Title

Identification of RCP interactive domain in CLR and its role in calcitonin gene-related peptide signaling

Date of Award

2004

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Physiology and Biophysics

First Committee Member

Ian M. Dickerson, Committee Chair

Abstract

The receptor for calcitonin gene-related peptide (CGRP) named the calcitonin-like receptor (CLR) requires two accessory proteins for function: receptor activity-modifying protein (RAMP1), and receptor component protein (RCP). RAMP1 regulates ligand binding. RCP is an intracellular peripheral membrane protein that couples the protein complex to the intracellular signaling pathway. An interaction between RCP and the second cytoplasmic loop of CLR was identified by yeast two-hybrid experiments. This interaction was confirmed by expressing in NIH3T3 cells the second cytoplasmic domain of CLR fused to the enhanced green fluorescent protein (EGFP), and demonstrating that this domain co-immunoprecipitated with RCP. In transfected cells the soluble CLR domain sequestered RCP in the cytoplasm away from the membrane-bound CLR + RAMP1 complex, inhibiting CGRP-mediated signal transduction by 85%. No inhibitory effect was observed by expressing EGFP alone, or by expressing the second cytoplasmic domain of the beta 2-adrenergic receptor, a G protein-coupled receptor that does not require RCP for signaling. Furthermore, a chimeric CLR containing the second intracellular domain of the beta2-adrenergic receptor also inhibited CGRP-mediated signaling. The data supports a direct interaction between RCP and the second cytoplasmic domain of CLR that allows the ligand-bound receptor to signal via G protein-coupled mechanisms. RCP expression correlates with CGRP efficacy in vivo, suggesting that RCP also regulates CGRP receptor function in vivo.

Keywords

Biology, Molecular; Biology, Neuroscience; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3141906