REGULATION OF SIGNALING BY G PROTEIN BINDING PROTEINS

描述(改编自申请人的摘要)：申请人辩解 激素和神经递质传递的跨膜信号需要 细胞表面受体、G蛋白和效应酶。其他内容 蛋白质是调节信号所必需的。例如，蛋白激酶和 Arrestin有助于受体的脱敏。我们知道的要少得多 关于促进G蛋白脱敏的蛋白质。这个 申请者的提案试图检验G蛋白经历 脱敏。具体地说，他将研究两种蛋白质，硫代蛋白 和Sst2，改变G蛋白功能。这些实验将会进行 在酵母菌株中，因为酵母表达受体和G 蛋白质与人类和酵母同源物中发现的蛋白质非常相似 最近在申请人的实验室中发现了一种硫代磷酸脱氢酶。在……里面 此外，酵母菌特别适合体内遗传分析。 初步实验表明，光导蛋白和Sst2与G蛋白结合。 在体外和体内脱敏所需。为了实现他的目标， 申请者提出了四个具体目标。 目标1将研究光导蛋白是如何影响G蛋白功能的。假说 是光导蛋白阻断了G蛋白亚单位关联并进一步 由酵母中的受体激活。的功能后果 光导蛋白与G蛋白的结合将通过测量其影响来评估 体内信息素敏感性(转录激活、生长停滞) 和G蛋白的体外活性(亚基结合、GTP水解) Aim 2将研究磷酸脱氢酶是如何调控的。假设是这样的 在体内，磷酸化的硫代蛋白不与G蛋白相互作用。至 检验这一假设，磷酸化位点(S)将被绘制出来，并且 防止修饰的突变体将被评估信息素的变化 体内信号转导和G蛋白体外激活。 Aim 3将研究Sst2如何影响G蛋白功能。假设是 Sst2通过刺激G蛋白GTPase活性促进脱敏。 为了验证这一假设，纯化的重组蛋白将用于 在存在和不存在Sst2的情况下测量G蛋白的活性。 Aim 4将研究Sst2是如何受到监管的。Sst2经历了一个 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 这一变化，修改的地点将被绘制出来，以及 将在体内测试信息素信号的变化 G蛋白的体外活性。

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.