The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH2 (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; similar to3nM) and is as potent as HP1 in stimulating GTPgammaS 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 similar to 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 Cys123 and Cys200 has been confirmed biochemically. (C) 2003 Elsevier Inc. All rights reserved.