©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Requirement of a Motif within the Carboxyl-terminal Domain of -Platelet-derived Growth Factor (PDGF) Receptor for PDGF Focus Forming Activity Chemotaxis, or Growth (*)

(Received for publication, January 26, 1995)

Jin-Chen Yu Weiqun Li Ling-Mei Wang Aykut Uren Jacalyn H. Pierce Mohammad A. Heidaran(§)

From the Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To determine the molecular basis for the transforming function of platelet-derived growth factor (PDGF)-A in NIH/3T3 cells, we have constructed chimerae consisting of the extracellular domain of the human CSF-1R (fms) linked to the cytoplasmic domain of the alphaPDGF receptor (alphaR) containing a series of deletion or point mutations. The ability of fms/alphaR chimerae to mediate CSF-1-dependent anchorage-independent growth, focus formation, and chemotaxis of NIH/3T3 cells was then examined. Our results provide evidence that a domain encompassing amino acid residues 977-1024 of the alphaPDGFR is required for ligand-dependent focus formation, but not chemotaxis or anchorage-independent growth, and that tyrosine residues within this domain constitute the major binding site for phospholipase C. Therefore, our findings suggest that: (i) the focus forming function of alphaPDGFR correlates well with the ability of the receptor to bind phospholipase C, and (ii) the mechanism of focus formation mediated by alphaPDGFR may be distinguished from that required for chemotaxis or anchorage-independent growth.


INTRODUCTION

Platelet-derived growth factor (PDGF) (^1)is a multifunctional molecule that does not only regulate DNA synthesis and cell division but also induces a variety of other biological effects that are implicated in neoplastic diseases, atherosclerosis, tissue repair, and inflammatory responses(1, 2) . PDGF comprises dimers of PDGF-A and PDGF-B chains encoded by distinct genes(3, 4) . PDGF dimeric isoforms have been shown to bind with different affinities to two different but related receptor molecules, designated alphaPDGFR and betaPDGFR, which are encoded by distinct genes(5, 6) . The PDGF-A homodimer mediates biological functions that include chemotaxis, anchorage-independent growth, and focus formation by activating the tyrosine kinase activity of alphaPDGFR in NIH/3T3 fibroblasts(7, 8, 9, 10) . Accumulating evidence indicates that many of the effector molecules involved in the PDGF-mediated signaling pathway interact with the kinase insert or carboxyl-terminal domains of PDGFRs(11) . In the present study, we sought to further examine the functional role of these domains in a physiologically relevant microenvironment in an attempt to better understand the molecular basis of biological responses mediated by alphaPDGFR.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human CSF-1 was obtained from Genetic Institute. Anti-CSF-1R antibody was purchased from Santa Cruz Biotechnology, Inc. The mouse monoclonal anti-phosphotyrosine (anti-Tyr(P)) antibody and anti-p85 were purchased from Upstate Biotechnology Inc. The anti-PLC was a gift of Dr. Sue Goo Rhee.

Generation of fms/alphaPDGFR Chimerae

The mutant alphaPDGFRs were generated using a combination of site-directed mutagenesis and polymerase chain reaction techniques. A detailed description will be provided upon request.

Chemotaxis, Focus Formation, and Anchorage-independent Growth Assays

Chemotaxis, focus formation, and anchorage-independent growth assays were performed as described elsewhere(14, 15) .


RESULTS AND DISCUSSION

To examine the functional role of distinct structural domains of alphaPDGFR in ligand-dependent biological responses, two carboxyl-terminal truncation mutants lacking amino acid residues 1025-1089 or 977-1089, designated alphaRDeltaC1 and alphaRDeltaC2, and a group of point mutations substituting phenylalanine for tyrosine residues 988, 993, or 1018, designated alphaR(Y988F), alphaR(Y993F), and alphaR(Y1018F), were generated (Fig. 1A). In addition, we have reported previously the construction of a alphaPDGFR deletion mutant lacking amino acid residues 710-789 of the kinase insert domain, designated alphaRDeltaki-1, and point mutations within this domain converting tyrosine 731, 742, or both to phenylalanine, designated as alphaR(Y731F), alphaR(Y742F), and alphaR(Y731F+Y742F), respectively(12, 13) . Since the NIH/3T3 line is a cell type that normally expresses both alpha and betaPDGFRs, it provides a physiologically relevant microenvironment for analyzing PDGFR signal transduction. To overcome activation of endogenous alphaPDGFR, the cytoplasmic domain of alphaPDGFR mutants were linked to the ligand-binding domain of the human CSF-1R (c-fms), which is not normally expressed in NIH/3T3 cells. Expression vectors containing the chimerae were transfected into NIH/3T3 cells, and the ability of these chimerae to mediate CSF-1-stimulated anchorage-independent growth, focus formation, and chemotactic responses in NIH/3T3 cells was studied.


Figure 1: Biochemical characterization of fms/alphaR chimerae in NIH/3T3 cells. A, schematic diagram of wild type alphaPDGFR, fms, and chimeric receptors generated between c-fms and alphaPDGFRs. Coding region of the alphaPDGFR is represented by an openbox. Coding region of c-fms is represented by a shadedbox. Blackboxes correspond to signal peptide (SP), transmembrane(TM), kinase insert (ki), and carboxyl-terminal (C) domains; B, comparison of CSF-1-induced tyrosine phosphorylation of fms/alphaR chimerae; C, comparison of the level of anti-CSF-1R recoverable fms/alphaR protein expressed in NIH/3T3 transfectants; D and E, comparison of CSF-1-induced association of each chimerae with PLC and p85 in CSF-1-stimulated NIH/3T3 transfectants. NIH/3T3 transfectants were either untreated(-) or treated (+) with CSF-1 (200 ng/ml) for 5 min at 37 °C. In panelB, 2 mg of clarified lysates were subjected to immunoprecipitation using anti-Tyr(P) followed by SDS-PAGE and subsequent blotting with anti-Tyr(P). In panels C-E, 2 mg of clarified lysate was immunoprecipitated with anti-CSF-1R antibody (Santa Cruz Biotechnology). The immune complexes were then subjected to immunoblotting with anti-alphaPDGFR (panelC), anti-PLC (panelD), or anti-p85 antibody (panelE).



We first examined the effect of these mutations on alphaPDGFR autophosphorylation. NIH/3T3 cells transfected with fms/alphaRWT, fms/alphaR(Y988F), fms/alphaR(Y993F), fms/alphaR(Y1018F), fms/alphaRDeltaC1, fms/alphaRDeltaC2, and fms/alphaRDeltaki-1 were untreated or treated with CSF-1. Total cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (anti-Tyr(P)). The proteins in immune complexes were electrophoretically separated, transferred to a membrane, and immunoblotted with anti-Tyr(P). As shown in Fig. 1B, while fms/alphaRDeltaC2 showed approximately 50% reduction in tyrosine phosphorylation, the other chimerae exhibited levels of tyrosine phosphorylation that were comparable to that of fms/alphaRWT. Consistent with previous findings, we did not detect tyrosine phosphorylation of endogenous alphaPDGFR upon CSF-1 triggering (Fig. 1B, lanes1 and 2). These data suggest that amino acids 977-1024 within the carboxyl-terminal domain of alphaPDGFR may contain relevant tyrosine autophosphorylation sites. Alternatively, the reduction in the level of fms/alphaRDeltaC2 tyrosine phosphorylation may be due to conformational changes induced by deletion of the carboxyl-terminal domain of alphaPDGFR. To examine the expression levels of chimeric receptors in the NIH/3T3 transfectants, immunoprecipitates were prepared from equivalent amount of cell lysates using antiserum directed against the extracellular domain of c-Fms (anti-CSF-1R). The immune complexes were then electrophoretically separated and immunoblotted with anti-alphaPDGFR antiserum raised against a synthetic peptide corresponding to amino acids 959-973 of the receptor. As shown in Fig. 1C, the levels of fms/alphaR chimeric proteins expressed were comparable among each NIH/3T3 transfectant. Under the same conditions, endogenous alphaPDGFR protein could not be detected in the anti-CSF-1R immune complex prepared from control NIH/3T3 cells (Fig. 1C, lanes 1). Cell surface expression levels of the chimeric proteins were also determined to be very similar by fluorescence-activated cell sorting analysis using a monoclonal antibody directed against the extracellular domain of the CSF-1R (data not shown). Together, these results suggest that the stoichiometry of CSF-1-induced tyrosine autophosphorylation of all mutant receptors, except fms/alphaRDeltaC2, was comparable to that of fms/alphaRWT.

We next examined the effect of these mutations on receptor association with its known substrates. The ectopic expression and biochemical analyses of alphaRDeltaC1 and alphaRDeltaC2 in 32D hematopoietic cells indicated that amino acid residues 977-1024 contain the major determinants necessary for PLC association and activation (data not shown). Therefore, the ability of mutant fms/alphaR chimerae to coimmunoprecipitate with PLC was compared using anti-CSF-1R antibody. As shown in Fig. 1D, this antibody coimmunoprecipitated comparable levels of PLC from NIH/3T3 cells expressing fms/alphaRWT and fms/alphaRDeltaC1. In contrast, the level of anti-CSF-1R- recoverable PLC was either reduced by about 90% in NIH/3T3 cells expressing fms/alphaR(Y988F), fms/alphaR(Y993F), or it was completely abolished in fms/alphaRDeltaC2 or fms/alphaR(Y1018F) transfectants after CSF-1 stimulation. Consistent with these results, the level of anti-Tyr(P)-recoverable PLC protein was also reduced (data not shown). As shown in Fig. 1E, each chimera associated to a similar extent with the 85 kDa regulatory subunit of PI 3-kinase (p85). Thus, our findings indicate that amino acid residues 977-1024 within the carboxyl terminus of alphaPDGFR contain the major binding site for PLC but not p85. These results are consistent with previous data demonstrating that tyrosine 1021 of the betaPDGFR is required for association with PLC, since the amino acid residues surrounding tyrosine 1018 of the alphaPDGFR are very similar to those surrounding tyrosine 1021 of the betaPDGFR (16) . Under the same conditions, fms/alphaRDeltaki-1, fms/alphaR(Y731F), and fms/alphaR(Y731F+Y742F) failed to coprecipitate p85, even though the ability of these mutants to associate with PLC was not impaired (data not shown).

To examine the effect of these mutations on ligand-stimulated biological responses, we next tested the ability of each transfectants to form colonies in semisolid medium. As shown in Fig. 2, NIH/3T3 expressing fms/alphaRWT formed colonies of sizes greater than 30 µm in diameter with an efficiency of around 25% (panelsC and D). Moreover, each transfectant exhibited a similar ability to form progressively growing colonies in the presence of CSF-1 except NIH/3T3 cells transfected with the vector alone (panelsA and B). These data suggest that ligand-stimulated association of PLC with alphaPDGFR is not required for colony formation in soft agar, since fms/alphaR(Y1018F), which failed to bind PLC completely, exhibited CSF-1-induced colony forming efficiency comparable to that of the fms/alphaRWT transfectant. In addition, neither deletion of 80 amino acids from the kinase insert nor truncation of 112 amino acids from the carboxyl terminus of alphaPDGFR detectably impaired the ability of CSF-1 to mediate colony formation. Taken together, our findings suggest that either the kinase insert or carboxyl-terminal domain is dispensable for anchorage-independent cellular growth.


Figure 2: CSF-1-stimulated cell growth of NIH/3T3 transfectants in semisolid media. NIH/3T3 cells (1 times 10^5) transfected with indicated DNAs were suspended in DMEM supplemented with 10% calf serum and 0.4% Seaplaque-agarose(15) . Cells were then fed with DMEM containing 10% calf serum in the presence or absence of CSF-1 (100 ng/ml) once per week. Photographs were taken by using light microscope after 2 weeks. Results are representative of at least three independent experiments.



CSF-1 has been shown to induce efficient focus formation of NIH/3T3 cells expressing fms/alphaRWT, fms/alphaR(Y731F), and fms/alphaR(Y731F+Y742F)(15) . Therefore, we sought to examine the ability of individual mutant chimera to mediate CSF-1-dependent focus formation of NIH/3T3 cells. As shown in Fig. 3, CSF-1 treatment induced approximately 140 foci in NIH/3T3 cells transfected with 1 µg of the fms/alphaRWT expression vector (panelsC and D). Truncation of 64 amino acid residues from the carboxyl terminus (fms/alphaRDeltaC1) or 80 amino acid residues from the kinase insert domain (fms/alphaRDeltaki-1) did not affect the level of ligand-stimulated focus formation, respectively (Fig. 3, panels E and F and panels O and P). In striking contrast, truncation of an additional 48 amino acid residues (fms/alphaRDeltaC2) resulted in 10-fold less focus forming activity in response to CSF-1 treatment (Fig. 3, G and H). These data suggest that amino acid residues 977-1024 within the carboxyl-terminal domain of the alphaPDGFR are important for mediating focus formation. Transfection of chimeric receptor containing mutation of tyrosine 988, 993, or 1018 within this domain each reduced CSF-1-induced focus formation by about 5-10-fold (Fig. 3, panels I and J, K and L, and M and N). A similar number of marker-selected colonies were observed in parallel plates, suggesting that equal levels of DNA were transfected into these cells (data not shown). Since the CSF-1-dependent focus formation of NIH/3T3 cells by various constructs correlated well with the ability of mutant receptors to bind PLC, our results suggest that PLC is probably involved in the focus forming function mediated by the alphaPDGFR. However, it is still possible that other putative substrates of alphaPDGFR that interact with this region may also be involved in mediating this response. In contrast to the mutations within the carboxyl-terminal domain, none of the mutations within the kinase insert domain of alphaPDGFR reduced ligand-induced focus-formation (data not shown and (15) ). These data suggest that substrates that bind to phosphotyrosine residues within amino acid residues 710-789 of the kinase insert domain of the alphaPDGFR, including p85 subunit of PI 3-kinase, are not required for efficient focus formation in NIH/3T3 cells.


Figure 3: CSF-1-stimulated focus formation of NIH/3T3 transfectants. NIH/3T3 cells were transfected by the calcium phosphate precipitation method, using 1 µg of indicated fms/alphaPDGFR chimeric receptor cDNA expression vectors and 40 µg of calf thymus DNA as carrier. Transfected cells were maintained in medium containing 5% calf serum and switched to medium containing 2% calf serum with or without 100 ng/ml human CSF-1 5 days post-transfection. Focus formation was scored 2-3 weeks after transfection. Parallel plates were also stained and photographed. Similar results have been obtained in two other independent experiments.



Ligand-stimulated alphaPDGFR is able to mediate chemotactic signaling in 32D hematopoietic and NIH/3T3 cells(9, 12, 17) . To compare the ability of the various fms/alphaR mutants to mediate chemotactic responses in NIH/3T3 cells, we examined the ability of each transfectant to migrate through a membrane toward the CSF-1 ligand (14) . As shown in Fig. 4, while deletions of amino acid residues 1025-1089 (fms/alphaRDeltaC1) or mutation of residues Tyr-988 or Tyr-993 did not impair CSF-1 dependent chemotactic response, deletion of amino acid residues 977-1089 (fms/alphaRDeltaC2) or mutation of Tyr-1018 inhibited chemotaxis by less than 50%. Therefore, our results suggest that mutation within the carboxyl terminus of alphaPDGFR did not impair chemotaxis to the same extent as focus formation. In contrast, deletion of 80 amino acids within the kinase insert domain or mutations of PI 3-kinase association sites reduced ligand-dependent chemotactic responses in NIH/3T3 cells by greater than 80%. Thus, our findings suggest that substrates interacting with the kinase insert domain of alphaPDGFR are required for efficient chemotactic responses in NIH/3T3 fibroblasts.


Figure 4: CSF-1-stimulated chemotactic response of NIH/3T3 transfectants requires alphaPDGFR association with PI 3-kinase activity. Cell migration was assayed by a modified Boyden chamber technique using a Transwell cell culture insert (Costar), which contained a filter with a pore size of 8 µm(14) . Briefly, 100 ng/ml CSF-1 ligand diluted in DMEM was first added to the bottom chamber. 1 times 10^5 NIH/3T3 cells were then added to the top chamber, and cells that had crossed the filter after 24 h of incubation at 37 °C were counted. Results are representative of at least three independent experiments.



Mutation of the PLC association site of the betaPDGFR or fibroblast growth factor receptors has been reported to have no effect on ligand-stimulated mitogenic responses(16, 18) . Consistent with these findings, ectopic expression of alphaRDeltaC1 or alphaRDeltaC2 in 32D hematopoietic cells indicates that amino acid residues 977-1089 encompassing the PLC binding site are not necessary for PDGF-induced mitogenic responses (data not shown). Moreover, our data indicate that fms/alphaR transfectants containing a mutation of the PLC binding site or even a larger truncation of amino acid residues 977-1089 of the carboxyl-terminal domain of alphaPDGFR did not reduce the ability of the receptor to mediate formation of colonies in the presence of the CSF-1. In contrast, analyses of carboxyl-terminal truncation mutants to mediate CSF-1-induced focus formation revealed that the presence of amino acids 977-1024 of the alphaPDGFR was necessary for this effect (see Fig. 3). Interestingly, mutation of Tyr-988, Tyr-993, or Tyr-1018 within this domain each reduced not only the CSF-1-induced focus formation but also receptor association with the PLC. These findings suggest that activation of PLC is likely to be involved in focus formation but not in anchorage-independent cell growth. We have observed that the inhibitory effect of Y1018F mutation on the CSF-1-induced focus formation or receptor association with PLC was more pronounced than that shown by mutation of either Y988F or Y993F. Although the biological significance of the difference observed remains to be established, our data suggest that Tyr-1018 may be differentially involved in receptor association with PLC and mediating the focus forming activity of alphaPDGFR. PDGF-induced focus formation of NIH/3T3 cells is characterized by the loss of contact inhibition and morphological alterations, which are considered to be the hallmark of neoplastic diseases. Interestingly, diacylglyceride and inositol trisphosphate, the enzymatic reaction products of PLC activation, have also been implicated in stimulating redistribution of cytoskeletal components in a variety of transformed cells(19, 20, 21) . In addition, a recent report suggests that protein kinase C- is involved in transformation of NIH/3T3 cells(22) . Together, these findings suggest that PLC and its associated proteins may mediate focus forming activity of PDGF by stimulating protein kinase C-dependent changes in the cytoskeletal architecture of NIH/3T3 cells.

In contrast to the mutations within the carboxyl-terminal domain, a double mutation within the kinase insert domain of alphaPDGFR that completely abrogated receptor-associated PI 3-kinase fully inhibited ligand-stimulated cell movement but not focus formation or colony formation of NIH/3T3 transfectants. This result is consistent with previous findings showing that PDGF-induced PI 3-kinase association with the betaPDGFR is required for chemotactic response(23) . Thus our findings indicate that the region of alphaPDGFR involved in ligand-dependent chemotactic signaling is distinct from that required for focus formation and anchorage-independent growth functions mediated by the alphaPDGFR. Finally, we would be remiss not to mention the possibility that differences between the signaling mechanisms uncovered in this study may potentially reflect the inherent property of CSF-1R/alphaPDGFR chimera utilized.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Cellular and Molecular Biology, NCI (37-1E24), National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-2778; Fax: 301-496-8479.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PLC, phospholipase C; PI 3-kinase, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium.


ACKNOWLEDGEMENTS

We thank S. A. Aaronson, S. Tronick, S. Gutkind, M. Chedid, and D. Mahadevan for helpful discussion. We also thank K. Myers for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.