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 expressed in CHO cells and characterized with regards to their binding profiles for orphanin FQ/nociceptin (OFQ/N) and a variety of opioids as well as their functional activities in cyclase studies. 125I[Tyr14]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/KOR‐3 chimera is quite good (K D 135 pM), it is slightly lower than the other two. Competition 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 than either of the chimeras (K i values > 350 nM). Conversely, the modest 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 ) chimeras. The remainder of the opioids tested have no appreciable affinity against any of the clones. Functionally, OFQ/N inhibits forskolin‐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/KOR‐3 chimera. Naloxone benzoylhydrazone 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 inactive in cyclase studies using the MOR‐1/KOR‐3 chimera, implying that the replacement of the first coding exon increases affinity while decreasing intrinsic activity.