GRP-R AND NMB-R SIGNALLING

纯化的异源三聚体GTP结合蛋白的重组研究 脂泡中的G蛋白表明GQ亚家族α 与Gs、Gi和Go子家族不同的是，亚单位激活 磷脂酶C-1在配体受体相互作用后，并不敏感 百日咳毒素。我们假设GQ，或结构相似 Galpha11对蛙皮素受体信号转导起重要作用。我们 通过观察GQ和Galpha11的影响来检验这一假设 非洲爪哇蛙毒蛋白介导反应的特异性反义分子 表达GRP-R或NMB-R的卵母细胞。合理设计反义基因 寡核苷酸，我们分离并测序了非洲爪哇的cdna克隆。 GQ和Galpha11。非洲爪哇的蛋白质氨基酸同源性超过90%。 它们的酸序列与它们的哺乳动物同源。这些序列被用来 反义硫代磷酸寡核苷酸的合成设计 将其注射到非洲爪哇卵母细胞中，表达GRP-R或NMB-R 观察它们对蛙皮素信号的影响。反义词 GQ特异性寡核苷酸抑制NMB-R信号转导，但不起作用 影响GRP-R信号转导。相比之下，Galpha11反义 寡核苷酸不改变GRP-R或NMB-R的信号转导。 这些研究表明，尽管信号转导途径为 GRP-R和NMB-R相似，两种受体的G蛋白不同 耦合特性。此外，GQ和Galpha11不是 可互换为NMB-R联轴器。我们假设， GQ家族成员的表达可能被用来调节受体 不同细胞的反应。

Reconstitution studies using purified heterotrimeric GTP-binding proteins (G-proteins) in lipid vesicles indicate that the Gq sub-family of alpha subunits, in contrast to the Gs, Gi, and Go subfamilies, activate phospholipase C-1 after ligand receptor interaction, and are insensitive to pertussis toxin. We postulate that Gq, or structurally similar Galpha11, are important for bombesin receptor signal transduction. We have tested this hypothesis by observing the effects of Gq- and Galpha11- specific antisense molecules on bombesin-mediated responses in Xenopus oocytes expressing GRP-R or NMB-R. To rationally design antisense oligonucleotides, we have isolated and sequenced cDNA clones for Xenopus Gq and Galpha11. The Xenopus proteins are over 90% identical in amino acid sequence to their mammalian counterparts. These sequences were used to design the synthesis of antisense phosphothiorate oligonucleotides which were injected into Xenopus oocytes expressing either GRP-R or NMB-R to observe their effects on bombesin signalling. The antisense oligonucleotide specific for Gq inhibited NMB-R signalling, but did not effect GRP-R signalling. In contrast, the Galpha11 antisense oligonucleotide did not alter signalling from either GRP-R or NMB-R. These studies indicate that, although the signal transduction pathway for GRP-R and NMB-R is similar, the two receptors differ in their G-protein coupling properties. In addition, Gq and Galpha11 are not interchangeable for NMB-R coupling. We hypothesize that the pattern of expression of Gq family members may be used to modulate receptor responses in different cells.