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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052328-02
Application #
6124818
Study Section
Endocrinology Study Section (END)
Program Officer
Sato, Sheryl M
Project Start
1999-01-01
Project End
2002-11-30
Budget Start
1999-12-01
Budget End
2000-11-30
Support Year
2
Fiscal Year
2000
Total Cost
$198,770
Indirect Cost
Name
University of Miami School of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Dickerson, Ian M; Bussey-Gaborski, Rhiannon; Holt, Joseph C et al. (2016) Maturation of suprathreshold auditory nerve activity involves cochlear CGRP-receptor complex formation. Physiol Rep 4:
Bawa, Zharain; Routledge, Sarah J; Jamshad, Mohammed et al. (2014) Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks. Microb Cell Fact 13:127
Sardi, Claudia; Zambusi, Laura; Finardi, Annamaria et al. (2014) Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis. J Neuroimmunol 271:18-29
Dickerson, Ian M (2013) Role of CGRP-receptor component protein (RCP) in CLR/RAMP function. Curr Protein Pept Sci 14:407-15
Egea, Sophie C; Dickerson, Ian M (2012) Direct interactions between calcitonin-like receptor (CLR) and CGRP-receptor component protein (RCP) regulate CGRP receptor signaling. Endocrinology 153:1850-60
Supowit, Scott C; Katki, Khurshed A; Hein, Travis W et al. (2011) Vascular reactivity to calcitonin gene-related peptide is enhanced in subtotal nephrectomy-salt induced hypertension. Am J Physiol Heart Circ Physiol 301:H683-8
Morara, Stefano; Wang, Li-Ping; Filippov, Vitaly et al. (2008) Calcitonin gene-related peptide (CGRP) triggers Ca2+ responses in cultured astrocytes and in Bergmann glial cells from cerebellar slices. Eur J Neurosci 28:2213-20
Tolun, Adviye A; Dickerson, Ian M; Malhotra, Arun (2007) Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli. Protein Expr Purif 52:167-74
Glaser, Shannon S; Ueno, Yoshiyuki; DeMorrow, Sharon et al. (2007) Knockout of alpha-calcitonin gene-related peptide reduces cholangiocyte proliferation in bile duct ligated mice. Lab Invest 87:914-26
Zhang, Zhongming; Dickerson, Ian M; Russo, Andrew F (2006) Calcitonin gene-related peptide receptor activation by receptor activity-modifying protein-1 gene transfer to vascular smooth muscle cells. Endocrinology 147:1932-40

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