REGULATION OF CELLULAR RESPONSES TO NEUROPEPTIDES

本研究提出了一种假设，即激动剂诱导的g蛋白偶联受体（gpcr）的转运是信号转导的起始和终止所必需的，并且缺陷导致信号转导

This proposal examines the hypothesis that agonist-induced trafficking of G-protein coupled receptors (GPCRs) is required for the initiation and termination of signal transduction, and that defects result in uncontrolled stimulation and disease. The molecular mechanisms and physiological roles of substance P (SP)- induced trafficking of the neurokinin 1 receptor (NKIR) will be examined at the level of the cell and the whole animal. It is important to understand the mechanisms of NK1R signaling in view of the pathophysiological roles of this receptor in neurogenic inflammation, pain and intestinal motility. NK1R regulation will be studied in transfected cell lines, endothelial cells and enteric neurons in short-term culture, and in knockout and transgenic mice. Aim 1 will define the molecular mechanism of SP-stimulated endocytosis and intracellular trafficking of the NK1R. The role of the clathrin adapter p-arrestin, and of dynamin and Rab GTPases in NK1R trafficking will be examined by expression of dominant negative mutants, and by studying neurons from p- arrestin knockout mice. The role of the cytoskeleton and of endosomal acidification and phosphatases will be examined using specific drugs Aim 2 will define the importance of NK1R trafficking for the initiation of signal transduction. The role of p-arrestins, Rabs and the cytoskeleton in NKlR-mediated MAP kinase activation will be determined using reagents developed in Aim 1. These studies will define the role of p-arrestins as molecular scaffolds that recruit and organize components of the MAPK cascade. Aim 3 will define the importance of NK1R trafficking in desensitization and resensitization of signaling. The role of G-protein receptor kinases (GRKs) and p-arrestins in desensitization will be determined by expression of dominant negative mutants and by studying neurons from knockout mice. The importance of Rabs, the cytoskeleton and endosomal sorting for resensitization will be examined using reagents from Aim 1. Aim 4 will determine whether defects in mechanisms of NK1R desensilization result in prolonged SP signaling and disease. SP signaling will be examined in mice deficient in GRKs or p-arrestins, or expressing a desensitization and internalization-defective mutant NK1R (NK1R8325, a naturally occurring NK1R variant). The effects of SP on intestinal motility and neurogenic inflammation will be examined, with the expectation that defects in desensitization will result in exaggerated motor and inflammatory responses. Together, the results of these experiments will provide new information about GPCR signaling, and will define how defects in signal transduction can cause disease.