title: Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.
abstract: Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.
authors.affiliation: From the School of Biological Sciences, the Maurice Wilkins Centre for Molecular Biodiscovery, and.
authors.forename: Harriet A
authors.initials: HA
authors.lastname: Watkins
authors.affiliation: From the School of Biological Sciences.
authors.forename: Madhuri
authors.initials: M
authors.lastname: Chakravarthy
authors.affiliation: From the School of Biological Sciences.
authors.forename: Rekhati S
authors.initials: RS
authors.lastname: Abhayawardana
authors.affiliation: From the School of Biological Sciences, the Maurice Wilkins Centre for Molecular Biodiscovery, and.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Gingell
authors.affiliation: From the School of Biological Sciences.
authors.forename: Michael
authors.initials: M
authors.lastname: Garelja
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Meenakshi
authors.initials: M
authors.lastname: Pardamwar
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: James M W R
authors.initials: JM
authors.lastname: McElhinney
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Alex
authors.initials: A
authors.lastname: Lathbridge
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Arran
authors.initials: A
authors.lastname: Constantine
authors.affiliation: the Maurice Wilkins Centre for Molecular Biodiscovery, and the School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Paul W R
authors.initials: PW
authors.lastname: Harris
authors.affiliation: the Maurice Wilkins Centre for Molecular Biodiscovery, and the School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Tsz-Ying
authors.initials: TY
authors.lastname: Yuen
authors.affiliation: the Maurice Wilkins Centre for Molecular Biodiscovery, and the School of Chemical Sciences, University of Auckland, Auckland 1010, New Zealand.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Brimble
authors.affiliation: the School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Barwell
authors.affiliation: the School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, United Kingdom.
authors.forename: David R
authors.initials: DR
authors.lastname: Poyner
authors.affiliation: the School of Clinical and Experimental Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Woolley
authors.affiliation: the School of Clinical and Experimental Medicine, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
authors.forename: Alex C
authors.initials: AC
authors.lastname: Conner
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Augen A
authors.initials: AA
authors.lastname: Pioszak
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom, reync@essex.ac.uk.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Reynolds
authors.affiliation: From the School of Biological Sciences, the Maurice Wilkins Centre for Molecular Biodiscovery, and dl.hay@auckland.ac.nz.
authors.forename: Debbie L
authors.identifier: 0000-0002-9558-5122
authors.initials: DL
authors.lastname: Hay
mesh_terms: D053607:Adrenomedullin / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D058286:Calcitonin Receptor-Like Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D058263:Receptor Activity-Modifying Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058264:Receptor Activity-Modifying Protein 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058265:Receptors, Adrenomedullin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058265:Receptors, Adrenomedullin; D053607:Adrenomedullin
keywords: G protein-coupled receptor (GPCR); GPCR; RAMP; adrenomedullin; allosteric regulation; cardiovascular disease; conformational change; extracellular loops; molecular modeling; receptor activity-modifying protein