G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins that bind to a diverse array of stimuli and are involved in a large number of physiological functions. The family A GPCRs are the largest and most comprehensively studied. The family B GPCRs are a small but important group of receptors (~15 members) that bind to peptide ligands and are involved in physiological processes that include vasodilation, stress, digestion and glucose homeostasis. The CGRP receptor is a unique member of this family as it is a heterodimer consisting of a GPCR subunit (calcitonin receptor-like receptor, CLR) and a single transmembrane accessory protein (receptor activity modifying protein, RAMP1). The extracellular loop two (ECL2) domain is involved in ligand binding and activation in a number of studied GPCRs. This makes it vital both with respect to receptor function and in the design of therapeutics. The main focus of this thesis is to study the structure and function of the ECL2 domain in the CGRP receptor. This was initially done through individual alanine substitutions of each ECL2 residue and measuring the effect of this on a number of receptor processes. Residues that were identified as important for receptor function through this investigation were selected for an extensive set of mutagenesis to identify the precise molecular interactions that were involved at each position. These experiments have shown that ECL2 is the most important domain of the CGRP receptor for ligand-based activation. The N-terminal half of ECL2 contains residues predicted to have structural function and the C-terminal half is predicted to be involved in direct ligand binding. These results have been used in collaboration to refine a computer model of receptor structure and ligand binding to predict specific ligand docking sites that can be used to design therapeutics for migraine, heart attack and hypertension. The final part of thesis produced preliminary data to support proof of concept for two techniques that can be used in the study of CGRP receptor function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640989 |
| Date | January 2014 |
| Creators | Woolley, Michael J. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/66794/ |
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