QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Conner, A. C. Articles by Poyner, D. R. Search for Related Content PubMed PubMed Citation Articles by Conner, A. C. Articles by Poyner, D. R.

A Key Role for Transmembrane Prolines in Calcitonin Receptor-Like Receptor Agonist Binding and Signalling: Implications for Family B G-Protein-Coupled Receptors

School of Life and Health Sciences, Aston University, Birmingham, United Kingdom (A.C.C., D.L.H., S.G.H.); Department of Metabolic Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom (D.L.H.); School of Biosciences, University of Birmingham, Birmingham, United Kingdom (J.S., M.W.); Metabolic Research, Boehringer Ingelheim Pharma KG, Biberach, Germany (M.S.); and AstraZeneca, CVGI, Macclesfield, Cheshire, United Kingdom (D.M.S.)

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in -helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241, Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC₅₀ for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP_8–37 and 1-piperidinecarboxamide, N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P, predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC₁ receptor.

Address correspondence to: David Poyner, School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK. E-mail: d.r.poyner{at}aston.ac.uk

This article has been cited by other articles:

				S. R. Hawtin, J. Simms, M. Conner, Z. Lawson, R. A. Parslow, J. Trim, A. Sheppard, and M. Wheatley Charged Extracellular Residues, Conserved throughout a G-protein-coupled Receptor Family, Are Required for Ligand Binding, Receptor Activation, and Cell-surface Expression J. Biol. Chem., December 15, 2006; 281(50): 38478 - 38488. [Abstract] [Full Text] [PDF]

				A. C. Conner, J. Simms, S. G. Howitt, M. Wheatley, and D. R. Poyner The Second Intracellular Loop of the Calcitonin Gene-related Peptide Receptor Provides Molecular Determinants for Signal Transduction and Cell Surface Expression J. Biol. Chem., January 20, 2006; 281(3): 1644 - 1651. [Abstract] [Full Text] [PDF]