title: Structure-function analysis of RAMP1 by alanine mutagenesis.
abstract: Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.
authors.affiliation: School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: John
authors.initials: J
authors.lastname: Simms
authors.forename: Debbie L
authors.initials: DL
authors.lastname: Hay
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bailey
authors.forename: Galina
authors.initials: G
authors.lastname: Konycheva
authors.forename: Graham
authors.initials: G
authors.lastname: Bailey
authors.forename: Mark
authors.initials: M
authors.lastname: Wheatley
authors.forename: David R
authors.initials: DR
authors.lastname: Poyner
mesh_terms: D053607:Adrenomedullin / Q000494:pharmacology; D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021381:Protein Transport; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D053607:Adrenomedullin; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide; D000409:Alanine