CCKA, bombesin (Bn) and m3 cholinergic (m3Ach) receptors are major regulators of the exocrine pancreas. At a superficial level the actions of these receptors appear identical. However, a variety of studies indicate that acinar cells respond differently to these three receptors. Characteristics which vary between these receptors include, the number of ligand binding affinity states, and characteristics of receptor regulation, including desensitization, internalization, and down- regulation. The focus of the current proposal is to identify the structural and functional basis of the differences in these receptors and thereby, to learn how the receptors act. The cDNAs for these three receptors have recently been cloned. Utilizing these clones and techniques of molecular and cell biology we will determine the specific receptor domains and cellular components which: l) determine receptor binding affinity states and their attendent signal cascades; 2) are involved in internalization and down-regulation of the receptors; and 3) are involved in rapid desensitization of the receptors. The principal approach will be one of constructing chimeric receptors by transferring homologous domains among the three receptors. Using this approach with these three receptors should yield novel and important information unavailable through standard deletion and mutagenesis studies of single receptors. We will focus particular attention on receptor G protein interactions and receptor phosphorylation because they are likely to influence several aspects of receptor function. We will identify specific G protein alpha-subunits which are able to interact with the receptors by co-expression of receptors and G protein alpha-subunits in tissue culture cells. To confirm physiologically relevant interactions receptors and G proteins from rat acinar cells will be co-immunoprecipitated. Phosphorylated residues in the receptors will be identified using anti- receptor antibodies and/or receptors tagged with epitopes recognized by monoclonal antibodies. Site-directed mutagenesis will then be used to investigate the roles of specific phosphorylation sites on receptor function and regulation. In general, analysis o potential mechanisms and interactions will be conducted in transfected cell lines using molecular techniques such as receptor chimeras and site-directed mutants. However, whenever possible, verification and analysis of physiological mechanisms will be conducted in normal rat pancreatic acinar cells. These studies will provide an understanding of the basis of the similarities and differences in binding affinities and receptor regulation between these three important receptors. Thereby, insight will be gained into the mechanisms and interactions involved in the regulation of other receptors and ultimately into the regulation of the exocrine pancreas.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK041225-06A1
Application #
2141647
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1989-04-15
Project End
1998-08-31
Budget Start
1994-09-30
Budget End
1995-08-31
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Physiology
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Owyang, Chung; Logsdon, Craig D (2004) New insights into neurohormonal regulation of pancreatic secretion. Gastroenterology 127:957-69
Zhang, Lizhi; Duan, Chao Jun; Binkley, Charles et al. (2004) A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A. Mol Cell Biol 24:2169-80
Ji, B; Bi, Y; Simeone, D et al. (2001) Human pancreatic acinar cells lack functional responses to cholecystokinin and gastrin. Gastroenterology 121:1380-90
Logsdon, C D (1999) The influence of the cellular context on receptor function: a necessary consideration for physiologic interpretations of receptor expression studies. Life Sci 64:369-74
Nicke, B; Detjen, K; Logsdon, C D (1999) Muscarinic cholinergic receptors activate both inhibitory and stimulatory growth mechanisms in NIH3T3 cells. J Biol Chem 274:21701-6
Dabrowski, A; Detjen, K M; Logsdon, C D et al. (1997) Stimulation of both CCK-A and CCK-B receptors activates MAP kinases in AR42J and receptor-transfected CHO cells. Digestion 58:361-7
Detjen, K; Yule, D; Tseng, M J et al. (1997) CCK-B receptors produce similar signals but have opposite growth effects in CHO and Swiss 3T3 cells. Am J Physiol 273:C1449-57
Grady, T; Liang, P; Ernst, S A et al. (1997) Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis. Gastroenterology 113:1966-75
Bragado, M J; Groblewski, G E; Williams, J A (1997) p70s6k is activated by CCK in rat pancreatic acini. Am J Physiol 273:C101-9
Blevins, G T; van de Westerlo, E M; Logsdon, C D et al. (1996) Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells. Regul Pept 61:87-93

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