Calcium-mediated degradation of epidermal growth factor receptor in dislodged A431 cells and membrane preparations.
Calcium-activated neutral protease has been purified partially from A431-V1 cells, a high passage variant of human epidermoid carcinoma A431 cells having extraordinary activity of this protease. This activity cleaves Mr = 160,000 epidermal growth factor receptors, converting them to a Mr = 145,000 form in detergent-treated membrane preparations, but not in intact cells or membrane vesicles. Properties of this activity, including molecular weight, resemble those of previously described Ca2+-activated neutral proteases. A431-V1 cell Ca2+-activated neutral protease action on epidermal growth factor receptors required 0.5 mM Ca2+ for half-maximal activity and the optimal pH was 6.5. Degradation was inhibited completely by Ca2+ chelators (EDTA and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and sulfhydryl group specific reagents (iodoacetate and 5,5'-dithiobis-(2-nitrobenzoic acid] and was inhibited partially by leupeptin. Degradation of epidermal growth factor receptors by Ca2+-activated neutral protease or trypsin was compared in intact cells, membrane vesicles, detergent-solubilized membranes, and membranes subjected to hypotonic shock in solutions containing protease. Results support the hypothesis that protease-susceptible domains of epidermal growth factor receptors, including the Ca2+-activated neutral protease-sensitive domain, are located on the cytosolic surface. The localization of Ca2+-activated neutral protease in A431-V1 cells is entirely cytosolic, making it available in cells to the protease-susceptible site on epidermal growth factor receptor substrates.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D066246:ErbB Receptors; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013724:Teratoma / Q000378:metabolism*
D002413:Cations, Divalent; D011956:Receptors, Cell Surface; D066246:ErbB Receptors; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium