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.