GRP-R AND NMB-R SIGNALLING

在脂质囊泡中使用纯化蛋白质的重构研究表明， α亚基的Gq亚家族，与Gs，Gi和 Go亚家族，在配体受体后激活磷脂酶C-β 1 相互作用，对百日咳毒素不敏感。我们假设Gq， 或结构相似的Galpha 11，对于蛙皮素受体是重要的 信号转导我们已经通过观察 Gq-和Galpha 11-特异性反义分子对蛙皮素的影响 在表达GRP-R或NMB-R的非洲爪蟾卵母细胞中介导的反应。到 合理设计反义寡核苷酸，我们分离并 非洲爪蟾Galpha 11的cDNA克隆测序。非洲爪蟾蛋白质 在氨基酸序列上与它们的哺乳动物对应物90%相同。 将特异性反义硫代磷酸寡核苷酸注射到 在非洲爪蟾卵母细胞中，它们特异性地消除了靶mRNA。使用 在这些条件下，将反义寡核苷酸注射到卵母细胞中， 表达GRP-R或NMB-R，观察其对蛙皮素的影响 信号装置. Gq特异性反义寡核苷酸抑制NMB-R 信号，但不影响GRP-R信号。相比之下，Galpha 11 反义寡核苷酸不改变来自GRP-R或 NMB-R。这些研究表明，虽然信号转导 GRP-R和NMB-R的通路是相似的，但这两种受体在其 G蛋白偶联特性。此外，Gq和Galpha 11不是 可互换用于NMB-R联轴器。我们假设 Gq家族成员的表达可用于调节受体应答 在不同的细胞。

Reconstitution studies using purified 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-beta1 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 Galpha11. The Xenopus proteins are over 90% identical in amino acid sequence to their mammalian counterparts. Specific antisense phosphothiorate oligonucleotides were injected into Xenopus oocytes where they specifically eliminated the target mRNAs. Using these conditions, antisense oligonucleotides were injected into 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.