Investigation of the activation mechanism and structural characterization of the CGRP receptor

  • Gabriel Kuteyi

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor
(GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying
protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other
homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino
acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health
conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode
of activation of this receptor could be key in developing therapeutic agents for associated health
conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this
receptor (in complex with an antagonist) has been published, the details of receptor-agonist
interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear.
Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8)
region, has not been well studied for its role in receptor signalling. This research project investigated
these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and
associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These
residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by
evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated
receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2
and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural
interaction study by surface plasmon resonance (SPR). Following expression and purification, these
receptor proteins were found to individually retain their secondary structures when analysed by
circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin
family receptor paradigm.
The research described in this thesis has produced novel data that contributes to a clearer
understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a
useful tool in determining novel interacting GPCR partners of RAMPs.
Date of Award6 Dec 2013
LanguageEnglish
SupervisorDavid Poyner (Supervisor) & Roslyn Bill (Supervisor)

Keywords

  • G protein-coupled receptor
  • calcitonin receptor-like receptor
  • receptor activity modifying protein
  • Pichia pastoris
  • extracellular domain
  • site-directed mutagenesis

Cite this

Investigation of the activation mechanism and structural characterization of the CGRP receptor
Kuteyi, G. (Author). 6 Dec 2013

Student thesis: Doctoral ThesisDoctor of Philosophy