This grant will examine the hypothesis that receptor-mediated endocytosis limits cellular responses to GRP both by receptor internalization and by intracellular lysosomal destruction of internalized GRP. The second hypothesis is that pH sensitive endosomal dissociation of ligand regulates receptor recycling and resensitization and thereby regulates responsiveness to activation of G protein coupled receptors (GPCRs). The local concentration of hormones or neuropeptides, and the number of responsive cell surface receptors, regulate receptor-mediated responses. The general phenomenon of receptor desensitization and resensitization is vital to all processes that are regulated by receptors. Just as it is necessary to have an intracellular signal transduction system to produce responses to extracellular ligands, it is also necessary to have mechanisms to terminate the responses or to maintain them. Using GRP receptor as a general model for gastrointestinal GPCRs, we will define the processes of endosomal uptake of GRP receptors, pH dependent intraendosomal dissociation of GRP lysosomal degradation of endocytosed GRP, and receptor recycling and reactivation by: a) using the rate of GRP degradation to measure turnover rates of functional GRP receptors; b) determining the role of acidification of endosomes and lysosomes for dissociation and enzymatic degradation of ligand and for recycling and resensitization of the receptor; c) determining the roles of phosphorylation and dephosphorylation in receptor desensitization and resensitization and characterizing the intracellular sites and pH at which these occur; and d) identifying interactions between receptors tagged with green fluorescent proteins, Cy3 labeled GRP, and other molecules that are colocalized at different stages of endocytosis and determine the effects of cytoskeletal elements and of colocalized substances on endocytosis and recycling. We also plan to characterize GRP receptor recycling and resensitization in cells that normally express GRP receptors: gastric G cells, Swiss 3T3 cells, and small cell lung carcinoma line NCI-H345 cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK035740-14
Application #
2905329
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
May, Michael K
Project Start
1985-08-01
Project End
2003-07-31
Budget Start
1999-09-30
Budget End
2000-07-31
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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