GQ IN BOMBESIN RECEPTOR SIGNALLING AND HUMAN TUMORS

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，激活磷脂酶C-β 1（Berstein等人，J. Biol. 267：8081 - 8088，1992）。 基于这些观察，我们假设Gq或结构上 与G11类似，对蛙皮素受体信号转导起重要作用。 我们 计划通过观察Gq特异性的影响来验证这一假设。 反义分子对蛙皮素介导的反应在两个不同的 生物背景下，非洲爪蟾卵母细胞和瑞士3T3成纤维细胞。 我们有 分离并测序小鼠和非洲爪蟾Gq和G11的cDNA克隆。 的 非洲爪蟾蛋白质在氨基酸序列上与它们的 哺乳动物的对应物。 这些克隆体将用于设计 合成将被注射到 表达GRP-R或NMB-R的非洲爪蟾卵母细胞观察其作用 蛙皮素信号 小鼠cDNA将用于稳定地产生 转染的Swiss 3T3成纤维细胞，其中表达反义Gq或 G11 RNA由地塞米松诱导型MMTV启动子指导。 抗血清 Gq/G11特异性将用于确定蛋白质水平是否 通过反义诱导减少，以及对GRP-R信号的影响 转导与蛋白水平相关。 G-α突变 亚基与人类垂体的转化有关 和甲状腺肿瘤 鉴于磷脂酶C激活的重要性 在细胞生长中，我们假设类似的突变改变了细胞的生长， Gq的功能在其他实体瘤中也可能很重要。 我们有 通过蛋白质印迹分析鉴定了一种结构修饰的Gq蛋白， 小细胞肺癌H82。 我们计划描述这个异常的Gq 通过分子克隆，并研究Gq的功能后果 在H82中发现的突变，以及在其他肺中发现的Gq突变， 癌细胞

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-beta 1 (Berstein et al., J. Biol. Chem. 267: 8081-8088, 1992) after ligand receptor interaction. Based on these observations, we postulate that Gq, or structurally similar G11, is important for bombesin receptor signal transduction. We plan to test this hypothesis by observing the effects of Gq-specific antisense molecules on bombesin-mediated responses in two different biological contexts, Xenopus oocytes and Swiss 3T3 fibroblasts. We have isolated and sequenced cDNA clones for mouse and Xenopus Gq and G11. The Xenopus proteins are over 90% identical in amino acid sequence to their mammalian counterparts. These clones will be used to design the synthesis of antisense oligonucleotides which will be injected into Xenopus oocytes expressing either GRP-R or NMB-R to observe their effects on bombesin signalling. The mouse cDNAs will be used to generate stably transfected Swiss 3T3 fibroblasts, where expression of antisense Gq or G11 RNA is directed by a dexamethasone-inducible MMTV promoter. Antisera specific for Gq/G11 will be used to determine if protein levels are diminished by antisense induction, and the effects on GRP-R signal transduction correlated with protein levels. Mutations in G-alpha subunits have been implicated in the transformation of human pituitary and thyroid tumors. Given the importance of phospholipase C activation in cell growth, we postulate that analagous mutations altering the function of Gq may also be important in other solid tumors. We have identified a structurally modified Gq protein by Western blot analysis in small cell lung carcinoma H82. We plan to characterize this abnormal Gq by molecular cloning, and investigate the functional consequences of Gq mutations found in H82, as well as Gq mutations found in other lung cancer cells.