Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors.
Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000302:isolation & purification*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors; D008168:Lung / Q000201:enzymology*; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D008565:Membrane Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D066246:ErbB Receptors; D014357:Trypsin; D002154:Calpain; D002118:Calcium