title: A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors.
abstract: 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 alpha-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 pEC(50) 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 VPAC(1) receptor.
authors.affiliation: School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Alex C
authors.initials: AC
authors.lastname: Conner
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Hay
authors.forename: John
authors.initials: J
authors.lastname: Simms
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Howitt
authors.forename: Marcus
authors.initials: M
authors.lastname: Schindler
authors.forename: David M
authors.initials: DM
authors.lastname: Smith
authors.forename: Mark
authors.initials: M
authors.lastname: Wheatley
authors.forename: David R
authors.initials: DR
authors.lastname: Poyner
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D058286:Calcitonin Receptor-Like Protein; D002522:Chlorocebus aethiops; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline; D018003:Receptors, Calcitonin / Q000819:agonists* / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015183:Restriction Mapping; D015398:Signal Transduction; D014162:Transfection
chemical_list: C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D017931:DNA Primers; D018003:Receptors, Calcitonin; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D011392:Proline; D015740:Calcitonin Gene-Related Peptide