REPLACEMENT OF GLN(280) BY HIS IN TM6 OF THE HUMAN ORL1 RECEPTOR INCREASES AFFINITY BUT REDUCES INTRINSIC ACTIVITY OF OPIOIDS

The ORL1 (Opioid Receptor-Like) receptor is the G protein-coupled rece ptor whose amino acid sequence is closest to those of opioid receptors , Residues that are conserved in ORL1 and the three types of opioid re ceptor, but also a residue, His in the sixth putative transmembrane (T M6) helix, which is present in all opioid receptor types but absent in ORL1, appear to play a key role in receptor recognition and/or activa tion, Here we have sought to create an opioid binding pocket in the no n-opioid ORL1 receptor by replacing residue Gln(280) in its TM6 by the corresponding His residue of opioid receptors, The mutation affects n either the affinity of nociceptin - the natural ORL1 agonist - for the receptor, nor the potency of nociceptin to inhibit adenylyl cyclase v ia ORL1, In contrast, we find that a few opioid ligands, the agonists lofentanil, etorphine and dynorphin A, and especially the antagonists diprenorphine and nor-BNI, bind the mutant Q280H receptor with substan tially (5- to > 100-fold) higher apparent affinity than they do the wi ld-type receptor, Moreover, lofentanil and etorphine no longer act as pure agonists, as they do at the native ORL1 receptor. but are endowed with clear antagonist properties at the mutant receptor, The mutation Q280H, which increases affinity while decreasing intrinsic activity o f opioids at ORL1, emphasizes the importance of the His residue for op ioid recognition and activation.