COMPARATIVE STUDIES OF SOMATOMEDIN AND INSULIN RECEPTORS

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.