Abstract

The amino-terminal domain of class B G protein-coupled receptors is critically important for natural peptide agonist binding and action. The precise role it plays and the molecular basis of the interaction between ligand and this domain are not well understood. In the current work, we have developed a new probe for affinity labeling the secretin receptor through a photolabile benzoyl-phenylalanine residue in position 13. This represented a high affinity ligand (K_i = 56 ± 8 nM) that was a potent full agonist to stimulate cellular cAMP (EC₅₀ = 236 ± 22 pM). It covalently labeled the secretin receptor saturably in a single site. This was localized to the amino-terminal domain near the first transmembrane segment using a series of chemical and enzymatic digestions. Edman degradation sequencing of radiolabeled cyanogen bromide and skatole digestion products that were attached to glass beads and further cleaved with endoproteinase Asp-N demonstrated that the labeled residue represented Val¹⁰³. This is in contrast with previous photoaffinity labeling through positions 6, 18, 22, and 26 of secretin that all labeled the distal end of the amino terminus of this receptor. Together, these five pairs of residue-residue approximations provide important constraints to better understand the molecular conformation of the agonist-bound receptor.