The third extracellular loop of the human calcitonin receptor-like receptor is crucial for the activation of adrenomedullin signalling
Version of Record online: 10 APR 2012
© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society
British Journal of Pharmacology
Special Issue: Themed Section: Secretin Family (Class B) G Protein-Coupled Receptors - from Molecular to Clinical Perspectives. Guest Editors: David R Poyner and Debbie L Hay
Volume 166, Issue 1, pages 137–150, May 2012
How to Cite
Kuwasako, K., Hay, D. L., Nagata, S., Hikosaka, T., Kitamura, K. and Kato, J. (2012), The third extracellular loop of the human calcitonin receptor-like receptor is crucial for the activation of adrenomedullin signalling. British Journal of Pharmacology, 166: 137–150. doi: 10.1111/j.1476-5381.2011.01803.x
- Issue online: 10 APR 2012
- Version of Record online: 10 APR 2012
- Accepted manuscript online: 5 DEC 2011 10:27AM EST
- Received; 11 March 2011; Revised; 25 October 2011; Accepted; 11 November 2011
- adrenomedullin receptor;
- calcitonin receptor-like receptor;
- extracellular loop;
- receptor activation;
- receptor activity-modifying protein
BACKGROUND AND PURPOSE The extracellular loops (ECLs) in Family A GPCRs are important for ligand binding and receptor activation, but little is known about the function of Family B GPCR ECLs, especially ECL3. Calcitonin receptor-like receptor (CLR), a Family B GPCR, functions as a calcitonin gene-related peptide (CGRP) and an adrenomedullin (AM) receptor in association with three receptor activity-modifying proteins (RAMPs). Here, we examined the function of the ECL3 of human CLR within the CGRP and AM receptors.
EXPERIMENTAL APPROACH A CLR ECL3 chimera, in which the ECL3 of CLR was substituted with that of VPAC2 (a Family B GPCR that is unable to interact with RAMPs), and CLR ECL3 point mutants were constructed and transiently transfected into HEK-293 cells along with each RAMP. Cell-surface expression of each receptor complex was then measured by flow cytometry; [125I]-CGRP and [125I]-AM binding and intracellular cAMP accumulation were also measured.
KEY RESULTS Co-expression of the CLR ECL3 chimera with RAMP2 or RAMP3 led to significant reductions in the induction of cAMP signalling by AM, but CGRP signalling was barely affected, despite normal cell-surface expression of the receptors and normal [125I]-AM binding. The chimera had significantly decreased AM, but not CGRP, responses in the presence of RAMP1. Not all CLR ECL3 mutants supported these findings.
CONCLUSIONS AND IMPLICATIONS The human CLR ECL3 is crucial for AM-induced cAMP responses via three CLR/RAMP heterodimers, and activation of these heterodimers probably relies on AM-induced conformational changes. This study provides a clue to the molecular basis of the activation of RAMP-based Family B GPCRs.
LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1