Determination of the role of site specific GPCR phosphorylation in cellular responses. (360G-Wellcome-047600_Z_96_C)

Studies on the Mechanisms and Functional Consequences of Phospholipase C-Coupled Receptor Phosphorylation Many neurotransmitter, neuromodulator, hormone and growth factor receptors are coupled, via G-proteins, to the activation of phospholipase C (PLC). The processes that regulate this signal transduction pathway are, therefore, central in the cellular responses mediated by a diverse group of biological ligands, in addition to being important for the action of many therapeutic drugs. This project proposes to investigate the involvement of receptor phosphorylation in the regulation of PLC-coupled receptors. The investigation is aimed at three levels; (i) identification of the kinases responsible for PLC-coupled receptor phosphorylation, (ii) the functional consequences of receptor phosphorylation and (iii) the relevance of phosphorylation to signalling in an intact tissue; airway smooth muscle. I have recently been successful in the purification of a novel receptor-specific kinase (MRK) that is able to phosphorylate the PLC-coupled m3-muscarinic receptor. One of the major aims of this project is to obtain a cDNA clone for MRK. Analysis of the modes of MRK regulation will also be conducted, in addition, to investigation of the receptor-substrate specificity of MRK. Functional studies on the role receptor phosphorylation plays in the regulation of receptor/PLC coupling will form an important part of the project. This will benefit from the excellent infra-structure already in place in the department, that allows for a mix of molecular approaches and detailed analysis of receptor-signalling properties. It is planned to identify the sites of phosphorylation on the PLC-coupled m1- and m3-muscarinic receptors and delete these sites by point mutagenesis. Detailed functional analysis of mutant and wild type receptors will then be pursued. This will be coupled with reconstitution studies, and transfection studies where muscarinic receptor subtypes will be co-expressed with receptor-specific kinases and dominant negative kinase mutants. Particular emphasis will be placed on the possible role phosphorylation plays in receptor desensitisation, and receptor/G-protein coupling.