Abstract

The goal of this proposal is to use morphological and biochemical approaches to analyze insulin receptor regulation. Ultrastructural studies on insulin-sensitive cells have shown significant cell-specific heterogeneity in the organization, distribution and mobility of insulin receptors. These morphological observations correlate with cell-specific differences found in biochemical binding studies on the same cells. Recent evidence suggests that cell-to-cell variability may exist in the processes involved in insulin receptor internalization and recycling.
The specific aims of this proposal are: (1) to qualitatively and quantitatively determine and compare insulin receptor organization and distribution on different cell types. Relationships, if any, will be established between occupied insulin receptor and specific cell surface structures and components. (2) to analyze the post-occupancy mobility of insulin receptors on cells that demonstrate occupancy-induced changes in receptor organization or distribution. Various chemical reagents will be used in attempts to block receptor microaggregation in order to determine the mechanisms involved and relationships to insulin action. (3) to determine the routes and subcellular structures involved in the internalization, degradation or processing, and recycling of insulin and insulin receptors. In addition to using monomeric ferritin-insulin for morphological studies, immunocytochemistry using both anti-insulin receptor antibody and biotinyl-labeled insulin binding to insulin receptors followed by antibody or avidin conjugated colloidal gold particles and/or peroxidase will be used to demonstrate the insulin receptors in intact as well as permeabilized cells. This laboratory is in a unique position to accomplish this proposal because of our proven capability to perform both quantitative morphological and correlative biochemical studies. These studies will contribute to our understanding of insulin action and of possible sites for post-binding defects in insulin resistant states.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK028143-08
Application #
3228611
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1981-01-01
Project End
1990-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
8
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104

  Publications

Smith, R M; Harada, S; Smith, J A et al. (1998) Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain. Cell Signal 10:355-62
Harada, S; Smith, R M; Smith, J A et al. (1996) Insulin-induced egr-1 and c-fos expression in 32D cells requires insulin receptor, Shc, and mitogen-activated protein kinase, but not insulin receptor substrate-1 and phosphatidylinositol 3-kinase activation. J Biol Chem 271:30222-6
Harada, S; Smith, R M; Hu, D Q et al. (1996) Dexamethasone inhibits insulin binding to insulin-degrading enzyme and cytosolic insulin-binding protein p82. Biochem Biophys Res Commun 218:154-8
Lee, Y H; Harada, S; Smith, R M et al. (1996) The expression of and insulin binding to cellular thyroid hormone binding protein, but not insulin degrading enzyme, is increased during 3T3-L1 adipocytes differentiation. Biochem Biophys Res Commun 222:839-43
Smith, R M; Zhang, S; White, M F et al. (1996) The role of receptor kinase activity and the NPEY960 motif in insulin-accelerated receptor-mediated insulin internalization. J Recept Signal Transduct Res 16:339-55
Konrad, R J; Young, R A; Record, R D et al. (1995) The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis. J Biol Chem 270:12869-76
Shah, N; Zhang, S; Harada, S et al. (1995) Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin. Endocrinology 136:2825-35
Harada, S; Smith, R M; Smith, J A et al. (1995) Insulin-induced egr-1 expression in Chinese hamster ovary cells is insulin receptor and insulin receptor substrate-1 phosphorylation-independent. Evidence of an alternative signal transduction pathway. J Biol Chem 270:26632-8
Harada, S; Smith, R M; Smith, J A et al. (1995) Demonstration of specific insulin binding to cytosolic proteins in H35 hepatoma cells, rat liver and skeletal muscle. Biochem J 306 ( Pt 1):21-8
Shi, C Z; Dhir, R N; Kesavan, P et al. (1995) Mouse embryonic stem cells express receptors of the insulin family of growth factors. Mol Reprod Dev 42:173-9

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