The somatomedins are GH-dependent, insulin-like peptides with metabolic and mitogenic actions for a widely variety of cell lines. Insulin and the IGFs share overlapping biological activities, which are presumably mediated through binding to specific receptors on target cell membranes. However, studies designed to characterize the method of action of these peptides, and to determine which receptor(s) mediates each metabolic and mitogenic effect, have been handicapped by the significant structural and functional homology of the peptides and their respective receptors. The development of antibodies specifically directed at each receptor has provided powerful new probes for the delineation of insulin and IGF action. To date, monoclonal and polyclonal antibodies have been generated against the human insulin, human IGF-I and rat IGF-II receptors. In this project, we will continue our efforts to evaluate the structural and functional interrelationships of the insulin, IGF-I and IGF-II receptors. Specifically, we plan to purify each receptor by detergent solubilization and affinity chromatography, and to generate and characterize anti-receptor antibodies produced by immunization with rat and human insulin, IGF-I and IGF-II receptors at various levels of purity. Specific antibodies will be developed which will immunoprecipitate each receptor, inhibit ligand binding, and/or block the """"""""effector"""""""" portion of the receptor. These antibodies will be employed, together with competitive binding and affinity cross-linking, to determine tissue and species specificity of the receptors, ontogeny of each receptor, and receptor phosphorylation and kinase activity. Receptor biosynthesis and post-translational processing will be evaluated by immunoprecipitation of cells which have been pulse-chase labeled with (35-S)-methionine following treatment with a variety of agents that interfere with glycosylation and peptide processing. Immunohistochemical studies will: 1) localize insulin and IGF receptors in various tissues, 2) determine the distribution of receptors within a cell, and 3) follow receptor processing during glycosylation, as well as """"""""up"""""""" and """"""""down"""""""" regulation. Finally, we will attempt to determine the specific roles of the insulin and IGF receptors in DNA synthesis, cell replication, amino acid transport, glucose transport, and protein synthesis in fetal and adult fibroblasts, L6 myoblasts and myotubes, leukemic lymphoblasts, pituitary cells, and IGF-II producing cell lines. It is anticipated that these studies will provide important new data on the fundamental biological functions of each of these growth factors and their receptors.
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