Pan Y X, Xu J, Ryan-Moro J, Mathis J, Hom J S, Mei J, Pasternak G W
The Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
FEBS Lett. 1996 Oct 21;395(2-3):207-10. doi: 10.1016/0014-5793(96)01023-x.
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.
构建了KOR-3嵌合体，其中KOR-3的第一个编码外显子被MOR-1（MOR-1/KOR-3）或DOR-1（DOR-1/KOR-3）的相应第一个编码外显子所替换。所有这三个克隆都在CHO细胞中表达，并针对它们对孤啡肽FQ/痛敏肽（OFQ/N）和多种阿片类药物的结合谱及其在环化酶研究中的功能活性进行了表征。125I[酪氨酸14]OFQ/N对KOR-3（解离常数KD为37 pM）和MOR-1/KOR-3嵌合体（KD为39 pM）的标记效果同样良好。尽管它对DOR-1/KOR-3嵌合体的亲和力相当不错（KD为135 pM），但略低于其他两者。竞争研究证实了OFQ/N对所有这三个克隆具有高亲和力。然而，几种竞争剂明显区分了这些嵌合体与KOR-3。OFQ/N(1-11)对KOR-3（抑制常数Ki为55 nM）的竞争能力比任何一种嵌合体都强6倍以上。（Ki值>350 nM）。相反，纳洛酮苯甲酰腙对KOR-3的适度亲和力（310 nM）在MOR-1/KOR-3（Ki为69 nM）和DOR-1/KOR-3（Ki为74 nM）嵌合体中都大大增加。所测试的其余阿片类药物对任何一个克隆都没有明显的亲和力。在功能上，OFQ/N在KOR-3和MOR-1/KOR-3嵌合体中均可使福斯可林刺激的环磷酸腺苷（cAMP）积累抑制近40%，IC50值处于低纳摩尔范围。对DOR-1/KOR-3嵌合体几乎没有活性。纳洛酮苯甲酰腙在KOR-3和DOR-1/KOR-3嵌合体中可抑制cAMP积累。尽管在结合研究中纳洛酮苯甲酰腙对MOR-1/KOR-3嵌合体的亲和力高于KOR-3本身，但在使用MOR-1/KOR-3嵌合体的环化酶研究中它没有活性，这意味着第一个编码外显子的替换增加了亲和力但降低了内在活性。
