Producing ligands to G protein coupled receptors (GPCRs) with the desired actions is one of the most active areas of research for therapeutic agents. Determining how GPCRs, like the CCK-A receptor, interact at the molecular level with their ligands will aid in the design of reagents for treating obesity, gallbladder disease and motility disorders. CCK-58 is a major endocrine form of cholecystokinin that has different biological activity from CCK-8. One major difference is that CCK- 58 does not induce pancreatitis at any dose while high CCK-8 is an accepted model for induction of pancreatitis. There is evidence that peptide ligands interact with their GCPR via a lipid-associated mechanism. Therefore, we hypothesize that different lipid-induced tertiary structures of cholecystokinin agonists determine the biological responses elicited via the CCKA receptor. A corollary is that the N-terminus of CCK-58 acts as a scaffold for the C-terminal active region when associated with lipids. Initially we will identify intramolecular associations of the N- and C-terminus of CCK-58 and test if these associations are important for function (CCK-A receptor binding and stimulation of pancreatic protein from acinar cells). Next the lipid-associated tertiary structures of six cholecystokinin analogs (CCK-8, CCK-33, JMV-180, CCK-58, CCK-8(ns) and CCK-58(ns)) will be determined. The six analogs will be tested for differences in their physiological actions and their capacity to cause pancreatitis in an awake rat model. We will identify the 3D conformation(s) that elicit a normal physiological response or pancreatitis. Next we will titrate the receptor segment (extracellular loop 3, amino acids 345-373) with the cholecystokinin agonists and determine the specific ligand-receptor interactions. Further we will determine 3D conformation of CCK-8 and CCK-58 bound to the receptor fragment. The data collected will be used to predict amino acid substitutions that could increase binding of agonists to the CCK-A receptor. We will mutate the receptor with substitutions predicted to enhance binding to test our model.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK033850-18
Application #
7320283
Study Section
Special Emphasis Panel (ZRG1-GMA-3 (01))
Program Officer
May, Michael K
Project Start
1984-04-01
Project End
2010-11-30
Budget Start
2007-12-01
Budget End
2010-11-30
Support Year
18
Fiscal Year
2008
Total Cost
$257,579
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Cooper, Marvis S; Reeve Jr, Joseph R; Raboin, Shannon J et al. (2008) Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex. Regul Pept 148:88-94

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