Four monoclonal antibodies were identified by their ability to bind to 12-I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microi'jection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.

In cultured vascular smooth muscle cells, angiotensin II

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105.82,79,77,73,45 and 40 kDa in time-and dose-dependent manners. Other vasoconstrictors such as [Arglvasopressin, 5-hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C-activating phorbol ester, phorbol 12 myristate 13-acetate, and the CaZ+ ionophorc, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.

Stimulation of the activity of protein kinase C by pretreatment of cells with phorbol esters was tested for its ability to inhibit signaling by four members of the insulin receptor family, including the human insulin and insulin-like growth factor-I receptors, the human insulin receptor-related receptor, and the Drosophila insulin receptor. Activation of overexpressed protein kinase C␣ resulted in a subsequent inhibition of the ligand-stimulated increase in antiphosphotyrosine-precipitable phosphatidylinositol 3-kinase mediated by the kinase domains of all four receptors. This inhibition varied from 97% for the insulin receptorrelated receptor to 65% for the Drosophila insulin receptor. In addition, the activation of protein kinase C␣ inhibited the in situ ligand-stimulated increase in tyrosine phosphorylation of the GTPase-activating protein-associated p60 protein as well as Shc mediated by these receptors. The mechanism for this inhibition was further studied in the case of the insulin-like growth factor-I receptor. Although the in situ phosphorylation of insulin-receptor substrate-1 and p60 by this receptor was inhibited by prior stimulation of protein kinase C␣, the in vitro tyrosine phosphorylation of these two substrates by this receptor was not decreased by prior stimulation of the protein kinase C␣ in the cells that served as a source of the substrates. Finally, the insulin-like growth factor-I-stimulated increase in cell proliferation was found to be inhibited by prior activation of protein kinase C␣. These results indicate that the ability of activated protein kinase C␣ to antagonize signaling by the human insulin receptor is shared by the other members of the insulin receptor family despite their considerable differences in amino acid sequence. Moreover, the present study shows that this antagonism is exerted at a very early step, the initial tyrosine phosphorylation of three distinct endogenous substrates. Finally, the present study indicates that this inhibition is not caused by an increased Ser/Thr phosphorylation of these two substrates.Most studies have documented a critical role for the intrinsic tyrosine kinase activity of the insulin receptor (IR) 1 in mediating subsequent biological responses (1, 2). After binding insulin, the receptor autophosphorylates on several specific tyrosines and then tyrosine-phosphorylates several endogenous substrates including insulin-receptor substrate (IRS)-1, an SH2-containing protein called Shc, and various 60-kDa proteins including one that is tightly bound by the GTPase-activating protein of Ras (3-10). The tyrosine phosphorylation of IRS-1 results in its being bound by several SH2-containing proteins including the phosphatidylinositol (PI) 3-kinase, a tyrosine phosphatase, and two SH2 linker proteins called Grb-2 and Nck (3, 11). A number of studies utilizing a variety of approaches have implicated the tyrosine phosphorylation of IRS-1 as being important in initiating several biological responses including stimulation of growth responses as well as stimulat...

A cytosolic insulin-degrading enzyme (Mr = 110,000) was found to be cross-linked to [125I]-insulin in intact human hepatoma cells, HepG2, incubated with the hormone and treated with the bifunctional cross-linker, disuccinimidyl suberate. The labeling of this protein was greatly increased by concurrent treatment of the cells with N-ethylmaleimide, to the extent that the amount of [125I]-insulin cross-linked to the enzyme in these cells was approximately 20 to 50% that cross-linked to the insulin receptor. The labeling of the insulin-degrading enzyme required the prior interaction of [125I]-insulin with its receptor as well as a temperature- and energy-dependent processing of the hormone. The present work therefore supports a role for this protease in the cellular processing of insulin.