Structure-Function Analysis of RAMP1 by Alanine Mutagenesis † John Simms, ‡,§,| Debbie L. Hay, ‡,⊥ Richard J. Bailey, ⊥ Galina Konycheva, ⊥ Graham Bailey, ⊥ Mark Wheatley, # and David R. Poyner* ,§ School of Life and Health Sciences, Aston UniVersity, Birmingham B4 7ET, U.K., Department of Pharmacology, UniVersity of Monash, Clayton 3800, Australia, School of Biological Sciences, UniVersity of Auckland, Auckland, New Zealand, and School of Biosciences, UniVersity of Birmingham, Birmingham B15 2TT, U.K. ReceiVed October 3, 2008; ReVised Manuscript ReceiVed NoVember 19, 2008 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. Receptor activity modifying proteins (RAMPs) 1 are a family of three single-pass transmembrane proteins that associate with certain G-protein coupled receptors (GPCR) (1). Their best studied actions are with the calcitonin receptor-like receptor (CLR). By itself this is only poorly expressed at the cell surface and interacts with no known ligand. However, when coexpressed with RAMP1, it forms a receptor for calcitonin gene-related peptide (CGRP), the CGRP receptor. In combination with RAMP2 or RAMP3 it forms two distinct receptors for adrenomedullin (AM), the AM 1 and AM 2 receptors (2). The three RAMPs can also interact with the calcitonin receptor, in each case producing a receptor with high affinity for the peptide amylin (2, 3). Interactions with other family B GPCRs have also been described, as well as with the calcium sensing receptor, a family C GPCR (4-6). Each of the three RAMPs has an N-terminal extracellular domain of around 100 amino acids. By contrast, the intracellular domain for each is only around 10 residues long. Thus, it is predicted that the extracellular domains of these proteins are crucial for their functions. This is supported experimentally in that chimeras have clearly established the significance of the N-termini of the RAMPs for ligand binding (7-9). The RAMPs presumably modify the confor- mation of the N-terminus of the GPCR and potentially influence ligand binding at this site (10). There are a number of conserved cysteines that are involved in disulfide bonds (11, 12). However, there is little information on the role of individual amino acids, and little work has been done to understand the detailed structure of RAMPs or to relate this to their pharmacological properties. A series of deletions and point mutations indicated that residues 91-103 contain amino acids which are required for high-affinity CGRP binding (13). It has also been shown that Trp-74 is required for binding of the nonpeptide antagonist BIBN4096BS to CGRP and amylin receptors (14-16); its equivalent in RAMP3 may be a contact point for adrenomedullin (15). To help to understand the molecular basis for RAMP modulation of GPCR biology, we produced an ab initio model of the extracellular domain of human RAMP1 that predicted that it consisted of three helices (17). The major † This work was supported by grants from the Biotechnology and Biological Sciences Research Council of the U.K. (C20090) to D.R.P. and M.W. D.L.H. was supported by the Auckland Medical Research Foundation, Maurice and Phyllis Paykel Trust, Health Research Council, and the New Zealand Lottery Health Fund. * To whom correspondence should be addressed. Tel: +44 (0)121 359 3611. Fax: +44 (0)121 359 5142. E-mail: D.R.Poyner@aston.ac.uk. ‡ J.S. and D.L.H. were equal contributors to this study. § Aston University. | University of Monash. ⊥ University of Auckland. # University of Birmingham. 1 Abbreviations: AM, adrenomedullin; BIBN4096BS, N-[2-[[5- amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5- dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo- 3(2H)-quinazolinyl)-1-piperidinecarboxamide; CGRP, calcitonin gene- related peptide; CLR, calcitonin receptor-like receptor; E max , maximum response; GPCR, G-protein coupled receptor; HA, hemagluttin; pEC 50 , -log(EC 50 ); RAMP, receptor activity modifying protein; TBS, Tris- buffered saline; WT, wild type. Biochemistry 2009, 48, 198–205 198 10.1021/bi801869n CCC: $40.75  2009 American Chemical Society Published on Web 12/12/2008