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J. Biol. Chem., Vol. 258, Issue 15, 9254-9261, Aug, 1983

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

RW Yeaton, MT Lipari and CF Fox

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.
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