Abstract

We have discovered a 148 aa protein named calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) that is required for G protein-coupled signal transduction at receptors for the neuropeptide CGRP. During the previous project period we determined that RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP1). CRLR has the stereotypical 7-transmembrane topology of a G protein-coupled receptor, and requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. CRLR did not function in antisense cell lines that inhibited RCP expression, and CRLR co-immunoprecipitated with RCP suggesting that the two proteins were together in complex. The specific hypotheses to be tested in this proposal are that a functional CGRP receptor is composed of at least three proteins: CRLR, RCP, and RAMP1, and that the accessory proteins RCP and RAMP1 can modulate CRLR function.
The specific aims of this proposal are to: 1) determine the sites of interaction between RCP and CRLR required for a functional CGRP receptor, using yeast two-hybrid assay and cell culture, 2) determine if overexpression of RAMP1 or RCP can increase CGRP receptor function in cell culture, 3) determine if loss of RCP by homologous recombination has increased inhibitory effects on CGRP receptor function in cell culture, 4) identify the cellular environment that regulates the CGRP receptor complex, focusing on lipid rafts and their effect on receptor function and receptor distribution in the membrane. ? ? Recent studies have found that in hypertension the vasculature is hyper-responsive to CGRP, suggesting regulation of CGRP receptor function. Thus, the role of RCP in regulating CGRP receptor function is important both for fundamental studies on G protein-mediated signal transduction in neuroendocrine cells, but also for future therapeutic strategies for vascular disorders.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK052328-09
Application #
7023795
Study Section
Endocrinology Study Section (END)
Program Officer
Jones, Teresa L Z
Project Start
1999-01-01
Project End
2007-02-28
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
9
Fiscal Year
2006
Total Cost
$283,758
Indirect Cost

  Institution

Name
University of Rochester
Department
Neurosciences
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627

  Publications

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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