SIGNAL TRANSDUCTION IN CELL PROLIFERATION

本提案的目的是确定和表征GTP结合 与细胞增殖相关的调节蛋白（G蛋白）。G 蛋白质通常在信号转导中起着强制性的作用， 信息从细胞表面受体传递到靶细胞 酶或通道。几种生长因子利用G蛋白， 这是它们发挥促有丝分裂作用的唯一手段，而其他 通过联合使用G蛋白依赖性和- 独立的道路。虽然G蛋白在免疫系统中起着重要作用， 扩散，那些真正与之交流的人的身份 生长因子受体或作为下游靶点的受体 修改不清楚。此外，这些G蛋白的功能是 不太了解。提出了四个实验目标。首先，G蛋白 直接与之相互作用，或者以某种方式被其激活， 将鉴定生长因子的受体。 战略包括： 受体免疫沉淀，G蛋白复合物，光亲和性 用[32 P] γ-叠氮苯胺基GTP标记，并测量生长 GDP/GTP比率的变化。本目标和后续目标的重点将 被放置在与凝血酶和蛙皮素/胃泌素相关的G蛋白上- 释放肽（GRP），两种广泛公认的有丝分裂原， 通过七螺旋受体和G蛋白。 第二，子集 G蛋白的活化或修饰是特别需要的 细胞复制的能力。实验重点将是 凝血酶和GRP在静止细胞中重新启动DNA合成， 单层培养策略包括显微注射抗体， 破坏G蛋白与受体或靶标的通讯， G蛋白的百日咳毒素抗性类似物，和显性阴性 调变第三，生长因子利用的G蛋白阵列将 相对于其他激动剂使用的药物进行检查， 寻找与有丝分裂相关的G蛋白。问题 包括什么-如所定义的G蛋白的水平利用， 随后的下游事件-将激动剂鉴定为促有丝分裂的，和 G蛋白通过什么样的分子事件来激活 MAP激酶。第四，G蛋白在多大程度上受到后 转化后的翻译修饰 将检查DNA合成的重新启动。最近两次修改 证明了G蛋白α亚基，酪氨酸磷酸化和 棕榈酰化，将构成重点。对G蛋白的理解 在生长因子作用的背景下， 与细胞相关的通路的身份和协调 复制的

The objective of this proposal is to identify and characterize GTP-binding regulatory proteins (G proteins) relevant to cell proliferation. G proteins serve often obligatory roles in signal transduction, the process by which information is conveyed from cell-surface receptors to target enzymes or channels. Several growth factors utilize G proteins as perhaps the sole means by which their mitogenic actions are exerted, while others act in synergy through conjoint use of both G protein-dependent and - independent pathways. Although G proteins serve fundamental roles in proliferation, the identities of those actually communicating with receptors for growth factors or those serving as targets for downstream modification are unclear. The functions of such G proteins, moreover, are poorly understood. Four experimental goals are proposed. First, G proteins that interact directly with, or in some fashion are activated by, receptors for growth factors will be identified. Strategies include co- immunoprecipitation of receptor.G protein complexes, photoaffinity labeling with [32P]gamma-azidoanilido GTP, and measurements of growth factor-altered GDP/GTP ratios. The focus in this and subsequent goals will be placed on G proteins relevant to thrombin and bombesin/gastrin- releasing peptide (GRP), two widely recognized mitogens that operate through heptahelical receptors and hence G proteins. Second, the subset of G proteins whose activation or modification is specifically required for cell replication will be identified. The experimental focus will be reinitiation of DNA synthesis by thrombin and GRP in quiescent cells in monolayer culture. Strategies include microinjection of antibodies that disrupt communication of G proteins with receptors or targets, the use of pertussis toxin-resistant analogues of G proteins, and dominant negative modulation. Third, the array of G proteins utilized by growth factors will be examined relative to those used by other agonists, and specific roles for the G proteins relevant to mitogenesis will be sought. Questions include what - as defined at the level of G proteins utilized and subsequent downstream events - identifies an agonist as mitogenic, and what are the molecular events by which G proteins achieve activation of MAP kinases. Fourth, the extent to which G proteins are subject to post- translational modification upon transformation and in relation to reinitiation of DNA synthesis will be examined. Two modifications recently demonstrated for G protein alpha subunits, tyrosine phosphorylation and palmitoylation, will constitute the focus. An understanding of G proteins in the context of growth factor action will provide substantial insight into the identities and coordination of pathways relevant to cell replication.