SEMISYNTHETIC IGF-I/INSULIN HYBRIDS AS RECEPTOR PROBES

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This proposal has long-term goals related to (a) identifying the structural attributes of insulin-like growth factor I (IGF-I) which determine its potency for interactions with type I IGF receptors, and its consequent overall biological effects, (b) evaluating the development of rules that might apply to the separate determinants of affinity and selectivity for receptor interactions within structurally related groups of ligands and receptors, and (c) considering the potential for development of IGF-I analogs that might be of therapeutic or experimental value. Specific aims for the project are summarized as follows: (a) To prepare hybrid IGF-I/insulin analogs with selected structural changes and amino acid replacements by chemical peptide synthesis and semisynthesis. (b) To evaluate the potencies of these analogs with regard to their abilities to bind to the type I IGF and insulin receptors through binding competition studies and direct analysis of radiolabeled ligand binding. (c) To consider the separate issues of receptor site affinity and selectivity in relation to the IGF-I and insulin systems through comparisons of analog interactions with both receptors. (d) To address aspects of potential differences in the solution structures of analogs by spectroscopic methods and chemical modification. (e) To evaluate the consequences of structural changes in these analogs in relation to their biological activities in vitro, by use of assays for 3H-thymidine incorporation into DNA and for additional metabolic responses. (f) To evaluate the potential biological implications of binding site heterogeneity in ligand interactions with the type I IGF receptor through the kinetic analysis of related binding studies. (g) To approach an understanding of type I IGF receptor heterogeneity at the molecular level through use of chemical and affinity methods. As aberrations in IGF-I physiology can be expected to result under different sets of circumstances in either inappropriately low or inappropriately high rates of tissue growth, and as the IGF-I system could be useful in by-passing the insulin system in states of insulin resistance, it is not difficult to envision the potential health-related importance of analogs of IGF-I that might (a) be selective for one or the other of its actions, (b) be even more potent than the natural hormone, (c) inhibit the action endogenous hormone by binding to receptor without inducing cellular responses, or (d) act through multiple receptor systems to produce desired metabolic effects. This proposal represents a study of the IGF-I system at a fundamental level, at this stage, and the application of the methods of chemical peptide synthesis and semisynthesis to the elucidation of relevant structure-function relationships.