Pan Y X, Cheng J, Xu J, Rossi G, Jacobson E, Ryan-Moro J, Brooks A I, Dean G E, Standifer K M, Pasternak G W
Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Mol Pharmacol. 1995 Jun;47(6):1180-8.
We have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned mu, delta, and kappa 1 receptors. Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to mu, delta, and kappa 1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation in COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native kappa 3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region. Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of kappa 3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The mu, delta, and kappa 1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block kappa 3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. In conclusion, the antibody and antisense studies strongly associate KOR-3 with the kappa 3-opioid receptor, although it is not clear whether it is the kappa 3 receptor itself or a splice variant.
我们已从小鼠大脑中鉴定出一种假定的阿片受体（KOR-3），它属于G蛋白偶联受体家族，与先前克隆的μ、δ和κ1受体不同。该克隆属于阿片受体家族是基于结构和功能研究得出的。其预测的氨基酸序列与其他阿片受体高度同源，尤其是在许多跨膜区域，其中有很长一段与μ、δ和κ1受体相同。环唑辛和烯丙吗啡均可抑制稳定表达该克隆的COS-7细胞中cAMP的积累。Northern分析表明，该mRNA存在于大脑中，但在许多其他器官中不存在。Southern分析提示存在一个编码该受体的单一基因。一种针对天然κ3受体的高选择性单克隆抗体在Western分析中可识别在COS-7细胞中表达的该克隆。体外翻译产物也可被该抗体标记。其他克隆显示存在多个内含子，包括一个在第二个细胞外环中，另一个在第一个跨膜区域中。用针对第二个细胞外环区域的寡脱氧核苷酸进行的反义研究显示，在体内可选择性阻断κ3镇痛作用，而基于反义序列的错配寡脱氧核苷酸则未观察到这种作用。μ、δ和κ1镇痛作用不受这种反义处理的影响。对第一个跨膜区域剪接位点下游的克隆进行反义定位显示，六种不同的反义寡脱氧核苷酸均能阻断κ3镇痛作用。相比之下，针对该剪接位点上游区域的另外六种不同反义寡脱氧核苷酸中只有一种有效。这两个区域之间的明显界限增加了该受体存在剪接变体的可能性。另一个克隆显示在3'非翻译区有一个插入片段。总之，抗体和反义研究有力地表明KOR-3与κ3-阿片受体相关，尽管尚不清楚它是κ3受体本身还是一个剪接变体。
