Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH 2

Abstract

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH 2 (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [ p-benzoyl- l-Phe (Bpa) 2]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa 2]-HP1 binds the receptor with high affinity (K; ∼3 nM) and is as potent as HP1 in stimulating GTPγS binding (50–60% of nociceptin maximal effect). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa 2]-HP1 results in the irreversible labelling of a glycoprotein of M r∼66 kDa, as determined by SDS–PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107–113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.