©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Domain within the Carboxyl-terminal Region of the Platelet-derived Growth Factor (PDGF) Receptor That Mediates the High Transforming Activity of PDGF (*)

(Received for publication, February 28, 1996)

Aykut Uren (1)(§) Jin-Chen Yu(§)(¶) Weiqun Li (1) Il-Yup Chung (**) Daruka Mahadevan Jacalyn H. Pierce (1) Mohammad A. Heidaran (1)(§§)

From the Laboratory of Cellular and Molecular Biology, National Cancer Institute (37-1E24), National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alphaPDGFR and the intracellular domain of the betaPDGFR (alphabetaR) was markedly more efficient than the wild type alphaPDGFR (alphaRWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of betaPDGFR that confers this higher transforming activity, we generated several additional alpha/betaPDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alphaPDGFR and the final 165 amino acids from the carboxyl-terminal of the betaPDGFR (alphabetaR) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alphabetaR. However, when chimeric PDGFRs in which either the kinase insert domain (alphabetaRKI) or the last 79 amino acids from the carboxyl-terminal end of the betaPDGFR (alphabetaR) were substituted into alphaPDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alphaRWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of betaPDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of betaPDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the betaPDGFR (alphabetaR) were substituted for those in the alphaPDGFR. Cotransfection experiments indicated that alphabetaR increased transforming activity of PDGF-A to similar extent as the alphabetaR or alphabetaR. Therefore, our findings define a critical domain within the noncatalytic region of betaPDGFR intracellular domain that confers the higher focus forming activity mediated by the betaPDGFR.


INTRODUCTION

Platelet-derived growth factor (PDGF) (^1)is a potent mitogen for connective tissue cells(1) . This growth factor is composed of dimers of A and B chains encoded by distinct genes. All three PDGF isoforms (PDGF-AA, PDGF-BB, and PDGF-AB) have been identified(2) . They bind with different affinities to two related receptor molecules, designated alphaPDGFR and betaPDGFR, which are also encoded by two distinct genes(3, 4, 5) . Accumulating evidence indicates that PDGF-induced receptor activation involves recruitment of receptor dimers(6, 7, 8, 9) . In fibroblasts where both PDGFRs are expressed, PDGF-BB binds alphaalpha, betabeta, or alphabeta PDGFR dimers, PDGF-AA binds only alphaalpha PDGFR homodimers, and PDGF-AB binds alphaalpha or alphabeta PDGFR dimers, but does not bind betabeta PDGFR dimers(1, 7, 10) .

Activation of the PDGFR tyrosine kinase domain leads to physical association and tyrosine phosphorylation of many substrates, including Src, the 85-kDa subunit of phosphatidylinositol 3-kinase (p85), Nck, Ras GTPase-activating protein (RasGAP), phospholipase C- (PLC), and Syp(11, 12, 13, 14, 15) . The specific tyrosine residues interacting with each of these substrates have been identified for the betaPDGFR. Src binds to tyrosines 579 and 581 within the juxtamembrane domain(16) , and p85 binds to tyrosines 740 and 751 within the kinase insert domain of betaPDGFR(17, 18, 19) . Tyrosine 751 is also the association site for Nck (20) . RasGAP binds to tyrosine 772 within the kinase insert domain of betaPDGFR(18, 19) . Syp and PLC bind to tyrosine 1009 and 1021, respectively, within the carboxyl-terminal domain of betaPDGFR(21, 22) . In addition, Shc and protein kinase C- have been shown recently to be phosphorylated on tyrosine following PDGF-BB stimulation(23, 24) . Among these substrates, both activated alphaPDGFR and betaPDGFR tyrosine-phosphorylate p85 and PLC with similar stoichiometry(25) . However, the betaPDGFR has been shown to associate more efficiently than the alphaPDGFR with the PLC in vivo(25, 26) . Moreover, RasGAP and Syp have been shown to be poor substrates for the alphaPDGFR in comparison to the betaPDGFR(26, 27) .

Although both PDGF-AA and PDGF-BB are mitogenic as well as chemotactic for cells possessing appropriate PDGFRs(28) , we and others have shown previously that PDGF-BB is 10-100-fold more efficient than PDGF-AA at inducing transformation of NIH/3T3 cells(29, 30) . It is known that PDGF-BB stimulates alpha- as well as betaPDGFRs, while PDGF-AA binds only to the alphaPDGFR. Thus, the greater transforming efficiency of PDGF-B could be due to a quantitative increase in the level of activated PDGFRs and/or differences in substrate specificity of alpha versus betaPDGFRs. We have observed previously that cotransfection of the PDGF-A chain gene with wild type alphaPDGFR (alphaRWT) increased PDGF-A chain transforming activity by approximately 2-fold. In contrast, cotransfection of a chimeric receptor possessing alphaPDGFR PDGF-A ligand binding domain and the remaining sequences, including the catalytic domain from betaPDGFR (alphabetaR), resulted in a 17-fold enhancement of PDGF-A chain transforming activity(25) . Thus, our previous findings indicated that the increased transforming activity of PDGF-B in comparison with PDGF-A could be due to distinct substrate specificities of the two PDGFRs. In the present manuscript, we have identified a specific region within the carboxyl-terminal of the betaPDGFR that is responsible for mediating the enhanced focus formation of PDGF-B.


EXPERIMENTAL PROCEDURES

Materials

PDGF-AA, PDGF-BB, and monoclonal anti-Tyr(P) antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-alphaPDGFR is a polyclonal antibody that was generated against bacterially expressed protein encoding amino acids 25-530 of the extracellular domain of alphaPDGFR(31) . mAb-alphaR1 is a monoclonal antibody raised by immunizing BALB/c mice with 32D cells expressing the human alphaRWT(32) .

Generation of Cytoplasmic alpha/betaPDGFR Chimerae

The alpha/beta PDGFR chimerae were generated by polymerase chain reaction. The entire coding region of each chimeric PDGFR was then subcloned into the LTR-2 mammalian expression vector(35) . A detailed description will be provided upon request.

For in vitro kinase assays, total cell lysates (about 2 mg) were subjected to immunoprecipitation using mAb-alphaR1. The immune complexes were extensively washed and incubated in 20 mM Tris (pH = 7.5), 10 mM MgCl(2), 5 mM MnCl(2), 10 µg/ml aprotinin, and 50 µCi of [-P]ATP (Amersham Corp.) for 15 min at room temperature. The immune complex reactions were then washed and electrophoretically separated on 8% SDS-polyacrylamide gel electrophoresis. The gels were dried and autoradiogrammed. The levels of PDGFRs and the kinase activities of each alpha/beta chimerae in the various transfectants were quantitated using scanning densitometer (PDI, Inc.) and expressed relative to the signal derived from the alphaRWT. Immunoblot and immunoprecipitation analyses, NIH/3T3 transfection and cotransfection assays were performed as described previously(34) .


RESULTS AND DISCUSSION

Generation of the Chimeric Receptors between the Cytoplasmic Domain of alpha- and betaPDGFR

We have reported that cotransfection of an expression vector containing the PDGF-A gene cDNA with another vector encoding a chimeric receptor possessing the first 340 amino acids of the alphaPDGFR fused to the remaining extracellular transmembrane and intracellular domain of the betaPDGFR (alphabetaR) enhanced the transforming activity of PDGF-A by 17-fold. In contrast, a similar cotransfection experiment utilizing a alphaRWT expression vector increased PDGF-A transforming activity by approximately 2-fold only(25) . Since the expression levels of alphaRWT and alphabetaR were shown to be comparable, these findings strongly suggested that the greater transforming activity of PDGF-B compare with that of PDGF-A is due to distinct substrate specificities of the alpha- and betaPDGFR. Sequence comparison between alphaPDGFR and betaPDGFR intracellular domains revealed that amino acids sequence within the juxtamembrane, tyrosine kinase 1 and tyrosine kinase 2 domains are 80-90% homologous, while the amino acid sequences within the kinase insert (KI) and the carboxyl-terminal domains are less than 30% homologous(4) . Therefore, we sought to more precisely define the region of betaPDGFR that mediates the greater transforming activity by generating expression vectors containing cDNA that encoded various alpha/betaPDGFR chimerae (Fig. 1). The chimeric cDNAs were comprised mostly of the alphaPDGFR sequences. One chimeric receptor contained the kinase insert domain of the betaPDGFR and another one contained most of tyrosine kinase 2 and all of the carboxyl-terminal domains of betaPDGFR (designated as alphabetaRKI and alphabetaR, respectively). From the remaining alpha/betaPDGFR chimerae, one chimera contained all of the carboxyl-terminal domain of betaPDGFR (designated as alphabetaR), while the other contained the final 79 amino acids of the betaPDGFR carboxyl terminus (designated as alphabetaR). Finally, we also constructed a chimera in which amino acid residues 942-1028 of the betaPDGFR were substituted for those in the alphaPDGFR (designated as alphabetaR).


Figure 1: Schematic diagram of alphaRWT, betaRWT, and the alpha/betaPDGFRs chimerae. The coding region of alphaPDGFR is represented by an open box. The coding region of the betaPDGFR is represented by a shaded box. The black box corresponds to the signal peptide (SP) and transmembrane (TM) domain. Tyrosine kinase domain (TK1 and TK2), kinase insert (KI), and carboxyl-terminal (CT) domains are indicated.



Comparison of the Ability of Each Chimera to Enhance PDGF-A Chain Transforming Activity

To compare the ability of each chimera to enhance PDGF-A chain transforming activity, we cotransfected 1 µg of expression vector containing the PDGF-A gene along with 1 µg of expression vector containing each chimeric alpha/betaPDGFR into NIH/3T3 cells. As summarized in Table 1, cotransfection of PDGF-A with the LTR-gpt vector alone resulted in formation of 10-20 small foci. Cotransfection of PDGF-A chain with alphaRWT only weakly enhanced the number of large foci formed. Consistent with previous results, cotransfection of PDGF-A with alphabetaR generated around 350-400 large foci.



Cotransfection of PDGF-A chain with alphabetaRKI resulted in production of similar numbers of foci as those induced by cotransfection of PDGF-A with alphaRWT. In contrast, cotransfection of alphabetaR with PDGF-A resulted in marked increase in transforming activity similar to that induced by cotransfection of PDGF-A with alphabetaR. Moreover, alphabetaR exhibited a similar ability to enhance the transforming activity of PDGF-A as that of alphabetaR. These results suggest that substitution of KI domain of the alphaPDGFR with the KI domain of the betaPDGFR does not enhance transforming activity. However, the replacement of the carboxyl-terminal domain of the alphaPDGFR with that of betaPDGFR is sufficient to confer the higher transforming activity. Finally, we also showed that cotransfection of PDGF-A with alphabetaR did not result in enhanced transformation, suggesting that the most distal 79 betaPDGFR amino acids are not responsible for conferring the high transforming activity mediated by the betaPDGFR. Since the expression vectors containing PDGF-A and alpha/betaPDGFR cDNAs each contain different drug-resistant markers(29, 33) , the number of cells expressing both cDNAs was also determined by analyzing the number of colonies that survived in the presence of both HAT/mycophenolic acid and geneticin. As shown in Table 1, each cotransfection resulted in similar numbers of double marker-selected colonies, suggesting similar levels of plasmid DNA were transfected into the cells. Together, these results predicted that the 86 amino acids (amino acids 942-1028) following tyrosine kinase 2 domain of the betaPDGFR may be responsible for its transforming activity.

Amino Acid Residues 942-1028 of the betaPDGFR Are Critical for Mediating Its Higher Transforming Potential

To confirm that amino acid residues 942-1028 of the betaPDGFR were sufficient for enhancing cellular transformation, we next generated a chimera designated alphabetaR by substituting these betaPDGFR residues for those normally expressed by alphaRWT. The ability of this chimera to increase PDGF-A transforming activity was then compared with that mediated by alphaRWT, alphabetaR, and alphabetaR. Results shown in Fig. 2indicate that alphabetaR increased the low transforming activity of PDGF-A to similar extent as that observed for the alphabetaR. Consistent with our previous finding, alphaRWT and alphabetaR each failed to appreciably enhance the low transforming activity of PDGF-A.


Figure 2: Comparison of the ability of alphaRWT and chimeric alpha/betaPDGFRs to enhance PDGF-A transforming function in NIH/3T3. NIH/3T3 cells were transfected with expression vectors containing PDGF-A (A) or cotransfected with PDGF-A and LTR-gpt (B), alphaRWT (C), alphabetaR (D), alphabetaR (E), or alphabetaR (F). Transformed foci were detected 3 weeks after transfection, and cells were fixed with 10% buffered formalin phosphate and stained with Giemsa stain.



Since the level of receptor expression and their kinase activities have been documented to affect transforming activity of receptor tyrosine kinases expressed in NIH/3T3 cells(35, 36) , we next sought to examine the level of human chimeric alpha/betaPDGFR protein and its kinase activity in each transfectant. Total cell lysates prepared from NIH/3T3 cells transfected with alphaRWT, alphabetaR, alphabetaR, alphabetaR, or vector alone were immunoprecipitated using a monoclonal antibody, mAb-alphaR1, which recognizes human alphaPDGFR, but not endogenous murine alphaPDGFRs present in NIH/3T3(31) . The immune complexes were then subjected to either immunoblot analysis using anti-alphaPDGFR serum or an in vitro kinase assay. Since the anti-alphaPDGFR is directed against the extracellular domain of the alphaRWT, it was possible to compare the expression levels of each chimera and the wild type alphaPDGFR using this antibody. As shown in Fig. 3A, anti-alphaPDGFR specifically detected proteins with molecular masses of approximately 190 kDa in lysates from cells transfected with the human alphaRWT or the alpha/beta chimerae. Note that under these experimental conditions, the murine alphaPDGFR naturally expressed in NIH/3T3 cells, was not observed (Fig. 3A, lane 1). Quantitation of the amounts of the human receptor detected from each transfectant revealed that NIH/3T3 cells transfected with alphabetaR, alphabetaR, and alphabetaR expressed about 1.7-, 0.7-, and 1.0-fold that of alphaRWT, respectively. Thus, the higher transforming activity of alphabetaR was not due to higher protein expression, since the steady state level of this chimera was found to be nearly identical to alphaRWT level in the transfectants.


Figure 3: Comparison of the protein levels of the alphaRWT and each chimera and their kinase activity in the NIH/3T3 transfectants. The levels of alpha/betaPDGFR chimera expressed in each transfectant and their kinase activity is shown. Cell lysates from marker-selected cultures were subjected to immunoprecipitation using monoclonal mAb-alphaR1 antibody that recognizes human but not the murine alphaPDGFR. Following SDS-polyacrylamide gel electrophoresis, proteins were transferred to Immobilon-P and immunoblotted with anti-alphaPDGFR antiserum (A). Similar amount of cell lysates was also subjected to immunoprecipitation using mAb-alphaR1 followed by an in vitro kinase assay as described under ``Experimental Procedures'' (B).



The level of ligand-induced receptor autophosphorylation is a good measurement for receptor tyrosine kinase activity(37) . Therefore, we next subjected immune complexes prepared using mAb-alphaR1 to an in vitro kinase assay. Quantitation of data presented in Fig. 3B demonstrated that the level of receptor autophosphorylation of alphabetaR, alphabetaR, and alphabeta R was 0.5-, 1.1-, and 0.5-fold that of alphaRWT, respectively. Since the alphabetaR exhibited lower autokinase activity as compared with that shown by alphaRWT, our findings suggest that the higher transforming activity of this chimera is not due to the higher level of receptor tyrosine kinase activity.

In previous studies we showed that the higher focus forming activity of PDGF-B was due to distinct biochemical properties of the betaPDGFR intracellular domain(25) . In the present report, we have utilized a PDGF-A focus formation enhancement assay to localize a region within the betaPDGFR carboxyl terminus that is responsible for this effect. This region designated as the minimal transforming domain of the betaPDGFR is 86 amino acids in length and most likely represents an independent functional domain, since attempts to further dissect this domain led to a reduction in transforming activity (data not shown). The minimal transforming domain of betaPDGFR has minimal effect on expression level or kinase activity of the receptors, suggesting that the difference in transforming activity mediated by alphaPDGFR and betaPDGFR may be due to their different substrate specificity. Activation of the betaPDGFR has been reported to induce significantly higher levels of tyrosine phosphorylation of RasGAP and Nck than alphaPDGFR(25, 27) . However, the binding sites within betaPDGFR for both Nck and RasGAP are within its kinase insert domain(13, 18, 19, 20) . Since substitution of alphaPDGFR KI domain with that of betaPDGFR did not confer the high transforming activity to the alphaPDGFR, our findings suggest that Nck or RasGAP are not likely to be important for PDGF-induced transformation. Furthermore, we have found that Shc, which is involved in mediating Ras activation(24, 38, 39) , is tyrosine-phosphorylated to a similar extent by activated alpha and betaPDGFR and each alpha/betaPDGFR chimerae expressed in 32D transfectants. (^2)Thus, the possibility that Shc is the major downstream signaling molecule that regulates the higher transforming activity of betaPDGFR versus alphaPDGFR is unlikely(40) .

The minimal transforming domain of the betaPDGFR contains four tyrosine residues. Among these tyrosines 966 and 970 are not phosphorylated in vivo.(^3)In contrast, tyrosine residues 1009 and 1021 have been shown to undergo ligand-dependent tyrosine phosphorylation and association with Syp and PLC, respectively(11, 12, 13, 14, 15) . The alphaPDGFR has been shown previously to interact with PLC and Syp with a lower efficiency(25, 27) . In addition we have shown that the low transforming activity of PDGF-A mediated by the alphaPDGFR can be abolished by mutation of the PLC binding site(41) . Therefore, our data indicate that PLC and/or Syp may be involved in the higher transforming activity of PDGF-B mediated by the betaPDGFR. Interestingly, it has been reported recently that Tyr and Tyr are also required for efficient ligand-induced ubiquitination of the activated betaPDGFR leading to proper degradation of the ligand-activated receptor (42) . In addition, the minimal transforming domain of the betaPDGFR contains a hydrophobic region that has been shown previously to be essential for proper ligand-induced internalization and down-regulation of activated betaPDGFRs(43) . Thus, it would be remiss not to consider the possibility that the higher transforming activity of the betaPDGFR may also be due to the difference in the ligand-induced post-translational regulation of the betaPDGFR. Further studies to dissect the role of this region in the ligand-induced receptor processing and substrate phosphorylation will potentially help us to elucidate the exact molecular basis for PDGF-induced transformation of NIH/3T3 cells by the betaPDGFR.


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.

§
The first two authors have made an equal contribution to this work.

Present address: COR Therapeutics, 256 East Grand Ave., South San Francisco, CA 94080.

**
Present address: Dept. of Biochemistry, Hangyang University, Ansan, Kyungki-do, 425-791, South Korea.

§§
To whom correspondence should be addressed: Laboratory of Cellular and Molecular Biology, National Cancer Institute (37-1E24), 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, platelet-derived growth factor receptor; PLC, phospholipase C-; PCR, polymerase chain reaction; KI, kinase insert.

(^2)
M. A. Heidaran, unpublished observation.

(^3)
A. Kazlauskas, personal communications.


ACKNOWLEDGEMENTS

We thank Drs. Stuart A. Aaronson and Steven R. Tronick for their support and helpful discussions; Dr. Andrius Kazlauskas for kindly providing us with the unpublished results; Drs. Ling-Mei Wang, William Wong, and Paolo Michieli for helpful discussions; and Charles Knicley, Nelson Ellmore, and Kimberly A. Meyers for excellent technical support.


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