R37 DK032878: The cholecystokinin (CCK) receptor is important as a prototypic Family A G protein-coupled receptor activated by a small peptide ligand and as a potentially important target for pharmacotherapy of normal and pathological tissues. In normal physiology, the type A CCK receptor plays multiple roles related to nutrient homeostasis, mediating gallbladder contraction, pancreatic exocrine secretion, gastrointestinal motility, and even post-cibal satiety. The type B CCK receptor has predominant roles in mediating gastric acid secretion and in neuronally-mediated motor and anxiety events. Tumors of the colon and pancreas have been reported to express these receptors as well. In the current application, we propose three broad aims to examine the variedstates of this receptor, the dynamic changes in conformation associated with these states, its quaternary structure including key molecular interactions, and to use new tools and insights to study the physiology and pathophysiology of CCKreceptors in health and disease. (1) Aim 1 is designed to explore the hypothesis that the CCK receptor undergoes dynamic changes in conformation, thereby exposing surfaces that are critical for initiation of signaling and for receptor regulation. We propose that an understanding of the full spectrum of conformational states can provide insights that can facilitate the development of new classes of drugs targeting this receptor that might activate or block selected signaling pathways and be responsive or resistant to selected regulatory mechanisms. The projects proposed for this aim will concentrate on photoaffinity labeling and fluorescence methodologies. These studies will utilize novel photolabile and fluorescent analogues of the full spectrum of CCK receptor ligands, agonists, partial agonists, and antagonists, situating the probes throughout the pharmacophore. Relevant distances will be measured using Fluorescence Resonance Energy Transfer (FRET) technology. Morphological FRET will be applied to explore whether receptor conformations are different in distinct cellular compartments. (2) Aim 2 will be focused on the quaternary structure of the CCK receptor, and is designed to explore the hypothesis that bimolecular interactions (in addition to the coupling of the receptor to its proximal G protein effector) are key for altering the properties of this receptor. These include homo- and hetero-oligomerization of GPCRs, as well as receptor interactions with structurally distinct molecules. These interactions will be probed using fluorescent techniques like FRET, and with biochemical and molecular biological approaches. Transmembrane peptides without and with modified faces will be utilized to examine which helical domains and which components of these domains contribute to particular molecular associations. The functional impact of bimolecular interactions will also be explored. (3) Aim 3 is designed to examine the hypothesis that understanding of the roles played by CCK receptors in health and disease can lead to novel manipulations to ameliorate disease processes. The areas to be explored include the interactions between the CCK receptor and its microenvironment within the lipid bilayer that can be altered in dyslipidemias and in obesity, and the alternative spliceoforms of CCK receptors that have been described to exist in various diseases. The goal of this aim is to make our increasingly detailed insights into CCK receptor structure and function more relevant to human disease, possibly providing new diagnostic and therapeutic tools for improving health and disease outcomes. PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
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