Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin.
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-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K(i) approximately 0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC(50) approximately 0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa(10),Tyr(14)]noc results in the irreversible labeling of a glycoprotein of approximately 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (approximately 6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of approximately 3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant receptor also yields a single labeled fragment of approximately 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 [Bpa(10)]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile(300) within the experimentally identified photoreactive region.
Moulédous
L
L
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 31077 Toulouse, France.
Topham
C M
CM
Mazarguil
H
H
Meunier
J C
JC
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008024:Ligands; D018847:Opioid Peptides / Q000737:chemistry* / Q000378:metabolism; D019920:Photoaffinity Labels; D011485:Protein Binding; D011957:Receptors, Opioid / Q000737:chemistry* / Q000378:metabolism; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D008024:Ligands; D018847:Opioid Peptides; D019920:Photoaffinity Labels; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor; D000097629:Nociceptin