Differences in substrate specificities of alpha and beta platelet-derived growth factor (PDGF) receptors. Correlation with their ability to mediate PDGF transforming functions

M.A Heidaran,J.F Beeler,J.C Yu,T Ishibashi,W.J Larochelle,J.H Pierce,S.A Aaronson

doi:10.1016/s0021-9258(18)98348-1

Abstract

Recombinant expression of either the alpha or beta platelet-derived growth factor (PDGF) receptors in 32D hematopoietic cells allows efficient coupling of PDGF with mitogenic and chemotactic signaling pathways inherently expressed by those cells. PDGF-BB stimulation of 32D-alpha R or beta R cells results in anti-P-Tyr recovery of cellular proteins possessing similar as well as distinct phosphotyrosine signals. Comparison of the ability of each receptor to couple with known second messengers revealed that both receptors associated with and/or tyrosine phosphorylated phospholipase C-gamma (PLC gamma) and phosphatidylinositol 3-kinase (p85) with similar stoichiometry. However, the beta platelet-derived growth factor receptor (PDGFR) was significantly more efficient at in vivo tyrosine phosphorylation of GTPase-activating protein (GAP). Similar differences in binding affinity for GAP were observed in NIH/3T3 cells which express both receptors. To quantitate the affinities of each receptor for GAP or PLC gamma, we utilized baculovirus-expressed alpha and beta PDGFRs purified by anti-P-Tyr affinity chromatography. Exposure of immunoblots containing bacterially expressed GAP or PLC gamma to activated alpha or beta PDGF receptors led to a comparable high affinity binding of each receptor to PLC gamma, while the beta PDGFR showed a 5-fold higher binding affinity for GAP. In an effort to correlate differences in their substrate specificities with biological properties of the receptors, we compare their abilities to enhance PDGF-A transforming function in NIH/3T3 cells. Cotransfection of PDGF-A with the alpha PDGFR increased PDGF-A transforming activity by approximately 2-fold. However, cotransfection with a chimeric receptor with the catalytic domain of the beta PDGFR but possessing alpha PDGFR ligand binding properties resulted in 17-fold enhancement of PDGF-A transformation. These findings argue that differences in alpha and beta PDGF receptor substrate specificity in NIH/3T3 fibroblasts correlate with greater transforming activity mediated by the beta PDGFR.

Highlights

Recombinant expression of either thea or B platelet- prised of dimers of A and B chains encoded by distinct genes derived growth factor (PDGF) receptors in 32D he- [3,4]
32D-aR and-/3R cells were measurement of in uiuo PDGFR-associated phosphatidylinositol 3- each challenged with saturating levels of Platelet-derived growth factor (PDGF)-BB which kinase activity, quiescent 32D transfectants were exposed to PDGFBB (100 ng/ml) for 5 min at 37 "C, incubated with 5mM diisopropyl fluorophosphate (DFP) at4 "C for 5 min, and lysed in a buffer containing 20 mM Tris. 137 mM NaCl, 2.7 mM KC1,l mM MgC12,1mM CaC12,1% Nonidet P-40, 10% glycerol, 1mM Naa V04,5 mM DFP, 1mM phenylmethylsulfonyl fluoride, 10 pg/ml aprotinin and leupeptin
I n Vitro Affinity for GAP Is Significantly Lower for a Compared to @ PDGFRs-In a n effort to directlycompare their affinities for GAP, we examined the ability of either not be attributed todifferences in levels of activated PDGFRs tyrosinephosphorylated a orPDGFR expressedin the,our results imply that the decreased level of GAP tyrosine phosphorylation by activated a PDGFRs rebaculovirus system to associatewithbacteriallyexpressed recombinant GAP or phosphorylatepdhospholipase Cy (PLCy) immobilized on a solid matrix

Summary

MATERIALS AND METHODS

Protein; DFP, diisopropyl fluorophosphate; PAGE, polyacrylamide Transfection Analysis-The interleukin-3-dependent mouse hegel electrophoresis; anti-P-Tyr; anti-phosphotyrosine. Transfection of NIH/3T3 fibroblasts was performed by the calcium- T method [40], (specific activity 1 X lo5counts/min/ng). Depending upon the DNA binding buffer (25 mM HEPES, 150 mM NaC1, 5 mM KCl, 1.2 mM construct, NIH/3T3 transfectantswere selected for survival in growth MgSO4, and 0.1% bovine serum albumin, pH 7.5) once and incubated medium containing either mycophenolic acid [32] or geneticin (750 for 2 h a t 4 "C with labeled PDGF-AA (1ng/ml) preincubated in the rg/ml). Stimu- analysis by the method of Scatchard [39]

RESULTS

PI20 4 P95 4 PB5

C PDGF-BB anti-GAP

DISCUSSION