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
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.