GQ IN BOMBESIN RECEPTOR SIGNALLING AND HUMAN TUMORS

在脂泡中使用纯化蛋白的重建研究表明 Gq亚家族的阿尔法亚单位，与Gs、Gi和 GO亚家族，激活磷脂酶C-β1(Berstein等人，J. 比奥尔。化学。267：8081-8088,1992)。 基于这些观察，我们假设GQ，或者说结构上 与G11类似，对蛙皮素受体信号转导也很重要。我们 计划通过观察GQ特定的影响来检验这一假设 反义分子对蛙皮素介导的两种不同反应的影响 生物学背景，非洲爪哇卵母细胞和瑞士3T3成纤维细胞。我们有 小鼠和非洲爪哇GQ和G11的cDNA克隆的分离和测序。这个 非洲爪哇蛋白的氨基酸序列与它们的 哺乳动物的对应物。这些克隆将被用来设计 注射用反义寡核苷酸的合成 表达GRP-R和NMB-R的非洲爪哇卵母细胞对其影响的观察 关于炸弹信号。小鼠的DNA将被用来稳定地产生 转染瑞士3T3成纤维细胞，反义GQ或 G11 RNA由地塞米松诱导的MMTV启动子引导。抗血清 GQ/G11的特异性将用于确定蛋白质水平是否 被反义诱导减弱及其对GRP-R信号的影响 转导与蛋白质水平相关。G-α基因突变 亚基参与了人类脑下垂体的转化 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 GQ的功能在其他实体肿瘤中也可能很重要。我们有 Western印迹分析鉴定了一种结构修饰的GQ蛋白 小细胞肺癌H82。我们计划对这种异常的GQ进行描述 通过分子克隆，并研究GQ的功能后果 在H82中发现突变，以及在其他肺中发现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.