Biosynthetic and glycosylation studies of cell surface platelet-derived growth factor receptors.
The cell surface pool of metabolically labeled platelet-derived growth factor (PDGF) receptors in BALB/c3T3 fibroblasts was studied using an antiphosphotyrosine antibody. Exposure of intact cells to PDGF stimulates autophosphorylation of surface PDGF receptors and allowed immunoaffinity purification of only PDGF-activated receptors. Pulse-chase experiments demonstrated appearance of newly synthesized receptors in a surface activatable pool within 30-45 min of synthesis. In the absence of exogenous PDGF, the apparent half-life of this pool was 2 h. The presence of both N- and O-linked oligosaccharide chains on cell surface PDGF receptors was demonstrated. Enzymatic removal of the N-linked oligosaccharide chains reduced the receptor's apparent Mr by approximately 40 kDa and removal of O-linked oligosaccharide caused approximately a 7-kDa reduction. Activation of receptor tyrosine autophosphorylation by PDGF did not require either processing of high-mannose N-linked oligosaccharides to complex forms or the presence of sialic acid on receptor oligosaccharide chains. Tryptic cleavage of PDGF-activated surface receptors in intact cells yielded two discrete phosphotyrosine-containing fragments of 107 and 85 kDa. Cleveland digest patterns from each fragment indicate that both are derived from the intact PDGF receptor. These data indicate that PDGF receptors are synthesized and turn over rapidly in the absence of ligand. Partial characterization of the extracellular domain oligosaccharide contribution to receptor function and trypsin susceptibility is provided.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006023:Glycoproteins / Q000096:biosynthesis*; D006026:Glycoside Hydrolases; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D017479:Receptors, Platelet-Derived Growth Factor; D014357:Trypsin
D006023:Glycoproteins; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D017479:Receptors, Platelet-Derived Growth Factor; D006026:Glycoside Hydrolases; D014357:Trypsin