Investigation of Effects on Adrenomedullin Pharmacology of Mutations in the Upper Transmembrane/Extracellular Loop Region of the Calcitonin Receptor-Like Receptor

HA Watkins1, R.S Abhayawardana1, M Chakravarthy1, J Barwell2, M. J. Woolley3, A.C. Conner3, DR Poyner2 and Debbie L Hay1.. 1 University of Auckland, Auckland, New Zealand, 2 Aston University, Birmingham, United Kingdom

3 University of Warwick, Coventry, United Kingdom

Adrenomedullin (AM) is a ligand for the AM1 and AM2 receptors. Activation of these receptors results in many downstream effects including angiogenesis of the blood and lymphatic vascular systems. The AM1 and AM2 receptors are heterodimers of the family B G protein-coupled receptor, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 2 or 3 respectively. The extracellular loops (ECLs) and the upper transmembrane helices (TMs) of GPCRs are involved in receptor activation (1). In this study we sought to determine the residues involved in ligand binding and receptor activation in this region of CLR in the AM1 and AM2 receptors.

An alanine/leucine scan of the upper TM/ECL regions (determined by molecular modelling (2)) was carried out. HEK293S cells were transiently transfected with each mutant receptor and their ability to elicit an AM mediated cAMP response was measured. Cell surface expression was verified by ELISA.

Table1, pharmacological parameters of mutant residues in the AM1 receptor. Blue; TM2, Red; TM3, Green; ECL2, Orange, TM6.

Table2, pharmacological parameters of mutant residues in the AM2 receptor. Blue; TM2, Red; TM3, Green; ECL2.For tables; *p<0.05,** p<0.01, ***p<0.001 by unpaired t-test against WT. Values are mean ± standard error of the mean.

At the AM1 receptor mutation of residues in TM2, TM3, TM6 and ECL2 resulted in decreased pEC50 and maximal cAMP accumulation (Emax) (Table1). L195A, C212A, L374A and E380A completely abolished the cAMP response. Mutation of residues in ECL1 and ECL3 had little effect on AM cAMP signalling. Fewer residues affected cAMP signalling at the AM2 receptor (Table 2). Where a change was observed, this was generally less than at the AM1 receptor. Most notably C212A and C282A, which form a stabilising disulphide bond between TM4 and ECL2, had no effect at the AM2 receptor. L195A which appears essential to receptor activation in the AM1 receptor had a much smaller effect at the AM2 receptor. In general the effects of the mutations were not due to decreased cell surface expression.

These data are the first complete scan in a single study of the ECLs of a family B GPCR and reveal changes in the pharmacological profile of residues in CLR depending on which RAMP is present. These may indicate a different conformation of the upper TM/ECL region and a different role for individual residues in the pharmacology of the AM1 and AM2 receptors.

(1) Dong M, et al, (2013) Br J Pharmacol: Jul 25 doi:10.111/bph.12293.

(2) Vohra S, (2012) J R Soc Interface; 10(79):20120846.