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Dissociation of affinity and efficacy in KOR‐3 chimeras
Author(s) -
Ryan-Moro Jennifer,
Mathis John,
Hom Judy S.H,
Pasternak Gavril W
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)01023-x
Subject(s) - chemistry , dissociation (chemistry) , chimera (genetics) , biophysics , computational biology , biochemistry , biology , organic chemistry , gene
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. 125 I[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/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 IC 50 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.