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 (KD 37 pM) and the MOR-1/KOR-3 chimera (KD 39 pM) equally well. Although its affinity for the DOR-1/KOR-3 chimera is quite good (KD 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 (Ki 55 nM) over 6-fold more potently than either of the chimeras. (Ki 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 (Ki 69 nM) and DOR-1/KOR-3 (Ki 74 nM) 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.
The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
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YX
Pan
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Xu
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J
Ryan-Moro
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Mathis
J S
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Hom
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J
Mei
G W
GW
Pasternak
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D005091:Exons; D007700:Kinetics; D051379:Mice; D008961:Models, Structural; D009294:Narcotics / Q000378:metabolism / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D018847:Opioid Peptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D014162:Transfection; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D005576:Colforsin; D000242:Cyclic AMP; D017931:DNA Primers; D009294:Narcotics; D009419:Nerve Tissue Proteins; D018847:Opioid Peptides; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; D000094942:Nociceptin Receptor; D000097629:Nociceptin