Structure-function analysis of calcitonin and amylin receptors

Abstract

The calcitonin receptor (CTR) is a peptide binding family B G protein-coupled receptor (GPCR). CTR alone has high affinity for calcitonin (CT) but when expressed with receptor activitymodifying proteins 1, 2 and 3 (RAMPs) it forms the AMY1, AMY2 and AMY3 receptors, which have increased affinity for amylin and CT gene-related peptide (CGRP). Family B GPCRs have a long N-terminal extracellular domain (ECD) that forms an important part of the peptide binding site. While peptide bound ECD structures are available for several family B GPCRs, there is no structural information available on the CTR. The precise binding sites of the peptides CT, amylin and CGRP within the ECD are unknown and it is also unclear if or how the presence of the RAMP changes the peptide binding site to allow high affinity amylin and CGRP binding. In this thesis, site-directed mutagenesis and functional assays were performed on the CTR ECD with the aim of identifying residues involved in peptide interactions. CTR ECD mutants were characterised with CT, CGRP and amylin in the presence and absence of RAMPs, to investigate how RAMPs influence peptide binding. In addition beta amyloid 1-42, was investigated as a potential novel agonist of AMY receptors. The isolated ECDs of the CTR and RAMP1 were also expressed with the aim of characterising them by X-ray crystallography. The mutagenesis data identified CTR ECD residues involved in CT, CGRP and amylin interactions. Interestingly there were differences in CT and amylin interactions with the CTR; alanine mutants W79A, F99A, D101A, F102A, W128A and Y131A, significantly reduced CT potency, however only W79A, F102A and Y131A reduced amylin potency. At the AMY receptors seven receptor mutants; W79A, F99A, D101A, F102A, R126A, W128A and Y131A resulted in a significant decrease in amylin potency, indicating that additional receptor residues become involved in amylin binding when RAMPs are present. This suggests that RAMPs may act indirectly to alter peptide binding at AMY receptors.