Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). It remains unclear how CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor. In the current study, a photoaffinity cross linker, p-azido L-phenylalanine (azF) was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimised to ensure efficient photolysis of azF residues near to the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of uv-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the 5th transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain (ECD) boundary of CLR. Reduced potency, Emax and affinity of [Leu16Ala]-human alpha CGRP is consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.
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