title: An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology.
abstract: G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein-protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class.
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.
authors.forename: Joseph
authors.initials: J
authors.lastname: J Gingell
authors.affiliation: School of Life and Health Sciences, Aston University , Birmingham, UK.
authors.forename: John
authors.initials: J
authors.lastname: Simms
authors.affiliation: School of Life and Health Sciences, Aston University , Birmingham, UK.
authors.forename: James
authors.initials: J
authors.lastname: Barwell
authors.affiliation: School of Life and Health Sciences, Aston University , Birmingham, UK.
authors.forename: David R
authors.initials: DR
authors.lastname: Poyner
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.
authors.forename: Harriet A
authors.initials: HA
authors.lastname: Watkins
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center , Oklahoma City, OK, USA.
authors.forename: Augen A
authors.initials: AA
authors.lastname: Pioszak
authors.affiliation: Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University , Parkville, VIC, Australia.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Hay
keywords: Amylin; CGRP; G protein-coupled receptor; RAMP; accessory protein