Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2.
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; approximately 3nM) and is as potent as HP1 in stimulating GTPgammaS binding (50-60% of nociceptin maximal effect). UV irradiation at 365nm 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) approximately 66kDa, 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.
Bes
Brice
B
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France.
Meunier
Jean-Claude
JC
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002918:Chymotrypsin / Q000494:pharmacology; D006224:Cricetinae; D003488:Cyanogen Bromide / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008027:Light; D008666:Metalloendopeptidases; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D019920:Photoaffinity Labels / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011957:Receptors, Opioid / Q000737:chemistry*; D014466:Ultraviolet Rays; D000094942:Nociceptin Receptor
D004220:Disulfides; D006023:Glycoproteins; D010455:Peptides; D019920:Photoaffinity Labels; D043562:Receptors, G-Protein-Coupled; D011957:Receptors, Opioid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010450:Endopeptidases; D002918:Chymotrypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D003488:Cyanogen Bromide; D000094942:Nociceptin Receptor