recycling and reorganization are altered by physiological signals and various drug treatments offers a means to modulate immune responses, malignant cell growth and cell death (Kaneko et al., 1997; Mollinedo and Gajate, 2006; Simons and Toomre, 2000). In the specific case of FR, one of the potential clinical significance of targeting raft recycling to internalize the receptor is to increase the uptake of folate compounds and novel antifolates by macrophages in rheumatoid arthritis and by ovarian or endometrial FR- positive tumor cells (Buist et al., 1995; Mantovani et al., 1994; Nagai et al., 2006; Nagayoshi et al., 2005; Theti et al., 2003; Veggian et al., 1989; Wu et al., 1999). In this study, we investigated the molecular mechanism by which FR recycling is regulated by PMA since its effects on physiological processes are known to stimulate cell 2 signaling through the second messenger diacylglycerol. We first attempted to identify which isoform(s) mediates the phorbol ester effect in MA104 cells especially since both classical and novel PKCs are activated by PMA. Our data suggest that PKCα, targeted to specific membrane microdomains, is one of the key players in mediating the phorbol ester effect on FR recycling in these cells. In order to identify other proteins that might also be involved in FR recycling, we purified FR-rich rafts using an immobilized biotinylated folate probe. Among the proteins identified, annexin II was required for the internalization of FR rafts and RACK1 mediated the effect of PMA on FR recycling in MA104 cells. Our hypothesis is that the population of FR rafts on the cell surface is increased by activators of PKCα as a result of targeting of PKCα to rafts by RACK1 and phosphorylation/inhibition of annexin II. These detailed studies provide a comprehensive molecular picture of the effect of PMA on FR recycling in MA104 cells. 3 LITERATURE The Glycosyl-phosphatidylinositol-anchored Folate Receptor (GPI-anchored FR) Folate receptor is bound to the plasma membrane via a GPI-anchor. The signal for the addition of a GPI anchor which is located in the C-terminal end of a newly translated polypeptide is cleaved off in the endoplasmic reticulum and replaced with a GPI moiety by a transamidase that covalently binds the ethanolamine of the GPI moiety to the new C- terminal end of the precursor protein destined to the plasma membrane (Bangs et al., 1985, 1986; Ferguson et al., 1986). The GPI signals of different proteins share a number of characteristics that mainly include an uncharged amino acid representing the GPI attachment (ω) site, separated by a spacer of 8 to 12 amino acids from a sequence (10 to 20 amino acids) that most importantly has a hydrophobic core of 6 to 8 amino acids (Cross, 1990; Ferguson and Williams, 1988; Moran et al., 1991) (Figure 1). Site-directed mutagenesis experiments of amino acids in the carboxyl-terminal regions of FRα and FRβ (Yan and Ratnam, 1995) allowed the characterization of the signal peptides of these two human isoforms that share 71% amino acid sequence identity with each other (Lacey et al., 1989; Ratnam et al., 1989). The third human FR isoform, FRγ is a secretory protein due to the lack of an efficient signal for GPI modification (Shen et al., 1995). Folate receptor α contains 257 amino acids (Lacey et al., 1989) and has a ω site at