The activation of G proteins by type 1alpha metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1alpha receptor has been studied by use of a [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding assay. 2. L-Glutamate increased the rate of [35S]-GTPgammaS binding in a concentration-dependent manner (-logEC50 (M) 5.25 +/- 0.07), with an optimal (62.4 +/- 1.6%) increase over basal binding being observed following 60 min incubation at 30 degrees C with 70 pM [35S]-GTPgammaS, 1 microM GDP, 10 mM MgCl2, 100 mM NaCl and 100 microg membrane protein ml(-1). The L-glutamate (100 microM)-stimulated increase in [35S]-GTPgammaS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 microM). 3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K(D)) for [35S]-GTPgammaS binding of 0.76 +/- 0.20 nM and a maximal number of GTPgammaS-liganded G proteins (Bmax) of 361 +/- 30 fmol mg(-1) protein. 4. Metabotropic glutamate receptor agonists, quisqualate (-logEC50 (M) 6.74 +/- 0.06), 1S,3R-ACPD (4.64 +/- 0.08) and (S)-3,5-dihydroxyphenylglycine (5.16 +/- 0.23) also increased [35S]-GTPgammaS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5. (+)-alpha-Methylcarboxyphenylglycine (300 microM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [35S]-GTPgammaS binding, allowing an antagonist equilibrium dissociation constant (K(D)) of 34.0 +/- 7.8 microM to be calculated for this mGluR antagonist. 6. Pretreatment of BHK-mGluR1alpha cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml(-1), 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [35S]-GTPgammaS binding (by 66.0 +/- 0.9%), and an altered concentration-effect relationship for agonist-stimulated [35S]-GTPgammaS binding by the residual PTX-insensitive G-protein population. 7. The modulation of [35S]-GTPgammaS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.
摘要
利用[35S]-鸟苷5'-[γ-硫代]三磷酸（[35S]-GTPγS）结合试验，研究了表达克隆大鼠mGluR1α受体的重组幼仓鼠肾细胞膜中1α型代谢型谷氨酸受体（mGluRs）对G蛋白的激活作用。2. L-谷氨酸以浓度依赖方式增加[35S]-GTPγS结合速率（-logEC50（M）5.25±0.07），在30℃下用70 pM [35S]-GTPγS、1 μM GDP、10 mM MgCl2、100 mM NaCl和100 μg膜蛋白ml-1孵育60分钟后，与基础结合相比，观察到最佳增加（62.4±1.6%）。在I组mGluR拮抗剂（S）-4-羧基-3-羟基苯甘氨酸（300 μM）存在下，L-谷氨酸（100 μM）刺激的[35S]-GTPγS结合增加完全被阻断。3. 对最大有效浓度L-谷氨酸激活的G蛋白的亲和力和数量进行定量分析，得出[35S]-GTPγS结合的平衡解离常数（K（D））为0.76±0.20 nM，GTPγS结合的G蛋白最大数量（Bmax）为361±30 fmol mg-1蛋白。4. 代谢型谷氨酸受体激动剂喹啉酸（-logEC50（M）6.74±0.06）、1S,3R-ACPD（4.64±0.08）和（S）-3,5-二羟基苯甘氨酸（5.16±0.23）也以浓度依赖方式增加[35S]-GTPγS结合，后两种试剂表现为部分激动剂。5. （+）-α-甲基羧基苯甘氨酸（300 μM）使[35S]-GTPγS结合的L-谷氨酸浓度-效应曲线平行右移，从而计算出该mGluR拮抗剂的拮抗剂平衡解离常数（K（D））为34.0±7.8 μM。6. 在制备膜之前，用显示为最大有效浓度（100 ng ml-1，24小时）的百日咳毒素（PTX）预处理BHK-mGluR1α细胞，导致激动剂刺激的[35S]-GTPγS结合显著降低（降低66.0±0.9%），并且残余的对PTX不敏感的G蛋白群体对激动剂刺激的[35S]-GTPγS结合的浓度-效应关系发生改变。7. 激动剂和拮抗剂对重组细胞膜中[35S]-GTPγS结合的调节提供了一个极好的系统，用于研究mGluR与对PTX敏感和不敏感的G蛋白的相互作用。
