Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin

Citation
L. Mouledous et al., Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin, J BIOL CHEM, 275(38), 2000, pp. 29268-29274
Citations number
44
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
275
Issue
38
Year of publication
2000
Pages
29268 - 29274
Database
ISI
SICI code
0021-9258(20000922)275:38<29268:DIOAPB>2.0.ZU;2-L
Abstract
The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogeno us agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor, An affinity labeling approach has been implemented to probe the interactio ns of the neuropeptide with the receptor using the photolabile nociceptin d erivative, [p-benzoyl-L-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 recept or, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K-i similar to 0.7 nM) and is as potent as nociceptin in the inhibition of forskolin-i nduced cAMP synthesis (EC50 similar to 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 similar to 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete dige stion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (similar to 6.5 kDa) that is readily cleaved by end oproteinase Glu-C to yield a labeled fragment of similar to 3.2 kDa, Kallik rein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant re ceptor also yields a single labeled fragment of similar to 6.5 kDa but is r esistant to further cleavage by endoproteinase Glu-C. Based upon the expect ed proteolytic fingerprint of the labeled receptor, the photoreactive regio n 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 tran smembrane 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 photoreact ive region.