Aharony D, Little J, Thomas C, Powell S, Berry D, Graham A
Department of Pharmacology, ZENECA Inc., Wilmington, Delaware 19897.
Mol Pharmacol. 1994 Jan;45(1):9-19.
Functional cDNA clones for hamster neurokinin-2 receptor (NK-2R) were isolated from hamster urinary bladder using a polymerase chain reaction-based methodology. The hamster NK-2R consists of 384 amino acids with a relative molecular weight of 43,418. Hamster NK-2R shares significant amino acid sequence homology with other tachykinin receptors, particularly with rat, bovine, and human NK-2R (94.3, 84.4, and 86.5%, respectively). To examine the pharmacology of cloned hamster NK-2R, we transfected mouse erythroleukemia cells with this receptor, prepared high speed membranes, and studied the receptor properties utilizing the ligand [4,5-3H-Leu9]NKA in a receptor-binding assay. For pharmacological comparison, we also transfected the human NK-2R into mouse erythroleukemia cells. [3H]NKA bound to hamster NK-2R receptor in a protein-dependent, high affinity (Kd1 = 4.14 +/- 0.31 nM), saturable (Bmax1 = 679 +/- 26 fmol/mg of protein), and highly specific manner (89 +/- 2%). A smaller population (10% density) of lower affinity receptors (Kd2 = 150 +/- 92 nM), was also observed in competition experiments. [3H]NKA bound to the human receptor with significantly higher affinity and overall greater receptor density (Kd1 = 0.37 +/- 0.11 nM, Bmax1 = 234 +/- 175 fmol/mg of protein; Kd2 = 9.0 +/- 2 nM, Bmax2 = 1989 + 990 fmol/mg of protein). [3H]NKA binding to both hamster and human receptors was enhanced greatly by divalent cations, whereas GTP analogs weakly inhibited binding to hamster receptor, but potently inhibited binding to the human receptor. Competition experiments with agonists demonstrated binding to high and low affinity states of NK-2 receptors, with identical order of potency in hamster or human NK-2R; NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) >> substance P >>> Senktide. However, remarkable differences were observed in studies with selective NK-2 antagonists (hamster, SR48,968 > L659,877 > R396 >> MEN10,376 versus human, SR48,968 > MEN10,376 > L659,877 > R396). The rank order of antagonist affinity is consistent with the observations of NK-2 receptor pharmacology in the native tissues.
采用基于聚合酶链反应的方法，从仓鼠膀胱中分离出仓鼠神经激肽 -2 受体（NK -2R）的功能性 cDNA 克隆。仓鼠 NK -2R 由 384 个氨基酸组成，相对分子质量为 43418。仓鼠 NK -2R 与其他速激肽受体具有显著的氨基酸序列同源性，特别是与大鼠、牛和人类的 NK -2R（分别为 94.3%、84.4%和 86.5%）。为了研究克隆的仓鼠 NK -2R 的药理学特性，我们将该受体转染到小鼠红白血病细胞中，制备高速膜，并在受体结合试验中利用配体[4,5 - 3H - Leu9]NKA 研究受体特性。为了进行药理学比较，我们还将人类 NK -2R 转染到小鼠红白血病细胞中。[3H]NKA 以蛋白质依赖性、高亲和力（Kd1 = 4.14 ± 0.31 nM）、可饱和（Bmax1 = 679 ± 26 fmol/mg 蛋白质）和高度特异性的方式与仓鼠 NK -2R 受体结合（89 ± 2%）。在竞争实验中还观察到一小部分（10%密度）低亲和力受体（Kd2 = 150 ± 92 nM）。[3H]NKA 与人类受体结合的亲和力显著更高，总体受体密度也更大（Kd1 = 0.37 ± 0.11 nM，Bmax1 = 234 ± 175 fmol/mg 蛋白质；Kd2 = 9.0 ± 2 nM，Bmax2 = 1989 + 990 fmol/mg 蛋白质）。二价阳离子极大地增强了[3H]NKA 与仓鼠和人类受体的结合，而 GTP 类似物对仓鼠受体的结合抑制作用较弱，但对人类受体的结合有强效抑制作用。激动剂的竞争实验表明与 NK -2 受体的高亲和力和低亲和力状态均有结合，在仓鼠或人类 NK -2R 中效力顺序相同；NKA > [Nle10]NKA(4 - 10) > [β - Ala8]NKA(4 - 10) >> P 物质 >>> 速激肽。然而，在选择性 NK -2 拮抗剂的研究中观察到显著差异（仓鼠，SR48,968 > L659,877 > R396 >> MEN10,376 与人类，SR48,968 > MEN10,376 > L659,877 > R396）。拮抗剂亲和力的顺序与天然组织中 NK -2 受体药理学的观察结果一致。
