Functional analysis of regulatory mechanism of G protein signal network

G蛋白由a、p和y三个亚基组成，通过分子开关的作用将多种信号从G蛋白偶联受体(GPCRs)传递到细胞内效应分子。我们证明了通过GQ和JNK的GPCR信号负性调节神经前体细胞的迁移。此外，我们还发现Ric-8和Flotillins是一种新型的GQ结合调节蛋白。RIC-8A促进G-α-Q的鸟核苷酸交换，并增强GQ介导的细胞反应，如细胞内钙动员和JNKK激活。结果表明，在受体刺激下，Ric-8A从胞浆移位到细胞膜。Fltillins是已知的脂筏制造蛋白，siRNA将其击倒，减弱了gpcr诱导的p38MAPK激活和酪氨酸磷酸化。用Src酪氨酸激酶抑制剂和胆固醇去除剂甲基-β-环糊精处理也能抑制GPCR诱导的p38MAPK活化和酪氨酸磷酸化。这些证据表明，GQ偶联受体通过脂筏和Src激活p38MAPK，其中Flotillins正向调节GQ信号。此前，我们证明了一种新的针对CDC42的Rhogef-FRG是在GQ偶联受体下游被激活的。在本研究中，我们发现FRG在CD47和Src下游的信号通路中发挥作用，并参与海马神经元中轴突和树突的发育。另一种RhoGEFP-Rex1是由G蛋白βγ亚基激活的，对中性粒细胞产生活性氧(ROS)是必不可少的。我们发现P-Rex1的分子内结构域相互作用在G-βγ诱导的激活和PKA诱导的抑制中起关键作用。

G proteins are composed of a, p, and y subunits, and transmit a variety of signals from G protein-coupled receptors (GPCRs) to intracellular effectors by acting as molecular switch. We demonstrated that the GPCR signaling through Gq and JNK negatively regulates the neural progenitor cell migration. Moreover, we found that Ric-8 and flotillins are a new type of Gq-binding regulatory proteins. Ric-8A promoted the guanine nucleotide exchange of Gαq, and amplified the Gq-mediated cellular responses, such as intracellular Ca mobilization and JNK activation. It was revealed that Ric-8A translocates from cytosol to membrane upon receptor stimulation. The knockdown of flotillins, which are known to be lipid raft maker proteins, by siRNA attenuated the GPCR-induced p38 MAPK activation and tyrosine phosphorylation. Treatment with Src tyrosine kinase inhibitors and cholesterol depleting agent methyl-beta-cyclodextrin also inhibited the GPCR-induced p38 MAPK activation and tyrosine phosphorylation. These lines of evidence suggested that a Gq-coupled receptor activates p38 MAPK through lipid rafts and Src, in which flotillins positively regulates the Gq signaling. Previously, we demonstrated that FRG, a novel RhoGEF for Cdc42, is activated downstream of Gq-coupled receptor. In this research, we found that FRG functions in the signaling pathway downstream CD47 and Src and is involved in the development of axons and dendrites in hippocampal neurons. Another RhoGEF P-Rex1 is activated by G protein βγ subunit and essential for reactive oxygen species (ROS) production in neutrophils. We found that intramolecular domain interaction of P-Rex1 is critical for Gβγ-induced activation and PKA-induced inhibition.