Characterization of the receptor for platelet-derived growth factor on human fibroblasts. Demonstration of an intimate relationship with a 185,000-Dalton substrate for the platelet-derived growth factor-stimulated kinase

Abstract

The receptor for platelet-derived growth factor (PDGF) on human foreskin fibroblasts has been characterized. The molecular weight of the PDGF-receptor complex was estimated by affinity labeling techniques to about 200,000, as determined by sodium dodecyl sulfate-gel electrophoresis performed under reducing conditions. Subtraction of the Mr of reduced PDGF (18,000 to 15,000) gives a Mr for the receptor proper of 185,000 (+/- 10,000). The mobility in sodium dodecyl sulfate-gel electrophoresis was similar whether or not reducing agents were present, suggesting that the receptor may be a single chain protein. The hydrodynamic size of the 125I-PDGF-receptor complex after solubilization with Triton X-100, corresponded to a Mr of approximately 320,000, as determined by gel chromatography. Subtraction of the Mr contributions from Triton X-100 and PDGF, respectively, gives a Mr of approximately 200,000 for the receptor itself, an estimate in good agreement with the value obtained from the affinity-labeling experiments. Several lectins were analyzed for their ability to inhibit binding of 125I-PDGF to its receptor. It was found that wheat germ agglutinin and a lectin from Crotalaria juncea were effective inhibitors and that their inhibitory effects could be neutralized by N-acetylglucosamine and galactose, respectively, suggesting that the receptor contains these sugars. The properties of the receptor were compared with those of a 185,000-Da component, being the major substrate for the membrane-bound PDGF-stimulated kinase. It was found that the 185,000-Da component behaved similar to the PDGF receptor in sodium dodecyl sulfate-gel electrophoresis, performed with or without reducing agents present. Further, the 185,000-Da component co-eluted with the PDGF receptor on a Sepharose 6B column, and had affinity for the same lectins that inhibited the binding of 125I-PDGF to its receptor. Finally, the 185,000-Da component had affinity for PDGF immobilized on Sepharose beads, suggesting that it has PDGF-binding activity. We conclude that the PDGF receptor and the 185,000-Da substrate for the PDGF-dependent kinase are intimately related and probably identical molecules.