Characterization of the Two-Domain Peptide Binding Mechanism of the Human CGRP Receptor for CGRP and the Ultrahigh Affinity ssCGRP Variant

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that functions in pain signaling and neuroimmune communication. The CGRP receptor, CGRPR, is a class B GPCR that is a drug target for migraine headache and other disorders. Here, we used nanoBRET receptor binding and cAMP biosensor signaling assays and theoretical modeling to characterize the CGRPR "two-domain" peptide binding mechanism. Single-site extracellular domain (ECD)-binding and two-site ECD/transmembrane domain (TMD)-binding peptides were examined for CGRP and a high-affinity variant "ssCGRP" with modifications in the C-terminal region. Wildtype and ssCGRP(27-37) bound the ECD with affinities of 1 μM and 0.5 nM, and residence times of 5 s and 8 min, respectively. The (8-37) antagonist fragments had affinities of 100 nM for wildtype and 0.5 nM for ss and exhibited behavior consistent with two-site ECD/TMD binding. ssCGRP(8-37) had a residence time of 76 min. CGRP(1-37) agonist had 25-fold higher affinity for the G protein-coupled state of the CGRPR (<i>K_i</i> = 3 nM) than the uncoupled state (<i>K_i</i> = 74 nM), and elicited short-duration cAMP signaling. In contrast, ssCGRP(1-37) had similar strong affinities for both receptor states (<i>K_i</i> = 0.2 to 0.25 nM), and induced long-duration signaling. An equilibrium reaction network mathematical model of CGRPR activation that includes peptide and G protein binding was developed. This captured wildtype CGRP binding experiments well, but the ssCGRP binding properties were not fully reproduced, suggesting that it may exhibit a distinct binding mechanism. Together, these results advance our quantitative understanding of the CGRPR two-domain mechanism and support the ss variants as potential long-acting therapeutics.