Abstract
Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this 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 optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-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 fifth 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 boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.
Highlights
Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1)
A crystal structure is available showing the C-terminal tail of a CGRP analogue bound to the extracellular domain (ECD) of CLR and RAMP1.10 Extensive mutagenesis data that have allowed the construction of models of how CGRP interacts with the TM domain of the receptor are available.[11−15] These studies have implicated extracellular loop 2 (ECL2) of CLR as being especially important for CGRP binding.[12−14] direct evidence of molecular contacts between the ligand and receptor is lacking
We have used unnatural amino acid mutagenesis to map key residues in and adjacent to ECL2 of CLR that contribute to the binding of CGRP
Summary
Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). With the use of genetic code expansion and unnatural amino acid mutagenesis,[17] it is possible to incorporate the photoaffinity cross-linker into the receptor, thereby eliminating the need for peptide mapping or sequencing to identify contact points on the receptor This general approach, known as targeted photo-cross-linking,[18] has been used to map ligand−receptor interactions in a number of GPCRs, including the neurokinin NK-1 receptor,[19] ghrelin receptor,[20] type 1 receptor for the corticotropin releasing factor (CRF1R),[21−23] and GLP-1 receptor.[24] This has not yet been extensively applied to a complex dimeric receptor, such as the CGRP receptor. In the study presented here, we have developed a modified targeted photo-cross-linking strategy to study ECL2 of CLR and have identified two major contact points for CGRP: I284 and L291
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