Overexpresssion of Tyrosine Kinase Discoidin Domain Receptor I (DDR1 ) in Transitional Cell Carcinoma

Overexpresssion of Tyrosine Kinase Discoidin domain receptor I (DDR1 ) in Transitional cell Carcinoma

Szu-Ting Chen,* and Shie-Liang Hsieh*

Institute and Department of Microbiology and Immunology, National

Yang-Ming University, Taipei, Taiwan;

To whom proofs are to be sent:

Shie-Liang Hsieh,

Department and Institute of Microbiology and Immunology, National

Yang-Ming University, Shih-Pai, Taipei 112, Taiwan.

E-mail address: [email protected]

Telephone number: 886-2-28267161

Fax number: 886-2-28277933

INTRODUCTION

DDR1, discoidin domain receptor 1, belongs to the novel subfamily of tyrosine

kinase receptor, which forms homodimer upon ligand engagement. DDR1 is distinguished from other receptor tyrosine kinase by the discoidin domain in their extracellular domain, which is a homology region originally identified in

Dictyostelium discoideum (slime mold) protein, discoidin I, and involves in cells

aggregation. Discoidin-1 has binding specificity toward galactose and N-acetyl

galactosamine and is essential for slime mold cells adhesion, migration and aggregation during its development, suggesting that DDR1 shared similar

biologically function in mammalian [1-3]. DDR1 has been found mainly

distributed and in human tissue epithelia, such as kidney, breast, lung [3],

bronchial [4] and keratinocytes [5]. Furthermore, DDR1 has been also reported

its expression in immune system like the monocyte-derived dendritic cells,

annotated as CD167 [6] and macrophage[7]. However, recently, the

overexpression of DDR1 has been detected in several human cancers, such as

primary breast cancer [1, 8, 9] ovarian[10, 11], brain[12], esophageal cancer [13],

and TCC (our data unpublished) in which raising the possibility that DDR1 may

play a important role in tumorigenesis [14]. Ligand binding to RTKs is though to induce receptors dimerization, and

dimerized receptors subsequently autophosphorylated specific tyrosine residues

in cytoplasmic domain each other. Collagen type I to IV can bind to discoidin

domain of DDR1 and activate this receptor; however, unlike other RTKs, the

activation process was so delayed that required ligand activation for 18 hrs to

reach maximal tyrosine phosphorylation [3, 15]. Collagen acts as a ligand for

DDR1, implying that DDR1 may involved in arterial wound repair and primary

vascular smooth muscle cells migration. The downstream genes MMP2 and

MMP9, which related to the extracellular matrix degradation and injury repair,

were further connected to DDR1 signaling pathway and regulated by activated

DDR1 [16, 17]. Recently, there has been hypothesized that DDR1 involved in

the regulation of leukocytes in response to inflammation microenvironment. By

degrading membrane basement, leukocytes extravasated the vascular endothelia

wall toward inflammatory site; and DDR1 expression was induced by

proinflammatory cytokine simultaneously. Due to basement destruction and

extracellular matrix exposure, the interaction between DDR1 and collagen,

which mediated the migration of tissue infiltrating leukocytes[7, 18]. In addition;

DDR1 also plays an essential role in coordinating other function of leucocytes, such like facilitating dendritic cells maturation, promoting macrophages differentiation [7, 19], and up-regulation of IL-1 and IL-8 production by human macrophages [20].

As mentioned previously, overexpression of DDR1 has been found in various types of cancer cells. Receptor overexpression mimicked the ligand engagement and resulted in the dimerization and autophosphorylation of these activated receptors. Therefore, basing on the constitutively activated form results from

DDR1 overexpression, we are interested to figure out the possibility of which results in tumorogenesis. According to the sequence similarity, there has been predicted that, tyrosine residues at 792, 796 and 797 may participate in autophosphorylation [21]. Owing to the phosphorylated tyrosine located in

DDR1 kinas domain (a.a. 610-915), implying 1) these tyrosine residues may involve in receptor activation; and 2) the expression of downstream gene may be regulated through these specific tyrosine residues in response to DDR1 activation.

Since the role of tyrosine residue in mediating DDR1 activation has not been illustrated yet, we characterized these specific tyrosine residues by using side-directed mutagenesis, and assayed the behavior of downstream gene in response to DDR1 activation. In the present study, constitutively activated DDR1 promoted IL-8 secretion and cells migration by modifying the tyrosine residues at site of 792, 796, and 797 of DDR1. These three tyrosine residues exhibited different level of contribution to mediating DDR1 activation, particularly the

Y796. This is a pilot experiment, in which supported the information of which

tyrosine should be chosen to lead the DDR1 constitutive activation. It will be a useful model in studying the mechanism of bladder cancer formation through the

DDR1 molecule.

EXPERIMENTAL PROCEDURES

Reagents, Cells and antibodies —DDR1 cDNA were cloned and PCR amplified

by using Pfu Turbo polymerase (Stratagene). The TransFastTM reagent was

purchased from Promega Biotechnology, Inc. (U.S.A.) in the utilization of all

cell type transient transfection. The human embryonic kidney 293 cells (with or

without large T antigen) and mouse fibroblast NIH3T3 were obtained from

American Tissue Culture Collection and cultivated under the recommended

conditions. Bovine type I collagen and all other reagents were purchased from

Sigma. Antibody to DDR1 (amino acids 894-913) were obtained from Santa

Cruz Biotechnology Inc. (Santa Cruz, CA), monoclonal anti-phosphotyrosine

antibody 4G10 was from Upstate Biotechnology, Inc. and monoclonal

anti-MMP2 antibody which recognized pro- and active MMP2 simultaneously was obtained form CHEMICON International Inc.

Plasmid construction and site-directed mutagenesis --DDR1 full-length cDNA

was cloned from U373MG cells with oligonucleotide primers harboring EcoRI

and BamHI restriction enzyme sequence. In this study, we used standard

overlapping PCR mutagenesis methods to construct the dominant active DDR1

expression vector. Briefly, DDR1-Y792E mutated fragments were PCR amplified with two primer pairs: (i) 5’-GTGAGG TCGACAGTCCCTCA-3’ sense primer harboring SalI site and the Y792E antisense primer, containing mutated bases shown in bold: 5’-CACACGGTAATAGTCCCCAGCCTCGAGG

TTCCGGCTCATGCCAAAG-3’, (ii) the Y792E sense, 5’-CTTTGGCATGAGC

CGAACCTCGAGGCTGGGGACTATTACCGTGTG-3’and 5’-TACGTGAGTT

GTGCCACACT-3’ primer containing an engineered BamHI site. PCR amplification condition was considered as usual, and these two fragments were overlapped in 15 cycles consisting of 1min at 95℃, 1 min at 55℃ and 1 min at

72℃. Overlapping PCR products were subjected to another amplification with the restriction enzyme harboring sense and antisense primers as previously described. The construction of DDR1 double-‘Y792/796E’ and triple-

‘Y792/796/797E’mutants were generated by replacing the DDR1wild type and

DDR1-Y792/796E mutant with the corresponding mutagenized fragments, respectively. Point mutation was introduced with the following oligonucleotides:

5’-CGGAACCTCGAGGCTGGGGAGTACCGTGTGCAGGGCCGGCAG-3’,

5’-CTGCCCGGCCCTGCACACGGTACTCGTCCCCAGCCTCGAGGTTCC-

3’were for Y792/796E and 5’-CCTCGAGGCTGGGGACGAGGACGTG

TGCAGGGCCGGGCAGTG-3’, 5’-CACTGCCCGGCCCTGCA1CACGTTCC

CGTCCCCACCTCGAGG-3’ were for Y792/796/797E. All cDNA were subcloned to pFlagCMV2 mammalian expression vector.

Transfection and western blotting---Semi-confluent 293T cells were independently transfected with wild type and tyrosine mutants of DDR1 full-length expression plasmids by using TransFastTM reagent. Sixteen hours later,

cells were transferred to serum-free media for another 20 hrs. Cells were

stimulated with 10μg/ml type I collagen for 90min and lysed with 1% Triton

X-100, 0.1% SDS, 150mM NaCl, 5mM EDTA, 50mM Tris-HCl (pH7.5), 10mM

NaF, 1mM sodium orthovanadate, 3μM H2O2 lysis buffer containing proteinase

inhibitor cocktail tablet. Equal amounts of total protein were subjected to

immunoprecipitation with anti-DDR1 antibody (C-20) for 4hrs at 4℃ on a

rotating wheel. The immunocomplex was washed three times and separated by

SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and

immuoblotted with anti-phosphotyrosine antibody diluted 1:1000 (4G10).

Western blots were developed using HRP-conjugated second antibody and

enhanced chemiluminescence (Amersham). For reprobing, the membrane was

stripped with strong re-probe kit (Chemicon) and immunoblotted with DDR1

c-20 antibody diluted 1:400.

DDR1 transient expression in 293 cell and IL8 secretion assay---Semi-confluent

293 cells (3×105) were seeded in 6-well plate and independently transfected with wild type and tyrosine mutants of DDR1 expression plasmids. Sixteen hours later,

the wild type transfectants were treated with or without 50μg/ml type I bovine

collagen. Harvesting the supernatants at 24, 36 and 48 hr after transfection

procedure. IL8 concentration was determined by using human IL8 ELISA set

(BD OptEIATM,BD Bioscience).

Cell migration assay---Mouse fibroblasts NIH3T3 were independently

transfected with wild type and tyrosine mutants of DDR1 expression plasmids.

Cells (5×104) were suspended in DMEM plus 1% fetal bovine serum and placed

onto the upper inserts of transwell (8-μm pores, o.33cm2, Costar), previously

coated with 0.01% gelatin (Sigma). DMEM containing 10% fetal bovine serum

in the lower compartment was used as chemoattractant. In some experiments,

cells were transferred directly on the uncoated inserts or in the presence of type I

collagen stimulation. These were allowed to migrate for 36 hours and then

non-migrated cells on the top of the filters were wiped off with cotton swabs.

Migrated cells attached to the bottom of filters were fixed with 4% paraformaldehyde followed by H&E staining. Migration was quantitated by counting the number of cells in four random ×200 field/filter and expressed as

the relative ratio in comparison with mock NIH3T3 transfectants.

Zymography analysis---MMP2 and MMP9 activity were determine by gelatin zymography with using 1mg/ml gelatin as substrate. The conditioned medium

was mixed with SDS-PAGE sample buffer in the absence of reducing reagent

and exectrophoresed in 8% polyacrylamide gel. After electrophoresis, the gel

were washed three times with 2.5% Triton X-100, 50mM Tris-HCl pH7.5 to

remove SDS and incubated in 0.1M Tris-HCl, 10mM CaCl2, 5mM ZnCl2 and

0.2% Brij35 for 24hr at 37℃. The gel was stained with Coomassie Blue R-250

and destained in 20% acetic acid, 20% methanol. Areas of MMP activity

appeared as clear bands.

Results

Lose of tyrosine phosphorylation in DDR 1mutants—There has been reported that the possibility of Y792, Y796, and Y797 in DDR1 may participate in receptor autophosphorylation [21] .We generate the dominant active construction of DDR1 by using site-directed mutagenesis, in which tyrosine residues was replaced with glutamate to mimic the negative charges of phosphate group on phosphotyrosine

(Fig1). As DDR1 has been reported to belong to the delay type of RTK activation, the intracellular tyrosine phosphorylation of DDR1 was delayed and peaked at

90min after collagen stimulation [15]. Therefore, we transfected wild type and mutant DDR1 expression vector in human 293T cells, and examined the phosphorylation level of different DDR1 mutants. As our prediction, wild type DDR1 achieved maximal phosphorylation at 90 min after collagen stimulation.

However, these three DDR1 mutants with different numbers of modified tyrosine

residues exhibited slight reducing in thelevel of tyrosine phosphorylation, which

compared with wild type DDR1 (Fig2). Due to the decreasing phosphorylation of

modified DDR1 after collagen stimulation, it seems that tyrosine site at 792 and 796

may required in the receptor autophosphorylation; the more modified tyrosine the

lower level of DDR1 phosphorylation. Furthermore, we observed that modified

Y797 seems not to further reduce phosphorylation level of DDR1-MT2, so we

suggest whether this tyrosine is essential in DDR1 autophosphorylation or it’s

change modified the DDR1 conformation and resulted in a little enhancement of

phosphorylation signal in DDR1-MT3? .

DDR1 mediated IL-8 secretion--It has been reported that collage-treated DDR1

would up-regulate chemokine production in the DDR1-infected human monocytic cell

line THP-1 [20]. Consequently, we performed this DDR1 transfection by using human

kidney 293 cells to evaluate whether the downstream IL-8 gene expression is also up-regulated by the collagen stimulation and DDR1 mutant. The secretion of IL-8 in

culture supernatants of each transfectant was quantified by ELISA analysis. In our

result, the IL-8 secretion of all mutant DDR1-overexpression cells were significantly

higher than mock control and wild type DDR1-transfected cells at 48 hr post-transfection. Also the result of IL-8 secretion at 36 hr post-transfection showed

the constant trend as 48 hrs does (Fig3 A.). Based on statistical analysis, the

DDR1-MT2 transfectant exhibited the highest IL-8 expression level in comparing

with other DDR1 mutants. However, the collagen-stimulated wild type DDR1

transfectants seem to not secret high level of IL-8 (Fig3 B.). According to these

results, we suggest that modification of tyrosine site at Y792, Y796 and Y797 by

replacement tyrosine with glutamate, which may involved in up-regulation of

downstream gene IL-8 expression via collagen independent manner in 293 cells

transient expression system, in particular the DDR1-MT2, containing the two

modified Y792/796E sites. As our prediction, modified Y797 would not further increase the IL-8 secretion of DDR1-MT2 transfectants.

Activation of DDR1 promotes fibroblast migration through gelatin basement--It has

been reported that DDR1 can mediate SMC cells migration in the presence of

collagen. To assess the role of different tyrosine residues of DDR1 in modulating

cells migration, mouse fibroblasts NIH3T3 were independently transfected and

measured the migration ability in the presence of chemoattractant. Migration assay

exhibited the significant differences between parental cells and DDR1-transfectants

at 36 hours. Thirty-six hours later, all DDR1-transfectants showed the more than

1.5- fold relative ratio compared with mock-transfected cells; however, there is no significant difference between DDR1 mutants and collagen–mediated wild type

DDR1 activation, except the DDR1-MT3, containing the modified Y797 (Fig.4 A.).

The in vitro invasion assay was performed by pre-coating gelatin gel on up-inserts of transwells. However, number of migrated cells in the presence of gelatin barrier was obviously less than the one directly seeded on the non-coating inserts (data not shown), but the overexpression of all wild type and DDR1 mutants still resulted in increasing cell motility compared with mock control as described previously.

Except our prediction, the DDR1-MT2 transfectants didn’t exhibit highest migration ability. Actually, the more modified tyrosine residues the higher invasion ability of cells was exhibited (Fig4 B.). The DDR1-MT2 which contain 792 and 796 modified tyrosine residues promoted 1.5-fold of invasion ability higher than the

DDR1 wild type-transfected cells, which similar to the collagen stimulated-wild type one. Nevertheless, the DDR1-MT3, which contains modified Y797, further promoted cells migrated through inserts of transwell, and exceeded the migration ratio of the collagen-stimulated DDR1-wild type transfectants. This observation was similar with our previous migration assay (Fig. 4 A&B).

Activated DDR1 and DDR1 mutants which, mediated cells migration in relation to the gelatinase activity--To further investigate whether the MMPs activation were related to the observed in vitro invasions, we performed gelatin zymography and western blotting of culture supernatants. Gelatin zympgraphy was mainly used to identify pro-MMP2, propMMP9 (72kD, 102kD) and active-MMP2, active-MMP9

(63kD, 83kD; Fig.5.A.). The western blotting identified the pro- and active MMP2 simultaneously. As our prediction, active-MMP2 was clearly increased in relation to the collagen mediated-DDR1 activation and tyrosine modified DDR1 mutants, in particularly the DDR1-MT2 (Fig5 B). Significant active MMP9 was seen in the collagen activated- DDR1 transfectants, surprisingly; also DDR1-MT2 containing two modified tyrosine residues induced significant MMP9 activity (Fig 5 A.). Based on MMPs analysis, the DDR1-MT3, which contains the modified Y797 didn’t further improve both MMP2 and MMP9 activity, and DDR1-MT3 is unlikely to have relationship with MMPs activation (Fig. 5. A&B). Results from these data,

The DDR1-MT2 mutant mediated the highest level of active MMP2 as well as the phenomenon of active MMP9, suggesting that Y796 is important in significant regulating gelatinase activation. In addition, to mimic the phosphate group in phosphorylated DDR1 by replacing both Y792 and 796 of DDR1 with glutamate residues is enough to mimic the constitutively activated DDR1. DISCUSSION

As a general mechanism, binding of ligand to receptor tyrosine kinase and subsequent conformational alteration of extracellular domain leads to receptor oligomerization.

The interactions between adjacent cytoplasmic domains lead to receptor autophosphorylation and activation kinase function by allosteric mechanisms [15, 22].

Autophosphorylation of receptor tyrosine kinase normaly occurs at a conserved tyrosine residues located in kinase domain that allosterically regulates the various tyrosine residues distributed along the intracellular portion [22]. Phsosphorylated tyrosine residues serve as docking sites for downstream signal transduction molecules containing either Src-homology or phosphotyeosine binding domains.[22-24]. DDR1 belongs to one subfamily of receptor tyrosine kinase, so far, there are five major isoforms of this receptor DDR1 protein have been characterized, which arise by alternative splicing. The longest DDR1 transcript codes for 919 amino acids long receptor (c-isoform), whereas the a –or b-isoform lack 37 or 6 amino acids in the juxtamembrane region, respectively [3, 9, 25]. As previous report, collagen bound and activated DDR1 to trigger the specific tyrosine residues in cytoplasmic domain. These tyrosine residues are embedded in consensus sequences, which on phosphorylation allow the binding of downstream effector molecules [15]. The activation of DDR1b induced phosphorylation of L-X-N-P-X-Y site in juxtamembrane of b-isoform and recruited Shc adaptor protein [3, 9, 25], however, activation of a-isoform created

another phosphorylation site at juxtamembrane region for FRS2 docking protein due

to lacking the L-X-N-P-X-Y motif [3, 26]. The tyrosine residue in DDR1 docking

sequence “L-X-N-P-X-Y ” was located at amino acid site of 513, and has been

reported in mediating complex downstream signal.[7, 18, 19]. According to sequence

similarity, tyrosine residues at 792, 796 and 797 may not only participate in

autophosphorylation but also locate in kinase domain of DDR1(a.a. 610-915). Since

DDR1 is a receptor tyrosine kinase, and so far, there are not any report about the

identification the role of these tyrosine residues in DDR1. So, we try to modify these

tyrosine residues and characterize the Y792, Y796 and Y797 in mediating DDR1

activation. In this study, we suggested that both Y792 and Y796 might participate in

receptor autophosphorylation after receptor activation. However, does Y797 play an

important role in DDR1 phosphorylation? From the data of phosphotyrosine western

blotting, we couldn’t answer this question precisely, because there were many reasons,

which can affect the phosphorylation level of DDR1. (i) Site Y797 just neighbors to

the Y796, which may disturb or enhance the recognition of anti-phosphotyrosine

antibody? (ii) The phosphorylation of DDR1-MT3 seemed a little enhance than the

DDR1-MT2, does it has the possibility to result in receptor conformation change and improving antibody recognition? All of these speculations need to further dissect by mass spectra. In IL-8 secretion, Y792 and Y796 involved in mediating downstream gene IL-8 expression. Modified Y792E and Y792/796E were sufficient to mimic constitutive activation of DDR1, in particular the Y796, the IL-8 secretion exhibited the highest expression level, and existed significant difference between

DDR1-MT1and DDR1-MT2 (p<0.01). However, except our prediction, the modified

Y797 at DDR1-MT3 decreased the IL-8 expression, and there is the significant

difference between DDR1-MT2 and DDR1-MT3 (p<0.05). Therefore, we suggested

that: (i) Y 797 is essential in supporting the constitutively activated DDR1, which is

modified by replacing Y to E at Y792 and Y796, (ii) Modifying Y797 to E797 may

raise the possibility of DDR1 conformation change. Unlike result of IL-8 secretion,

there were no difference between wide type, DDR1-MT1 and DDR1-MT2 in

mediating cells migration, except to the DDR1-MT3. Modified Y797 prompted the

cells migration; even relative migration ratio of DDR1-MT3 exceeded the collagen

stimulated -wild type one. When cells migrated through gelatin basement, number of

migrated wide type transfectants was obviously less than the one directly seeded on

the non-coating inserts, but once cells were stimulated with collagen, it seems to

restore the reducing in migration ability in the presence of gelatin barrier. Due to the

gelatin barrier, we suggested that major gelatinase-MMP2 and MMP9 may involved

in mediating cells migrated through gelatin gel. As our prediction, collagen stimulated-wild type DDR1 transfectants migrated through the gelatin barrier by secretion of active MMPs and gelatin digestion. Also the other DDR1 mutants such as

DDR1-MT1 and DDR1-MT2 exhibited higher relative migration ratio in gelatin

-coated transwells, which compared with the wide type one in the absence of collagen stimulation. Therefore, we suggested that Y792 and Y796 played an important role in regulating active MMP expression and as well as downstream IL-8 expression, modified E792 and E796 were sufficient to mimic the activation of DDR1 in response to cells migration through gelatin barrier, in particular the Y796. In this study, Y797 is still a controversial issue, we observed that, modified Y797E reduced the downstream

IL-8 expression but promoted cells migration in the presence or absence the gelatin barriers. However, there isn’t any relationship between the cells migration and active

MMPs expression, which mediated by the DDR1-MT3. DDR1-MT3 triggered no active MMP9 and less active MMP2 expression, compared with the DDR1-MT2, which contains only two modified tyrosine residues. Taken together, we thought whether the modified Y797E supported the positive regulation on cells migration but served a negative regulation in mediating IL-8 expression. DDR1-MT3 performed the positive regulation in cells migration, which excluded the gelatin digestion by active

MMPs expression. Due to DDR1 shares various biological functions, there were very complex signal molecules, which may be the associated protein or other transcription factor involved in signal transduction and gene expression. For example, (i) DNA damage induced a p53-dependent DDR1 response associated with activation of its tyrosine kinase, and DDR1 also activated the MAPK cascade in response to DNA damage [27]. (ii) DDR1 also plays an important role in controlling immune cells by regulating chemokine production, macrophage differentiation and demdritic cells maturation through the p38 MAPK kinase and NF-kB molecules [7, 19, 20]. (iii) Also the DDR1 modulated cells adhesion by down-regulated p42/p44 MAPK kinase activity [28]. However, there is no signal molecule, which is involved in DDR1 mediated- cells migration, reported until recently. Like DDR1, receptor with different associated protein in cytoplasmic domain resulted in different downstream signals.

Therefore, we suggested whether the Y797 played an essential role in maintaining conformation for certain kind of associated protein to adapt to cytoplasmic domain of this receptor, or the modified Y797E supported the suitable conformation change for other kind of adapted protein association. Finally, we identified that tyrosine residues Y 792,Y796 and Y797 indeed involved in regulating downstream signals of

DDR1. The Y792 and Y796 may directly participate tyrosine phosphorylation and these modified residues Y792/796E resulted in constitutively activation of DDR1 and the Y797 is still required to further characterize it’s role in DDR1 activation and conformation maintenance. In cancer cells, overexpression of DDR1 has been reported in various types of cancer cells, receptor overexpression mimicked the ligand

engagement and resulted in the dimerization and autophosphorylation of these

receptors. Our constitutively activated DDR1 mutants exhibited the promotion in cells migration under gelatin barriers, which hints that the cancer cells with over-expressed

DDR1 may have relationship with metastasis of these tumor cells. In addition, these

constitutively activated DDR1 mutants could further be studied the role in cancer

formation or cells metastasis in vivo by generating DDR1-MTs transgenic mice.

Reference

1. Johnson, J.D., J.C. Edman, and W.J. Rutter, A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain. 1993. 90(12): p. 5677-5681. 2. Lai, C. and G. Lemke, Structure and expression of the Tyro 10 receptor tyrosine kinase. 1994. 9(3): p. 877-883. 3. Vogel, W., Discoidin domain receptors: structural relations and functional implications. 1999. 13 Suppl: p. S77-S82. 4. Sakamoto, O., et al., Expression of discoidin domain receptor 1 tyrosine kinase on the human bronchial epithelium. 2001. 17(5): p. 969-974. 5. Di Marco, E., et al., Molecular cloning of trkE, a novel trk-related putative tyrosine kinase receptor isolated from normal human keratinocytes and widely expressed by normal human tissues. J Biol Chem, 1993. 268(32): p. 24290-5. 6. Lapteva, N., et al., Profiling of genes expressed in human monocytes and monocyte-derived dendritic cells using cDNA expression array. 2001. 114(1): p. 191-197. 7. Matsuyama, W., et al., Interaction of discoidin domain receptor 1 isoform b (DDR1b) with collagen activates p38 mitogen-activated protein kinase and promotes differentiation of macrophages. 2003. 17(10): p. 1286-1288. 8. Barker, K.T., et al., Expression patterns of the novel receptor-like tyrosine kinase, DDR, in human breast tumours. 1995. 10(3): p. 569-575. 9. Perez, J.L., S.Q. Jing, and T.W. Wong, Identification of two isoforms of the Cak receptor kinase that are coexpressed in breast tumor cell lines. 1996. 12(7): p. 1469-1477. 10. Laval, S., et al., Isolation and characterization of an epithelial-specific receptor tyrosine kinase from an ovarian cancer cell line. 1994. 5(11): p. 1173-1183. 11. Heinzelmann-Schwarz, V.A., et al., Overexpression of the cell adhesion molecules DDR1, Claudin 3, and Ep-CAM in metaplastic ovarian epithelium and ovarian cancer. 2004. 10(13): p. 4427-4436. 12. Weiner, H.L., et al., Consistent and selective expression of the discoidin domain receptor-1 tyrosine kinase in human brain tumors. 2000. 47(6): p. 1400-1409. 13. Nemoto, T., et al., Overexpression of protein tyrosine kinases in human esophageal cancer. 1997. 65(4): p. 195-203. 14. Weiner, H.L. and D. Zagzag, Growth factor receptor tyrosine kinases: cell adhesion kinase family suggests a novel signaling mechanism in cancer. 2000. 18(6): p. 544-554. 15. Vogel, W., et al., The discoidin domain receptor tyrosine kinases are activated by collagen. 1997. 1(1): p. 13-23. 16. Hou, G., W. Vogel, and M.P. Bendeck, The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair. 2001. 107(6): p. 727-735. 17. Hou, G., W.F. Vogel, and M.P. Bendeck, Tyrosine kinase activity of discoidin domain receptor 1 is necessary for smooth muscle cell migration and matrix metalloproteinase expression. 2002. 90(11): p. 1147-1149. 18. Kamohara, H., et al., Discoidin domain receptor 1 isoform-a (DDR1alpha) promotes migration of leukocytes in three-dimensional collagen lattices. 2001. 15(14): p. 2724-2726. 19. Matsuyama, W., M. Faure, and T. Yoshimura, Activation of discoidin domain receptor 1 facilitates the maturation of human monocyte-derived dendritic cells through the TNF receptor associated factor 6/TGF-beta-activated protein kinase 1 binding protein 1 beta/p38 alpha mitogen-activated protein kinase signaling cascade. 2003. 171(7): p. 3520-3532. 20. Matsuyama, W., et al., Activation of discoidin domain receptor 1 isoform b with collagen up-regulates chemokine production in human macrophages: role of p38 mitogen-activated protein kinase and NF-kappa B. 2004. 172(4): p. 2332-2340. 21. Perez, J.L., et al., Identification and chromosomal mapping of a receptor tyrosine kinase with a putative phospholipid binding sequence in its ectodomain. 1994. 9(1): p. 211-219. 22. Ullrich, A. and J. Schlessinger, Signal transduction by receptors with tyrosine kinase activity. 1990. 61(2): p. 203-212. 23. Cantley, L.C., et al., Oncogenes and signal transduction. 1991. 64(2): p. 281-302. 24. Koch, C.A., et al., SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. 1991. 252(5006): p. 668-674. 25. Alves, F., et al., Identification of two novel, kinase-deficient variants of discoidin domain receptor 1: differential expression in human colon cancer cell lines. 2001. 15(7): p. 1321-1323. 26. Foehr, E.D., et al., Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembrane domains in PDGFR/DDR/TrkA chimeric receptors. 2000. 14(7): p. 973-981. 27. Ongusaha, P.P., et al., p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop. 2003. 22(6): p. 1289-1301. 28. Curat, C.A. and W.F. Vogel, Discoidin domain receptor 1 controls growth and adhesion of mesangial cells. 2002. 13(11): p. 2648-2656. LEGENDS

Fig. 1. Schematic representation of constitutively active form of DDR1 cloned into

pFlagCMV2 vector. The tyrosine residues amino acid at 792, 796, and 797 located at

the tyrosine kinase domain were replaced with glutamate by site-directed mutagenesis.

Abbreviation: WT; wild type, MT1, mutation at aa 792; MT2: double mutation at a.a.

792 and 796; MT3: triple mutation at a.a. 792, 796 and 797.

Fig. 2. Human 293T cells were transfected with wild type DDR1 and its mutants,

respectively, followed by incubation with 10μg/ml type I collagen for 90 min to

activated DDR1. Cell lysates were immunoprecipitated by anti-DDR1 mAb, followed

by Western blot analysis using anti-phosphotyrosine (anti-pY) mAb or anti-DDR1

mAb.

Fig. 3. DDR1 mediated up-regulation of IL8 secretion. (A) Human kidney 293 cells

were transfected with wild type and DDR1 mutant. The secretion of IL8 at 24, 36,

48hrs post-transfection of 293 cells was quantified by ELISA. To evaluate the effect of collagen in IL8 up-regulation, wild type-transfectants were also stimulated with

type I collagen. (B) Statistical analysis of IL8 secretion at 48hrs post-transfection was

performed by one-way ANOVA test. *, p<0.05, n=5； **, p<0.01, n=5.

Fig. 4. Dominant active DDR1 mutants promote NIH3T3 cell migration. (A). NIH3T3 cells (5×104) transfected with wild type DDR1 and DDR1 mutants were placed onto upper inserts of transwell. Lower compartments contained medium plus 10% fetal bovine serum as chemoattractant. After 36 hours, cells which migrated through the filters were fixed and stained with H&E. (B) Upper-inserts were pre-coated with 0.01% gelatin before seeding of transfectants for invasion assay. To evaluate collagen effect, transfectants were also stimulated with 10μg/ml type I collagen as control. Migration was quantified by counting the numbers of cells in four random ×200 fields/filter and displayed as the relative migration ratio. Statistic analysis was performed by one-way

ANOVA test. *, p<0.05, n=3； **, p<0.01, n=3.

Fig 5. Expression and activation of gelatinase in response to DDR1 activation. Mouse fibroblasts were transfected with wild type and DDR1 mutants, while the activities of

MMP2 and MMP9 was determined by gelatin zymography (A), while the pro-active form of MMP2 (Pro-MMP2) and active MMP2 contained in the culture supernatants was detected by Western blot using anti-MMP2 antibodies. Band intensities were analyzed with ImageQuant ® 5.2 software. The ratio of active MMP2 versus total

MMP2 is listed in the bottom.

Figure 1.

MT1

MT2

MT3

Figure 2.

PFlagCMV-DDR1

Mock WT Mt1 Mt2 Mt3

Collagen + + + + +

130 IB: anti- pY

130 IB: anti-DDR1

792 - Y E E E 796 - Y Y E E 797 - Y Y Y E

Figure 3 A.

IL8 secretion 350 24hr 300 36hr 250 48hr 200 150 pg/ml 100 50 0

mock DDR1

DDR1/mt1 DDR1/mt2 DDR1/mt3

collagen-DDR1

B. **

** 350 300 * 250

200

pg/ml 150

100 50 0 * 1 R mock DDR1 DD DDR1/mt1 DDR1/mt2 DDR1/mt3 eat

collagen tr

A. Motility of NIH3T3

** 2.0

1.6

1.2

0.8

relative unit

Migration ratio 0.4

0.0

k -1 t2 oc mt -m m DDR1 - DDR1 n- e DDR1 DDR1 DDR1-mt3 g la ol B. c

Invasion of NIH3T3

** 2.5 * 2.0

1.5

1.0 relative unit

Migration ratio 0.5

0.0 k 1 1 c o R1 R -mt- m DD 1 DR DR1-mt2 DR1-mt3 en-DD D D D g colla A.

pFlagCMV-DDR1

Mock WT WT -C Mt1 Mt2 Mt3 Pro- MMP9 Active MMP9

Pro- MMP2 Active MMP2

pFlagCMV-DDR1

Mock WT WT -C Mt1 Mt2 Mt3

72kD Pro-MMP2 63kD Active MMP2

46 49 60 65 80 60 % Active MMP2