We have recently discovered a novel low molecular weight protein which appears to be required for signal transduction at receptors for calcitonin gene-related peptide (CGRP), and potentially at other G protein-coupled receptors. This small hydrophilic protein which has been named the Receptor Component Protein (RCP) is required for CGRP- mediated signal transduction in NIH3T3 cells. We have demonstrated the requirement for RCP in CGRP receptor function by making stable NIH3T3 cell lines which express RCP antisense cDNA, and have observed a loss of RCP protein with a concomitant loss of CGRP receptor activity. RCP was discovered in the context of the CGRP receptor, but its effects may not be limited to receptors for CGRP. In an effort to learn more about the function of this novel protein we have focused our initial studies on CGRP receptor activation. We do not believe that RCP represents a receptor itself, as transfection of RCP into COS fibroblast cells does not yield functional CGRP receptors. We instead hypothesize that RCP works in conjunction with a membrane-spanning, ligand-binding protein to form a functional CGRP receptor. Two CGRP receptors have recently been identified, but cotransfection of RCP with either of these receptors into COS fibroblasts fails to reconstitute CGRP receptor function, implicating a novel receptor working in conjunction with RCP in NIH3T3 cells. Our hypothesis is that RCP effects either targeting of receptor to the cell surface, or coupling of the receptor to signal transduction molecules. The results from the experiments outlined in this proposal will discern between these two possibilities.
The Specific Aims of this proposal are to: 1) Determine if the loss of RCP in NIH3T3 cells inhibits receptors other than CGRP. 2) Determine if RCP functions in receptor sorting or receptor coupling. 3) Identify the receptor(s) in NIH3T3 cells that require RCP for function. We are using the CGRP receptor present in NIH3T3 cells as a model for RCP-dependent receptors to determine the mechanism of RCP function. CGRP is one of the most potent vasodilators known, and CGRP binding sites are distributed widely throughout the cardiovascular system. Characterization of the proteins involved in CGRP receptor activation is important for developing CGRP receptor model systems, which will facilitate development of therapeutic ligands for treatment of cardiovascular disease such as hypertension.
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