The long-term goals of this proposal are to understand the functional relevance of neuropeptide signalling systems within the central nervous system and how these signalling systems are regulated at a molecular level. As a model system, we will analyze substance P receptor responses and aspects of substance P receptor regulation. We have recently cloned, expressed and molecularly characterized the rat substance P receptor, and now we want to examine its structural features as related to function. The substance P receptor couples to G-regulatory proteins and. upon ligand binding, activates a G-protein cycle which results in the activation of the effector enzyme phospholipase C to increase the production of inositol trisphosphate. In many physiological systems, the substance P receptor rapidly desensitizes upon agonist administration and this desensitization response may be mediated by phosphorylation of the multiple serine and threonine residues located in discrete structural regions. In this proposal, we shall: 1) create substance P receptor expressing cell lines containing the wild type, truncated and chimeric receptor forms in CHO cells and examine receptor expression and second messenger responses in these created cell lines and in native cells expressing this receptor (astrocyte and lymphocyte), 2) examining desensitization of substance P receptor responses in the various cell lines after agonist, partial agonist and antagonist administration, 3) determine structure-function relationships of substance P receptor responses using substance P receptor based peptides to assess the roles of these sequences in substance P-mediated desensitization, and 4) examine the effects of agonists, partial agonists and antagonists on substance P receptor mRNA expression in the native cell lines. These studies should provide insights into the understanding of receptor function as related to structure, and they will define the mechanisms by which long-term changes in neuropeptide receptor responsiveness and expression can occur as a result of ligand interaction.
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