Newman-Tancredi A, Audinot V, Moreira C, Verrièle L, Millan M J
Department of Psychopharmacology, Institut de Recherches Servier, Croissy-sur-Seine (Paris), France.
Mol Pharmacol. 2000 Nov;58(5):1042-9. doi: 10.1124/mol.58.5.1042.
This study evaluated the influence of receptor/G-protein (R:G) stoichiometry on constitutive activity and the efficacy of agonists, partial agonists, and inverse agonists at human (h) 5-hydroxytryphamine 1B (5-HT(1B)) receptors. Two Chinese hamster ovary cell lines were used; they expressed 8.5 versus 0.4 pmol h5-HT(1B) receptors/mg (determined by [(3)H]GR125,743 saturation analysis) and 3.0 versus 1.5 pmol receptor-activated G-proteins/mg [determined by guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) isotopic dilution], respectively. Thus, they displayed R:G ratios of approximately 3.0 (RGhigh) and approximately 0.3 (RGlow), respectively. In competition-binding experiments, the agonists, 5-HT and sumatriptan, displayed fewer high-affinity (HA)-binding sites and the partial agonists, BMS181, 101 and L775,606, displayed decreased affinity in RGhigh versus RGlow membranes. In contrast, the inverse agonists, SB224,289 and, to a lesser extent, methiothepin, showed increased affinity. In G-protein activation experiments, both basal and 5-HT-activated [(35)S]GTPgammaS binding were higher in RGhigh than in RGlow membranes. Constitutive activity (determined by inhibition of basal [(35)S]GTPgammaS binding with GTPgammaS in the absence of receptor ligands) was more pronounced in RGhigh versus RGlow membranes, as revealed by the >5-fold greater proportion of HA sites. Correspondingly, the negative efficacy of inverse agonists was strikingly augmented, inasmuch as they suppressed approximately two-thirds of HA [(35)S]GTPgammaS binding in RGhigh membranes, but only approximately one-third in RGlow membranes. Furthermore, the efficacy of partial agonists was greater at RGhigh versus RGlow membranes, as estimated by their ability to enhance [(35)S]GTPgammaS binding. In conclusion, an increase in R:G ratios at h5-HT(1B) receptors was associated with an increase in relative efficacy of partial agonists and, most notably, an increase in both constitutive G-protein activation and negative efficacy of inverse agonists.
本研究评估了受体/G蛋白（R:G）化学计量对人（h）5-羟色胺1B（5-HT(1B)）受体组成活性以及激动剂、部分激动剂和反向激动剂效力的影响。使用了两种中国仓鼠卵巢细胞系；它们分别表达8.5与0.4 pmol h5-HT(1B)受体/毫克（通过[(3)H]GR125,743饱和分析测定）以及3.0与1.5 pmol受体激活的G蛋白/毫克[通过鸟苷-5'-O-(3-[(35)S]硫代)-三磷酸([(35)S]GTPγS)同位素稀释测定]。因此，它们分别显示出约3.0（RG高）和约0.3（RG低）的R:G比率。在竞争结合实验中，激动剂5-HT和舒马曲坦在RG高的膜中显示出较少的高亲和力（HA）结合位点，部分激动剂BMS181、101和L775,606在RG高的膜中与RG低的膜相比亲和力降低。相反，反向激动剂SB224,289以及在较小程度上的甲硫噻嗪显示出亲和力增加。在G蛋白激活实验中，RG高的膜中基础和5-HT激活的[(35)S]GTPγS结合均高于RG低的膜。组成活性（通过在无受体配体时用GTPγS抑制基础[(35)S]GTPγS结合来测定）在RG高的膜中比RG低的膜中更明显，这通过HA位点比例高出5倍以上得以揭示。相应地，反向激动剂的负效力显著增强，因为它们在RG高的膜中抑制了约三分之二的HA [(35)S]GTPγS结合，但在RG低的膜中仅抑制了约三分之一。此外，部分激动剂在RG高的膜中与RG低的膜相比效力更大，这通过它们增强[(35)S]GTPγS结合的能力来估计。总之，h5-HT(1B)受体处R:G比率的增加与部分激动剂相对效力的增加相关，最显著的是与组成性G蛋白激活以及反向激动剂负效力的增加相关。
