cAMP-independent G protein signaling in yeast

假菌丝和侵袭性生长需要一个由G蛋白β亚基Gpa2p及其偶联受体Gpr1介导的信号通路。Gpr1p是一种低亲和力的葡萄糖受体，对环境中高浓度的葡萄糖做出反应。从Gpr1p到Gpa2p的信号传递导致与高水平的细胞内cAMP相关的表型。我们最近发现KRH1和KRH2是编码Gpa2p信号通路组成部分的基因。我们已经证明Krh1p和Krh2p作用于腺苷环化酶下游，通过一个不涉及细胞内cAMP产生的过程来抑制蛋白激酶A(PKA)。Gpa2p的激活被认为可以缓解 Krh1p和Krh2p抑制PKA活性，导致PKA活性升高。该项目的长期目标是：1)获得组成Gpa2p信号转导途径的分子过程的完整描述；2)从整个细胞群体经历假菌丝生长的角度了解Gpa2p途径的生物学功能。 这个项目的第一个具体目标是确定Krh1p和Krh2p如何调节PKA。这些研究将调查Krh1p和Krh2p是否直接与PKA结合，以及结合是否抑制PKA激酶活性。第二个特异性目的是确定Krh1p和Krh2p是否通过影响PKA的定位来控制信号转导。第三个特异性目的是通过确定GPA2的非激活等位基因和构成等位基因对Krh1p和Krh2p功能的影响，来研究Gpa2p的GTP结合形式是否阻断了Krh1p和Krh2p的抑制功能。第四个具体目的是测试Gpa2p是否在生长菌落边缘的细胞中被特异性激活，以抑制应激和饥饿反应，促进生长和假菌丝发育。此目标的目标是开发一种 允许在整个菌落经历的水平上检测激活的Gpa2p的报告系统 假菌丝生长。这些研究将提供有关G蛋白介导的信号通路的新信息，这些通路对于人类许多生理过程的正常运作至关重要。

Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae require a signaling pathway that is mediated by the G protein beta-subunit Gpa2p and its coupled receptor Gpr1. Gpr1p is a low affinity glucose receptor that responds to high concentrations of glucose in the environment. Transmission of a signal from Gpr1p to Gpa2p results in phenotypes associated with high levels of intracellular cAMP. We have recently identified KRH1 and KRH2 as genes that encode components of the Gpa2p signaling pathway. We have shown that Krh1p and Krh2p act downstream of adenylyl cyclase to inhibit protein kinase A (PKA) by a process that does not involve production of intracellular cAMP. Activation of Gpa2p is thought to relieve the inhibition of PKA by Krh1p and Krh2p, resulting in high levels of PKA activity. The long-term objectives of this project are: 1) To obtain a complete description of the molecular processes that comprise the Gpa2p signal transduction pathway; 2) To understand the biological function of the Gpa2p pathway in terms of an entire cell population undergoing pseudohyphal growth. The first specific aim of this project is to determine how Krh1p and Krh2p regulate PKA. These studies will investigate whether binding of Krh1p and Krh2p to PKA is direct and whether binding inhibits PKA kinase activity. The second specific aim is to determine whether Krh1p and Krh2p control signaling by affecting the localization of PKA. The third specific aim is to investigate whether the GTP-bound form of Gpa2p blocks the inhibitory function of Krh1p and Krh2p by determining the effects of non-activatable and constitutive alleles of GPA2 on Krh1p and Krh2p function. The fourth specific aim is to test whether Gpa2p is specifically activated in cells at the edge of a growing colony in order to suppress stress and starvation responses and to promote growth and pseudohyphal development. The goal of this aim is to develop a reporter system to allow detection of activated Gpa2p at the level of a whole colony undergoing pseudohyphal growth. These studies will provide new information about G protein-mediated signaling pathways, which are essential for the proper functioning of many physiological processes in humans.