The GOALS of the proposed research are: to determine how the interaction of insulin with its cell-surface receptor triggers the pleiotropic metabolic response, and to determine how target cells control their responsiveness (and resistance) to insulin. The insulin receptor system of 3T3-L1 adipocytes will serve as an experimental model. This system is ideal because 3T3-L1 adipocytes possess a large number of insulin receptors, are highly responsive to insulin and can be studied under the controlled conditions of cell culture. Moreover, homogeneous insulin receptor has been purified from 3T3-L1 cells and a large body of background information has already been amassed on the metabolic pathway and epression of the receptor in these cells. The systematic approach to investigate insulin receptor action and regulation in 3T3-L1 adiopocytes will include the following SPECIFIC AIMS: I. to characterize the structural features of the Alpha- and Beta-subunits of the pure insulin receptor, in particular, the tyrosine-specific protein kinase autophosphorylation sites of the Beta-subunit. II. to elucidate the mechanism by which the receptor-insulin interaction triggers signal transmission for the activation of certain cellular processes; in particular, the role of the receptor tyrosine kinase will be investigated. Attempts will be made to reconstruct a cell-free system, responsive to insulin (e.g. insulin-activated glucose transport), with which the minimal requirements for insulin activation can be defined. III. to purify and characterize a recently-discovered polypeptide, distinct from insulin, which activates glucose transport in both insulin-responsive (3T3-L1) and non-responsive (3T3-C2) cells. IV. to identify the steps and characterize the intermediates in the post-translational processing pathway which gives rise to functional insulin receptor. V. to determine how the 3T3-L1 adipocyte controls its cellular level of functional insulin receptor. The mechanisms of ligand-induced down-regulation of receptor level and of the control of expression of the receptor will be investigated.
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