Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation.
Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000502:physiology*; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007328:Insulin; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin