DISSOCIATION OF AFFINITY AND EFFICACY IN KOR-3 CHIMERAS

KOR-3 chimeras were constructed in which the first coding exon of KOR- 3 was exchanged for the corresponding first coding exon of either MOR- 1 (MOR-1/KOR-3) or DOR-1 (DOR-1/KOR-3). All three clones were expresse d in CHO cells and characterized with regards to their binding profile s for orphanin FQ/nociceptin (OFQ/N) and a variety of opioids as well as their functional activities in cyclase studies, I-125[Tyr(14)]OFQ/N labels both KOR-3 (K-D 37 pM) and the MOR-1/KOR-3 chimera (K-D 39 pM) equally well, Although its affinity for the DOR-1/KOR3 chimera is qui te good (K-D 135 pM), it is slightly lower than the other two, Competi tion studies confirm the high affinity of OFQ/N for all three clones. However, several competitors clearly distinguish the chimeras from KOR -3, OFQ/N(1-11) competes KOR-3 (K-i 55 nM) over 6-fold more potently t han either of the chimeras (K-i values > 350 nM), Conversely, the mode st affinity of naloxone benzoylhydrazone for KOR-3 (310 nM) is greatly increased in both the MOR-1/KOR-3 (K-i 69 nM) and DOR-1/KOR-3 (K-i 74 nM) chimeras. The remainder of the opioids tested have no appreciable affinity against any of the clones. Functionally, OFQ/N inhibits fors kolin-stimulated cAMP accumulation in both the KOR-3 and the MOR-1/KOR 3 chimera by almost 40%, with IC50 values in the low nanomolar range. Little activity is seen against the DOR-1/KOR3 chimera, Naloxone benzo ylhydrazone inhibits cAMP accumulation in the KOR-3 and the DOR-1/KOR- 3 chimera. Although naloxone benzoylhydrazone has higher affinity for the MOR-1/KOR-3 chimera in binding studies than KOR-3 itself, it is in active in cyclase studies using the MOR-1/KOR-3 chimera, implying that the replacement of the first coding exon increases affinity while dec reasing intrinsic activity.