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.
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