title: Mapping the CGRP receptor ligand binding domain: tryptophan-84 of RAMP1 is critical for agonist and antagonist binding.
abstract: The calcitonin receptor-like receptor (CLR) associates with the accessory protein RAMP1 to form a receptor for the neuropeptide calcitonin gene-related peptide (CGRP). Multiple lines of evidence have implicated CGRP in the pathophysiology of migraine headache making the CGRP receptor an attractive target for development of small-molecule antagonists as a novel treatment for this debilitating condition. The CGRP receptor antagonists telcagepant and olcegepant (BIBN4096BS) have demonstrated clinical efficacy in the treatment of migraine and there is now a need to better understand how these molecules interact with the receptor. Previous work has shown the extracellular portion of RAMP1 to be important for binding of these antagonists, with tryptophan-74 being a key interaction site. The crystal structure of the extracellular portion of human RAMP1 placed tryptophan-74 in a hydrophobic patch hypothesized to interact with CGRP receptor ligands and also identified nearby residues that may be important for ligand binding. In this study we explored the role played by these residues of RAMP1 using an alanine replacement strategy. We confirmed a role for tryptophan-74 in antagonist binding and also identified arginine-67 as being important for binding of telcagepant but not compound 3, a close analog of BIBN4096BS. We also identified tryptophan-84 as being critical for both high-affinity binding of the non-peptide antagonists as well as the peptides CGRP and CGRP(8-37). These data for the first time pinpoint a specific RAMP1 residue important for both antagonist and agonist potency and are consistent with the N-terminal domain of RAMP1 forming the binding pocket interface with CLR.
authors.affiliation: Merck Research Laboratories, Department of Pain & Migraine Research, West Point, PA, USA. eric_moore2@merck.com
authors.forename: E L
authors.initials: EL
authors.lastname: Moore
authors.forename: J J
authors.initials: JJ
authors.lastname: Gingell
authors.forename: S A
authors.initials: SA
authors.lastname: Kane
authors.forename: D L
authors.initials: DL
authors.lastname: Hay
authors.forename: C A
authors.initials: CA
authors.lastname: Salvatore
mesh_terms: D001381:Azepines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D058286:Calcitonin Receptor-Like Protein; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008881:Migraine Disorders / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011799:Quinazolines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000819:agonists / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
chemical_list: D001381:Azepines; C545578:CALCRL protein, human; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D058286:Calcitonin Receptor-Like Protein; D004151:Dipeptides; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D010879:Piperazines; D011799:Quinazolines; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D014364:Tryptophan; C525458:telcagepant; C406305:olcegepant