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Multiple residues within the second extracellular loop of the C1 receptor are required for receptor activation by CGRP Calcitonin-gene related peptide (CGRP) is a 37 amino acid neuropeptide which acts as a potent vasodilator. A CGRP receptor consists of a heterodimer of a family B G-protein coupled receptor, called the calcitonin receptor-like receptor (CL) and a single-pass transmembrane protein known as the receptor activity modifying protein 1 (RAMP1) (McLatchie et al., 1998). Little is known of the residues within the CL receptor which are crucial for the binding and activation of the receptor by CGRP. In this study, the second extracellular loop was investigated by means of an alanine scan. Residues from R252 to H267 were replaced by alanine, either individually or in pairs. Point mutations were created using the Stratagene Quick-change mutagenesis method. The genes encoding the mutant and wild-type (WT) CL receptors contained within the pcDNA3.1- mammalian expression vector were transiently co-transfected with RAMP1 into Cos7 cells using 10mM polyethylenimine. Measurement of CGRP-mediated cAMP-signalling was by a cAMP radio-receptor assay after challenge with human a-CGRP from 10pM to 100nM (Poyner et al., 1998; Conner et al., 2005). pEC50 values were calculated using Graphpad Prism version 4.00. Statistical testing was by paired t-tests or Dunnett’s test after a one-way ANOVA if multiple comparisons were being made; values are quoted as means ±s.e.m. Table 1 pEC 50 values for mutant receptors
Table 1 shows; the majority of the loop is required for CGRP mediated-cAMP production, with 50 to 75% of the residues implicated. Further work is required to split the double mutants and establish whether they are required for CGRP binding or receptor activation. This work was funded by the Wellcome Trust and the British Heart Foundation (PG 15278).
Conner A.C. et al., (2005), Mol. Pharmacol., 67, 20-31. |
