Abstract
Heterologous regulation of the epidermal growth factor (EGF) receptor by platelet-derived growth factor (PDGF) was studied in FS4 human skin fibroblasts. The addition of PDGF to FS4 cells inhibited high affinity binding of 125I-EGF and stimulated phosphorylation of the EGF receptor. Phosphopeptide analysis by high performance liquid chromatography revealed that PDGF treatment of cells increased phosphorylation at several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine 654, a residue previously shown to be phosphorylated when protein kinase C is activated. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated phosphorylation of the same peptides from the EGF receptor as PDGF, and, in addition, induced phosphorylation of threonine 654. TPA inhibited both high and low affinity 125I-EGF binding by these cells. PDGF treatment of cells had no effect on EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was inhibitory. TPA, but not PDGF, stimulated phosphorylation of a Mr = 80,000 protein, known to be a substrate for protein kinase C, even though PDGF appeared to mediate breakdown of phosphoinositides. These data suggest that regulation of EGF receptor function by PDGF and TPA are distinct in these cells, even though some elements of regulation are shared. The results differ from those previously reported for a human lung fibroblast isolate, indicating that cell type-specific differences may exist in metabolism of the EGF receptor.
Highlights
The additionof PDGF to FSc4ells inhibited high affin-such as Epidermal growth factor (EGF)[12,13].Exposure to PDGFinhibits high affinity ity bindingof ‘*‘I-EGF and stimulated phosphorylation binding of Iz5I-EGFby several types of fibroblasts [14,15]
These data receptor metabolism in human skin fibroblasts are different suggest that regulation of EGF receptor function by from results previously reported for human lung fibroblasts PDGF and TPA are distinct in thcelsles, even though [16, 17]. some elements of regulation a r e shared
The data presented are from experiments examining the effects of PDGF on metabolism of the EGF receptor in FS4 humanskin fibroblasts [27], similar results were found with three other independenhtuman foreskin fibroblast isolates
Summary
Effects of TPA, PDGF, and EGFon binding of EGF receptor monoclonalantibody 528 to FS4 cells. Binding of antibody and 1Z5-I-proteinA to FS4 cells were performed as described under “Experimental Procedures.”. The data presented arethe mean andstandard error of resultsobtained from three separate experiments. Were treated as above except nonimmune mouse IgG was included in place of antibody 528. Two-dimensional thin layer phosphopeptide analysis, phosphoamino acid analysis, assay of [3H]thymidineincorporation, and SDSpolyacrylamide gel electrophoresis were performed as previously described [20]
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