CGRP-Receptor Component Protein (RCP)
CGRP has several important physiologic roles: (1) CGRP is a potent vasodilator, and can affect the force and rate of heart beat. (2) CGRP can modulate acetylcholine receptor function at the neuromuscular junction. (3) CGRP has been demonstrated to block tolerance to morphine. (4) CGRP can modulate antigen presentation in Langerhans cells in the skin. Despite these important physiologic functions, therapeutic strategies using CGRP have been impeded due to the lack of a cloned CGRP receptor with which ligands could be developed.
Most neuropeptide receptors are members of the superfamily of proteins characterized by the presence of seven hydrophobic, putative membrane-spanning domains. These receptors are linked to intracellular G-proteins for signal transduction, and can participate in multiple signal transduction pathways, depending on which G-protein is present.
We have recently cloned the cDNA for a novel protein we named the CGRP-receptor component protein (RCP), which is required for CGRP receptor activation. RCP is a novel protein required for signal transduction, and may represent the prototype for a new class of molecules that interact with G-protein coupled receptors. Cloning RCP required a multi-disciplinary approach, involving molecular biology and oocyte electrophysiology (carried out in collaboration with Dr. Gerhard Dahl, Department of Physiology, University of Miami). We expression-cloned the RCP cDNA using a novel assay employing the cystic fibrosis transmembrane conductance regulator (CFTR) as a sensor for cAMP production in Xenopus laevis oocytes. RCP is required for CGRP receptor activation in the oocyte-CFTR expression assay and in tissue culture cells, and is co-localized with CGRP in cerebellum and cochlea. RCP is a 146 amino acid hydrophilic protein, which we hypothesize works in conjunction with a membrane-spanning protein to form a functional CGRP receptor. RCP thus represents the first example of an accessory protein required for activation of a G protein-coupled receptor, and implies an additional level of receptor regulation unique to the CGRP system.
Oocyte-CFTR assay. Addition of peptide results in activation of G-protein, and subsequently of adenylate cyclase and protein kinase A (PKA), resulling in a peptide-specific chloride current.
Dose response curve of oocytes co-injected with RCP cRNA and CFTR cRNA; EC50=14 nM.
To confirm the function of RCP, an antisense oligonucleotide was synthesized based on the sequence of the RCP cDNA. Co-injection of this antisense oligonucleotide with either RCP cRNA or cerebellar mRNA abolished CGRP receptor activity in the oocyte-CFTR assay, indicating that the cerebellum requires RCP for CGRP signal transduction.
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Record of CFTR chloride·currents from oocytes injected with CFTR cRNA plus a) guinea cerebellar mRNA or b) cerebellar mRNA plus antisense oligonucleotides made against RCP. Arrows indicate applicatoin and washout of ligand.
The recent identification by others of two orphan G protein-coupled receptors (RDC1 and CGRP1) as candidate CGRP receptors provides two substrates on which RCP may act. Our hypothesis is that RCP is required for CGRP receptor activation, working in combination with membrane-spanning proteins (either the candidate CGRP receptors RDC1 or CGRP1, or with other unidentified receptors) to form a functional CGRP receptor.
Putative roles for RCP, either at the level of receptor activation, or at subsequent steps in signal transduction.
It has been reported in the literature that CGRP and calcitonin cross-react at each other's receptor. To investigate this possibility, we have cloned the guinea pig calcitonin receptor. We found that the calcitonin receptor was not responsive to CGRP when tested in the oocyte-CFTR assay, or when transfected into COS fibroblast cells (determined by cAMP response or displacement of 125I-calcitonin binding). Similarly, oocytes injected with RCP were not responsive to calcitonin in the oocyte-CFTR assay. From these experiments we have concluded that the calcitonin receptor does not interact with CGRP, and have since focused our efforts on delineating the roles of RCP and the candidate CGRP receptors (RDC1 and CGRP1) in forming high affinity CGRP receptors.
Future Projects
We are now focusing on how RCP works by identifying:
- The subcellular localization of RCP, utilizing E.M. immunohistochemistry and subcellular fractionation followed by Western blot.
- Which proteins interact with RCP by co-immunoprecipitation of RCP from tissue and cell lines, by yeast two-hybrid cloning, and by co-expression in cell culture.
- Reconstituion and antisense experiments in tissue culture cells and in Xenopus oocytes.
Identification of the CGRP-receptor component protein (RCP) is an exciting break-through for the field of CGRP research. RCP is a critical component of the CGRP receptor complex, and will immediately aid experiments to reconstitute CGRP receptors in cell culture, thereby facilitating the development of therapeutic CGRP ligands.
This research has been supported by grants from NIH, the American Heart Association (Florida Affiliate) and Merck Research Laboratories to I.M.D