Abstract
Using human-specific antibody reagents, we have examined the biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells. Four Mr species (Mr = 70,000, 95,000, 135,000, and 145,000) are detected when cells are subjected to a brief pulse of L-[35S]methionine; an Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel. In pulse-chase experiments, the four lower Mr species appear to bear a precursor relation to the Mr = 165,000 protein. The molecule acquires N-linked oligosaccharide cotranslationally, and two of the species (Mr = 95,000 and 145,000) are susceptible to digestion with endo-beta-N-acetylglucosaminidase H. The Mr = 145,000 and Mr = 165,000 proteins, which become labeled with 125I-epidermal growth factor after treatment of intact cells with a bifunctional cross-linking reagent, are phosphorylated at serine and threonine on identical tryptic peptides.
Highlights
Are subjected to a brief pulse of ~-[~~S]methioninane; Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel
The functionalcomplexity of the EGFreceptor raises questions regarding the events thatlead to the appearanceof this protein at the cell surface. The first of these is whether a relation exists between the molecular processing of this protein and its biological function; that is, does the acquisition of a particular structural moiety during biosynthesis confer specific function upon the molecule? The second question is acetylglucosaminidase H
165,000 proteins, which become labeled with lZ5I-epi- structural domains defined by its orientation in the plasma dermal growth factor after treatment of intact cells membrane
Summary
Dermal and mesodermal origin [2,3,4,5]. The late effects of this Identification of EGF Receptor Protein Species-The cl 21 hormone invitro include induction of DNA synthesis andcell antiserum immunoprecipitates the human EGF division and, in the case of human diploid fibroblasts,in- receptor in the form of a protein doublet of M , = 145,000-. Degradation of the receptor in disrupted cells is mediated by for 60 rnin Other bands, notably those representing proteins a cytosolic Caz+-activated neutral proteas(e11-13), suggesting of M , = 45,000, 80,000, and 200,000, are present if extracts that some receptor processing may occur prior to thedeposi- areimmunoprecipitatedwithpreimmunenormal mouse tion of ligand-receptor complexes in lysosomes [14,15]. Notably those representing proteins a cytosolic Caz+-activated neutral proteas(e11-13), suggesting of M , = 45,000, 80,000, and 200,000, are present if extracts that some receptor processing may occur prior to thedeposi- areimmunoprecipitatedwithpreimmunenormal mouse tion of ligand-receptor complexes in lysosomes [14,15] It has serum (Fig. 1A) and presumably represent nonspebeen hypothesized that internalization and/or processing of cific contaminants.
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