Characterization of a platelet-derived growth factor receptor on Swiss 3T3 cells by affinity crosslinking.
Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205,000 and Mr 190,000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable p-azidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190,000 band represents a Mr 175,000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205,000 species the same Mr 175,000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150,000, while pretreatment with neuraminidase resulted in a shift of the Mr 205,000 and 190,000 bands by 5,000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr approximately 135,000 proteinaceous core and a Mr approximately 40,000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37 degrees C performed 125I-PDGF receptor complexes disappear from the cell surface with a t1/2 approximately 8 min.
Hosang
M
M
Pharmaceutical Research Department, F. Hoffmann-La Roche & Co., Ltd., Basel, Switzerland.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents; D005347:Fibroblasts / Q000378:metabolism*; D006031:Glycosylation; D051379:Mice; D009439:Neuraminidase / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor; D012795:Sialoglycoproteins / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D012795:Sialoglycoproteins; D014415:Tunicamycin; D017479:Receptors, Platelet-Derived Growth Factor; D009439:Neuraminidase