Gardner B R, Hall D A, Strange P G
Department of Biosciences, The University, Canterbury, England, U.K.
J Neurochem. 1997 Dec;69(6):2589-98. doi: 10.1046/j.1471-4159.1997.69062589.x.
Mechanisms of agonist action at the G protein-coupled D2(short) dopamine receptor expressed in Chinese hamster ovary cells have been investigated. Agonist binding was assayed in the presence and absence of GTP (100 microM). Data in the absence of GTP were fitted best by a two-site model (apomorphine, dopamine, 10,11-dihydroxy-N-n-propylnorapomorphine hydrochloride, and quinpirole) or a one-site model [bromocriptine, dihydroergocristine, and (-)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride], whereas in the presence of GTP a one-site model was the best fit for all compounds. Agonist binding parameters were used to provide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding for a range of agonist concentrations was measured and the EC50 and maximal effects determined. The initial rates of [35S]GTPgammaS binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist-stimulated [35S]GTPgammaS binding and receptor occupancy by spiperone was determined. Agonist inhibition of forskolin-stimulated cyclic AMP accumulation was determined for a range of agonist concentrations and the EC50 and maximal inhibition recorded. The data on the maximal agonist responses showed that it was possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencies of agonists to elicit the functional responses showed that different extents of amplification of response were seen for different agonists in both assays. The maximal activity data have been compared with the stabilisation of the agonist/receptor/G protein ternary complex as measured in binding assays.
已对在中国仓鼠卵巢细胞中表达的G蛋白偶联D2（短）多巴胺受体的激动剂作用机制进行了研究。在存在和不存在GTP（100 microM）的情况下测定激动剂结合。在不存在GTP的情况下，数据最适合用双位点模型（阿扑吗啡、多巴胺、10,11-二羟基-N-正丙基去甲阿扑吗啡盐酸盐和喹吡罗）或一位点模型[溴隐亭、二氢麦角隐亭和（-）-3-（3-羟苯基）-N-丙基哌啶盐酸盐]拟合，而在存在GTP的情况下，一位点模型最适合所有化合物。激动剂结合参数用于衡量激动剂稳定激动剂/受体/G蛋白三元复合物的能力。测量了一系列激动剂浓度下激动剂对[35S]鸟苷5'-O-（3-硫代三磷酸）（[35S]GTPγS）结合的刺激作用，并确定了EC50和最大效应。还记录了最大刺激激动剂浓度诱导的[35S]GTPγS结合的初始速率。测定了舒必利对激动剂刺激的[35S]GTPγS结合和受体占据的同时抑制作用。测定了一系列激动剂浓度下激动剂对福斯高林刺激的环磷酸腺苷积累的抑制作用，并记录了EC50和最大抑制作用。关于最大激动剂反应的数据表明，在两种功能测定中都可以检测到激动剂效力的一系列情况（部分激动和完全激动）。关于激动剂引发功能反应的表观效力的数据表明，在两种测定中，不同激动剂的反应放大程度不同。已将最大活性数据与结合测定中测量的激动剂/受体/G蛋白三元复合物的稳定性进行了比较。
