Audinot V, Newman-Tancredi A, Millan M J
Department of Psychopharmacology, Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy-sur-Seine, Paris, France.
Neuropharmacology. 2001;40(1):57-64. doi: 10.1016/s0028-3908(00)00104-0.
Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT(1D) receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]-GTPgammaS) binding to CHO-h5-HT(1D) membranes whereas methiothepin and the 5-HT(1B/1D)-selective ligand, SB224,289, exerted robust inhibition of basal [(35)S]-GTPgammaS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT(1B/1D) ligand, S18127. Constitutive activity was investigated by homologous inhibition of [(35)S]-GTPgammaS binding to CHO-h5-HT(1D) membranes with unlabelled GTPgammaS. Under 'basal' conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [(35)S]-GTPgammaS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT(1D) receptor-G-protein coupling. Thus, at 5-HT(1D) receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPgammaS versus [(35)S]-GTPgammaS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT(1D) receptors and, possibly, other GPCRs, inverse agonists will be detectable by [(35)S]-GTPgammaS binding if a HA component is present under basal conditions.
尽管许多G蛋白偶联受体（GPCRs）可能表现出组成性活性，但迄今为止，它们的检测依赖于反向激动剂的使用。本研究采用了一种新方法来研究稳定表达于中国仓鼠卵巢（CHO）细胞中的重组人（h）5-羟色胺（5-HT）5-HT(1D)受体的组成性活性。5-HT适度刺激鸟苷-5'-O-(3-[(35)S]硫代)-三磷酸（[(35)S]-GTPγS）与CHO-h5-HT(1D)细胞膜的结合，而甲硫噻平以及5-HT(1B/1D)选择性配体SB224,289对基础[(35)S]-GTPγS结合具有强烈抑制作用（反向激动作用）。这些作用具有特异性，因为新型选择性5-HT(1B/1D)配体S18127可逆转这些作用。通过用未标记的GTPγS对CHO-h5-HT(1D)细胞膜上[(35)S]-GTPγS结合的同源抑制来研究组成性活性。在“基础”条件下（不存在受体配体），观察到双相等温线。大多数（80%）[(35)S]-GTPγS结合位点处于等温线的高亲和力（HA）与低亲和力（LA）组分中。HA结合被5-HT增强（至155%；相对于基础值 = 100%），但被甲硫噻平降低（至23%）以及被SB224,289降低（至67%）。相反，LA结合未改变。此外，未转染的CHO细胞膜仅表现出LA结合位点，表明后者与h5-HT(1D)受体 - G蛋白偶联无关。因此，在该CHO细胞系中表达的5-HT(1D)受体，基础条件下在同源抑制实验（GTPγS与[(35)S]-GTPγS）中检测到的HA结合提供了组成性G蛋白激活的一种度量。因此，有人提出对于h5-HT(1D)受体以及可能的其他GPCRs，如果基础条件下存在HA组分，反向激动剂将可通过[(35)S]-GTPγS结合检测到。
