Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa10,Tyr14]Nociceptin

Abstract

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-L-Phe10,Tyr14]nociceptin ([Bpa10,Tyr14]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa10,Tyr14]noc binds the receptor with high affinity (Ki ∼0.7 nM) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC50 ∼0.5 nM). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa10,Tyr14]noc results in the irreversible labeling of a glycoprotein of ∼65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (∼6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of ∼3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu295 → Asp mutant receptor also yields a single labeled fragment of ∼6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296-302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa10]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile300 within the experimentally identified photoreactive region.