REGULATION OF SIGNALING BY G PROTEIN BINDING PROTEINS

描述（改编自申请人摘要）：申请人认为 激素和神经递质的跨膜信号需要一个 细胞表面受体、G蛋白和效应酶。 额外 需要蛋白质来调节信号传导。 例如，蛋白激酶和 抑制蛋白有助于受体的脱敏。 知之甚少 关于促进G蛋白脱敏的蛋白质。 的 申请人的建议试图检验G蛋白经历 脱敏。 具体来说，他将研究两种蛋白质， 和Sst 2，改变G蛋白功能。 这些实验将在 在酵母菌株S.因为酵母表达受体和G 与人类中发现的蛋白质非常相似， 最近在申请人的实验室中鉴定出了一种新的糖蛋白。 在 此外，酵母特别适合于体内遗传分析。 初步实验表明，在细胞中，Adducin和Sst 2与G蛋白结合。 体外和体内脱敏所需。 为了达到目的， 申请人提出了四个具体目标。 目的1将研究糖蛋白如何影响G蛋白功能。 的假设 Ducin阻断G蛋白亚基结合， 酵母中的受体激活。 的功能性后果 将通过测量其作用来评估与G蛋白结合的糖蛋白 对体内信息素敏感性的影响（转录激活，生长停滞） 和体外G蛋白活性（亚基缔合，GTP水解） 目标2将研究如何调节Ducin。 前提是 磷酸化的糖蛋白在体内不与G蛋白相互作用。 到 测试这一假设，将绘制磷酸化位点， 防止修改的突变体将被评估信息素的变化 体内信号传导和体外G蛋白活化。 目的3将研究Sst 2如何影响G蛋白功能。 这个假设是 Sst 2通过刺激G蛋白GT3活性促进脱敏。 为了检验这一假设，纯化的重组蛋白将用于 在存在和不存在Sst 2的情况下测量G蛋白活性。 目标4将研究Sst 2是如何调节的。 Sst2经历了 信息素刺激的电泳迁移率的变化，反映了一些 翻译后修饰 确定的性质和后果 这种变化，修改的网站将被映射，和突变体， 块修饰将测试信息素信号在体内的变化 和G蛋白活性。

DESCRIPTION (Adapted from the applicant's abstract): The applicant argues that transmembrane signaling by hormones and neurotransmitters requires a cell surface receptor, a G protein, and an effector enzyme. Additional proteins are needed to modulate signaling. For example, protein kinases and arrestin contribute to desensitization of receptors. Much less is known about proteins that promote desensitization of the G proteins. The applicant's proposal seeks to test the hypothesis that G proteins undergo desensitization. Specifically, he will examine how two proteins, phosducin and Sst2, alter G protein function. These experiments will be carried out in the yeast strain S. cerevisiae because yeast express receptors and G proteins remarkably similar to those found in humans and a yeast homologue of phosducin has been recently identified in the applicant's laboratory. In addition, yeast is particularly well suited for in vivo genetic analysis. Preliminary experiments reveal that phosducin and Sst2 bind to G protein in vitro and are required for desensitization in vivo. To achieve his goals, the applicant proposes four Specific Aims. Aim 1 will examine how phosducin affects G protein function. The hypothesis is that phosducin blocks G protein subunit association and further activation by the receptor in yeast. The functional consequences of phosducin binding to G proteins will be evaluated by measuring its effects on pheromone sensitivity in vivo (transcriptional activation, growth arrest) and G protein activity in vitro (subunit association, GTP hydrolysis) Aim 2 will examine how phosducin is regulated. The hypothesis is that phosphorylated phosducin does not interact with the G protein in vivo. To test this hypothesis, the phosphorylation site(s) will be mapped, and mutants that prevent modification will be evaluated for changes in pheromone signaling in vivo and G protein activation in vitro. Aim 3 will examine how Sst2 affects G protein function. The hypothesis is that Sst2 promotes desensitization by stimulating G protein GTPase activity. To test this hypothesis, purified recombinant proteins will be used to measure G protein activity in the presence and absence of Sst2. Aim 4 will examine how Sst2 is regulated. Sst2 undergoes a pheromone-stimulated change in electrophoretic mobility, reflecting some post-translational modification. To determine the nature and consequence of this change, the sites of modification will be mapped, and mutants that block modification will be tested for changes in pheromone signaling in vivo and G protein activity in vitro.