pubs.acs.org/Biochemistry Published on Web 01/25/2010 r 2010 American Chemical Society 1862 Biochemistry 2010, 49, 1862–1872 DOI: 10.1021/bi901848m Refolding and Characterization of a Soluble Ectodomain Complex of the Calcitonin Gene-Related Peptide Receptor Christopher M. Koth, ‡, ) Norzehan Abdul-Manan, ‡ Christopher A. Lepre, ‡ Peter J. Connolly, ‡ Sanghee Yoo, § Arun K. Mohanty, ‡ Judith A. Lippke, ‡ Jacque Zwahlen, ‡ Joyce T. Coll, ‡ John D. Doran, ‡ Miguel Garcia-Guzman, § and Jonathan M. Moore* ,‡ ‡ Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, Massachusetts 02139, and § Vertex Pharmaceuticals San Diego LLC, 11010 Torreyana Road, San Diego, California 92121. ) Present address: Genentech, Inc., 1 DNA Way, MS 27, South San Francisco, CA 94080. Received October 28, 2009; Revised Manuscript Received January 22, 2010 ABSTRACT: The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC 50 of 12 μM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K D < 0.02 μM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 μM) was observed. The 37-amino acid calcitonin gene-related peptide (CGRP) 1 is the most potent endogenous vasodilator known (1). In the brain, it is released from cell bodies in the trigeminal ganglia, where it functions locally with its receptors to cause dilation of cerebral blood vessels. Enhanced release of CGRP exacerbates cranial vasodilation and is thought to be a primary determinant in the pathogenesis of migraine headaches (2, 3). Several key observa- tions support the role of CGRP in migraine. For example, early studies detected CGRP at elevated levels in jugular venous blood, but not in peripheral blood (4), and intravenous administration of CGRP rapidly induced headache and other migraine-like disorders in migraine-susceptible patients (5). The strongest evidence, however, comes from recent clinical trials in which the potent and selective CGRP receptor antagonists olcegepant (BIBN4096BS) (6) and telcagepant (MK-0974) (7) were effica- cious in treatment of acute migraine. The receptor for CGRP is a heterodimer of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a member of the class B (type II) G-protein-coupled receptor (GPCR) family that includes recep- tors for parathyroid hormone, glucagon, secretin, gastric inhibi- tory peptide, and corticotropin releasing factor. RAMPs are accessory membrane proteins required for the proper sub- cellular localization, function, and selectivity of certain class B GPCRs (8). When expressed alone, CLR is not known to bind any ligand (9). However, when coexpressed with RAMP1, it functions as a cell surface receptor for CGRP and has moderate affinity for adrenomedullin (8). CLR is also capable of hetero- dimerizing with two other RAMP proteins (RAMP2 and RAMP3), which generates receptors with selectivity for adreno- medullin (reviewed in ref 10). Several classes of CGRP receptor antagonists have been identified. The first peptide antagonists were simply N-terminal truncations of the CGRP peptide (11-14). Later, more potent peptide antagonists included non-natural C-terminal fragments of CGRP (15, 16). The prototype nonpeptide antagonist, olce- gepant (Figure 1, compound 1), was discovered through cellular- based screens and subsequent chemical optimization. Olcegepant is a selective antagonist of the human CGRP receptor (K i = 0.014 nM) (17). Although the compound displays poor oral bioavailability, early clinical trials provided proof of concept linking CGRP receptor antagonism with successful treatment of migraine (18). More recently, the first orally bioavailable CGRP receptor antagonist, telcagepant (also known as MK-0974), has shown efficacy in migraine treatment (7). *To whom correspondence should be addressed: Vertex Pharmaceu- ticals Inc., 130 Waverly St., Cambridge, MA 02139. E-mail: Jonathan_Moore@vrtx.com. Phone: (617) 444-6444. Fax: (617) 444- 6688. 1 Abbreviations: BSA, bovine serum albumin; CGRP, calcitonin gene- related peptide; CRFR2, corticotropin releasing factor type 2; CLR, calcitonin receptor-like receptor; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; ECD, extracellular domain; EDTA, ethylenediaminete- traacetic acid; GPCR, G-protein-coupled receptor; HEPES, 4-(2-hy- droxyethyl)-1-piperazineethanesulfonic acid; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement; PEI, polyethyle- neimine; P20, polyoxyethylene-20-sorbitan monolaurate; RAMP, re- ceptor activity-modifying protein; SPR, surface plasmon resonance; TRIS, tris(hydroxymethyl)aminomethane; TROSY, transverse relaxa- tion-optimized spectroscopy.