Watson M A, Yamada M, Yamada M, Cusack B, Veverka K, Bolden-Watson C, Richelson E
Department of Psychiatry and Psychology, Mayo Foundation, Jacksonville, Florida 32224.
J Neurochem. 1992 Nov;59(5):1967-70. doi: 10.1111/j.1471-4159.1992.tb11035.x.
To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.
为了研究由克隆的大鼠神经降压素受体介导的第二信使合成，我们构建了一个稳定表达该受体的细胞系。将该受体的cDNA克隆亚克隆到pcDNA1neo表达载体中。然后用该构建体转染中国仓鼠卵巢（CHO）-K1细胞。选择对硫酸G-418具有抗性的集落克隆进行分离并单独培养。对19个单独的克隆进行筛选，以检测总的[3H]神经降压素结合情况，以此作为神经降压素受体表达的指标。对显示出最高水平特异性[3H]神经降压素结合的克隆（CHO-rNTR-10）进行了进一步表征。对于完整细胞，特异性[3H]神经降压素结合的平衡解离常数（KD）为18 nM，最大结合位点数（Bmax）为900 fmol/mg蛋白质或740 fmol/10(6)个细胞（细胞表面约4.4×10(5)个位点）。虽然KD与在其他细胞系统中发现的值相似，例如小鼠神经母细胞瘤克隆N1E-115，但Bmax超过了先前报道的值。用神经降压素孵育完整的CHO-rNTR-10细胞会导致肌醇磷酸以剂量依赖性方式释放（EC50 = 3 nM），结果表明表达的转染受体具有功能。神经降压素不会抑制福司可林刺激的环磷酸腺苷水平。与其他系统一样，神经降压素（8-13）比神经降压素更有效。神经降压素介导的肌醇磷酸释放是在转染细胞系中表达的该受体的第二信使合成的首次报道。这些结果表明，在转染细胞系中可以很容易地研究神经降压素受体的结构与功能之间的关系。
