REGULATION OF SIGNALING BY G PROTEIN-BINDING PROTEINS

跨膜信号传导通常需要细胞表面受体、G蛋白和效应酶。 RGS蛋白通过加速GTP水解和G蛋白失活来调节信号传导。信号传导也可以通过翻译后蛋白质修饰来调节。 这一提议将检验RGS和G蛋白也在产后调节的假设。 这些实验将在酿酒酵母中进行，酿酒酵母表达与人类相似的G蛋白信号传导装置。 酵母系统的使用将允许体外生物化学方法和体内遗传策略以协调的方式使用，以解决以下四个目标：目标1：RGS蛋白是如何调节的？以前，我们使用质谱法显示酵母中的RGS（Sst 2）在体内在Ser-380和Ser-539处被磷酸化。Ser-539在响应信息素刺激时被磷酸化，需要MAP激酶，并稳定蛋白质。Ser-380磷酸化尚未表征。 表达阵列文库将用于鉴定使Ser-380磷酸化的激酶。 激酶破坏和磷酸化位点突变体，然后将用于确定如何改变这种修饰G蛋白的催化活性在体外，和G蛋白的信号转导活性在体内。 目的2：G蛋白a亚基是如何调控的？ 已经确定酵母中的Galpha（Gpa 1）是泛素化的，但这种修饰的位置和功能作用尚不清楚。 质谱法将用于鉴定泛素化氨基酸。 泛素化和蛋白水解途径的突变体将用于确定G蛋白降解的机制。 最后，基因破坏和泛素化位点突变体将用于确定这一过程如何改变G蛋白的活性。 目的3：G蛋白B亚基是如何调控的？ 已知酵母中的G β（Ste 4）响应于信息素刺激而磷酸化。 有间接证据表明，Ste 4-磷酸化有助于信号脱敏。 将使用质谱法鉴定磷酸化位点。 表达阵列文库将用于鉴定Ste 4-激酶。 激酶破坏和磷酸化位点突变体，然后将被用来确定如何在体外和体内改变这种修饰G蛋白的活性。目的4：还有哪些修饰调控RGS和G蛋白？随着质谱的最新进展，现在应该可以识别感兴趣的蛋白质的所有修饰。将检测RGS和G蛋白发生额外（尚未鉴定）修饰的可能性。 将使用RGS和G蛋白功能的标准测定来评价每种修饰的分子和细胞后果。

Transmembrane signaling typically requires a cell surface receptor, a G protein, and an effector enzyme. RGS proteins modulate signaling, by accelerating GTP hydrolysis and G protein inactivation. Signaling can also be modulated through post- translational protein modifications. This proposal will test the hypothesis that RGS and G proteins are also regulated post- translationally. These experiments will be carried out in the yeast S.cerevisiae, which expresses a G protein signaling apparatus similar to those in humans. The use of the yeast system will allow in vitro biochemical methods and in vivo genetic strategies to be used in a coordinated manner, to address the following four aims: Aim 1: How is the RGS protein regulated? Previously we used mass spectrometry to show that the RGS in yeast (Sst2) is phosphorylated at Ser-380 and Ser-539 in vivo. Ser-539 is phosphorylated in response to pheromone stimulation, requires a MAP kinase, and stabilizes the protein. Ser-380 phosphorylation has not been characterized. An expression array library will be used to identify the kinase that phosphorylates Ser-380. Kinase-disruption and phosphorylation-site mutants will then be used to determine how this modification alters G protein catalytic activity in vitro, and G protein signaling activity in vivo. Aim 2: How is the G protein a subunit regulated? It is established that the Galpha in yeast (Gpa1) is ubiquitinated, but the location and functional role of this modification are unknown. Mass spectrometry will be used to identify the ubiquitinated amino acid. Mutants in the ubiquitination and proteolysis pathways will be used to determine the mechanism of G protein degradation. Finally, gene disruption and ubiquitination-site mutants will be used to determine how this process alters G protein activity. Aim 3: How is the G protein B subunit regulated? It is known that the Gbeta in yeast (Ste4) is phosphorylated in response to pheromone stimulation. There is indirect evidence that Ste4-phosphorylation contributes to signal desensitization. Mass spectrometry will be used to identify the site of phosphorylation. The expression array library will be used to identify the Ste4-kinase. Kinase-disruption and phosphorylation-site mutants will then be used to determine how this modification alters G protein activity in vitro and in vivo. Aim 4: What other modifications regulate RGS and G proteins? With recent advances in mass spectrometry, it should now be possible to identify all modifications of a protein of interest. The likelihood that the RGS and G protein undergo additional (as yet unidentified) modifications will be tested. The molecular and cellular consequences of each modification will be evaluated using standard assays of RGS and G protein function.