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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK014574-19
Application #
3225234
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1975-01-01
Project End
1989-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
19
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Ezaki, O; Flores-Riveros, J R; Kaestner, K H et al. (1993) Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice. Proc Natl Acad Sci U S A 90:3348-52
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Lane, M D; Flores-Riveros, J R; Hresko, R C et al. (1990) Insulin-receptor tyrosine kinase and glucose transport. Diabetes Care 13:565-75
Kaestner, K H; Christy, R J; McLenithan, J C et al. (1989) Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes. Proc Natl Acad Sci U S A 86:3150-4
Flores-Riveros, J R; Sibley, E; Kastelic, T et al. (1989) Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit. J Biol Chem 264:21557-72
Janicot, M; Lane, M D (1989) Activation of glucose uptake by insulin and insulin-like growth factor I in Xenopus oocytes. Proc Natl Acad Sci U S A 86:2642-6

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