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Calcium-mediated degradation of epidermal growth factor receptor in dislodged A431 cells and membrane preparations.

1983; Elsevier BV; Volume: 258; Issue: 15 Linguagem: Inglês

10.1016/s0021-9258(17)44660-6

1083-351X

Robin W. Yeaton, Michael T. Lipari, Colin Fox,

Chemical Synthesis and Analysis

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 M, = 160,000 epidermal growth factor receptors, converting them to a M, = 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(&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 re- ceptors 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 proteasesusceptible domains of epidermal growth factor receptors, including the Ca2+-activated neutral proteasesensitive 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.The receptor for the mitogenic polypeptide hormone EGF' is an integral membrane glycoprotein.EGF receptors from human placenta, human and murine cell strains and lines, and plasma membrane preparations from these cells have