The cholecystokinin-B/gastrin receptor (CCK-BR) is a seven transmembrane domain peptide hormone receptor. Among its most important physiologic functions, the CCK-BR modulates acid secretion and mucosal proliferation in the stomach as well as anxiety and pain perception in the central nervous system. The potential clinical relevance of this receptor has generated considerable interest in understanding the molecular basis of ligand - CCK-BR interactions. Mutational analysis of the receptor, carried out in our laboratory, suggests he existence of a ligand binding pocket comprised of transmembrane domain residues. Non-peptide benzodiazepine-based ligands appear to occupy this putative pocket . Minor structural modifications either of these compounds or of the amino acids which comprise the receptor pocket , can influence whether these ligands act as agonists or antagonists. In addition to these findings with non-peptides, we have obtained evidence that affinity for the endogenous peptide agonist, gastrin, is conferred by an interaction between trans-membrane and extracellular domain amino acids. In the current application, we propose to further explore the molecular determinants of ligand binding to the CCK-BR, and of ligand-induced receptor activation, comparing peptide and non-peptide compounds. The relative roles of both the extracellular and transmembrane domains will be explored.
Specific Aim 1 of this grant is directed toward defining CCK-BR amino acids which confer ligand affinity.
Specific Aim 2 addresses which CCK-BR -ligand interactions influence the ability of the ligand to induce second messenger signaling.
Specific Aim 3 will exploit the recent discoveries in our laboratory of a constitutively active CCK-BR together with a compound that functions as an inverse agonist and as such attenuates ligand-independent signaling. These novel tools will be utilized to explore the receptor-ligand interactions which result in inverse agonism. A combination of molecular (generation of chimeric and mutant receptors, transient expression of recombinant proteins) and pharmacologic methods (radioligand binding, second messenger signaling assays) will be utilized to address these objectives. The proposed studies of the CCK-BR will establish a framework for understanding how non-peptide ligands mimic the activity of endogenous peptides. This information should expand current knowledge of structure-function relationships of peptide hormone receptors in general and may therefore be useful in developing new therapeutic options for a wide range of diseases mediated by this class of proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK046767-09S1
Application #
6449646
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Hamilton, Frank A
Project Start
1992-09-30
Project End
2003-05-31
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
9
Fiscal Year
2001
Total Cost
$119,655
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02111
Whited, K L; Thao, D; Lloyd, K C Kent et al. (2006) Targeted disruption of the murine CCK1 receptor gene reduces intestinal lipid-induced feedback inhibition of gastric function. Am J Physiol Gastrointest Liver Physiol 291:G156-62
Beinborn, Martin; Worrall, Christine I; McBride, Edward W et al. (2005) A human glucagon-like peptide-1 receptor polymorphism results in reduced agonist responsiveness. Regul Pept 130:1-6
Bi, Sheng; Scott, Karen A; Kopin, Alan S et al. (2004) Differential roles for cholecystokinin a receptors in energy balance in rats and mice. Endocrinology 145:3873-80
Wang, David Q-H; Schmitz, Frank; Kopin, Alan S et al. (2004) Targeted disruption of the murine cholecystokinin-1 receptor promotes intestinal cholesterol absorption and susceptibility to cholesterol cholelithiasis. J Clin Invest 114:521-8
Beinborn, Martin; Ren, Yong; Blaker, Michael et al. (2004) Ligand function at constitutively active receptor mutants is affected by two distinct yet interacting mechanisms. Mol Pharmacol 65:753-60
Giacobini, Paolo; Kopin, Alan S; Beart, Philip M et al. (2004) Cholecystokinin modulates migration of gonadotropin-releasing hormone-1 neurons. J Neurosci 24:4737-48
Doran, Amanda C; Wan, Yieh-Ping; Kopin, Alan S et al. (2003) Established theory of radiation-induced decay is not generalizable to Bolton-Hunter labeled peptides. Biochem Pharmacol 65:1515-20
Ren, Yong; Blaker, Michael; Seshadri, Lakshmi et al. (2003) Conserved cholecystokinin receptor transmembrane domain IV amino acids confer peptide affinity. J Mol Neurosci 20:115-24
Kopin, Alan S; McBride, Edward W; Chen, Ci et al. (2003) Identification of a series of CCK-2 receptor nonpeptide agonists: sensitivity to stereochemistry and a receptor point mutation. Proc Natl Acad Sci U S A 100:5525-30
Chen, Duan; Zhao, Chun-Mei; Al-Haider, Wisam et al. (2002) Differentiation of gastric ECL cells is altered in CCK(2) receptor-deficient mice. Gastroenterology 123:577-85

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