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Molecular Mechanism of activity dependent translocation of RGS8

RGS8活性依赖性易位的分子机制

基本信息

批准号：
13680730
负责人：
SAITOH Osamu
金额：
$ 2.37万
依托单位：
Tokyo Metropolitan Institute for Medical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680730/
关键词：
RGS G protein Receptor Desensitization Translocation Neuron Pukinje cell Cerebellum

项目摘要

RGS (regulators of G protein signaling) proteins comprise a large family of more than 30 members, which modulate heterotrimeric G protein signalling. We initially identified RGS8 as a brain specific RGS, then RGS8 was found to be specifically expressed in cerebellar Purkinje cells. Since the apparent site of action of RGS8 is considered to be adjacent to G proteins at the plasma membrane, we examined the subcellular distribution of the RGS8 protein by expression of RGS8 in non-neural DDT1MF2 cells. We found that RGS8 was concentrated in the nuclei, and that co-expression of constitutively active Gao (GaoQL) resulted in the translocation of RGS8 to the plasma membrane. Concerning detail distribution and the mechanism regulating the subcellualr distribution of RGS8 protein, here, we investigated the following four points.1. Various Gα subtypes were co-expressed with RGS8. Inactive and active forms were expressed for each Gα subunit, and their effects on the distribution of RGS8 protein w … More ere investigated. Only when the Gαi family was expressed, did the membrane translocation of RGS8 occur. Expression of the inactive or active form of the Gαi family showed similar effects. By generating and using a point mutant RGS8(L153F) that does not bind to Gαo, we determined whether the membrane recruitment of RGS8 might be the direct result of physical association with the Gαi family. This RGS8(L153F) was co-expressed with an inactive or active form of the Gαi family. Only active Gαo caused the membrane shift of RGS8(L153F). These results demonstrated that activation of Go can specifically induce membrane-translocation of RGS8 without their direct interaction.2. The cellular distribution of the RGS8 protein in cerebellar Pukinje cells was studied in detail using cultured Purkinje cells and frozen sections of the cerebellum. It was shown that the protein is excluded from the nuclei and distributed in the cell body and dendrites except the axons of Purkinje cells.3. We examined the possibility that distribution of RGS8 protein is differently regulated in neuron. The subcellular distribution of RGS8 protein in neuronally differentiated P19 cells was studied. We observed nuclear distribution and Gα dependent membrane translocation of RGS8, both of which were quite similar to observation in nonneural cells.4. We identified a new short isoform of RGS8, RGS8S, that arises by alternative splicing. RGS8S cDNA encodes a N- terminus of 7 amino acids instead of aa 1-9 of RGS8, and 10-180 of RGS8. We examined the effects of RGS8 and RGS8S on Gq-mediated signaling. RGS8 decreased the amplitude of the response upon activation of ml muscarinic or substance P receptors, but did not remarkably inhibit signaling from m3 muscarinic receptors. In contrast, RGS8S showed much less inhibition of the response of either of these Gq-coupled receptors. Thus, we found that 9 amino acids in the N-terminus of RGS8 have contribution to the function to inhibit Gq-coupled signaling in a receptor type-specific manner, suggesting that a certain type of Gq-coupled receptors may recruit RGS8 to the plasma membrane by direct interaction. Less

RGS （G蛋白信号的调节因子）蛋白包括一个有30多个成员的大家族，它们调节异源三聚体G蛋白信号。我们最初确定RGS8是脑特异性RGS，然后发现RGS8在小脑浦肯野细胞中特异性表达。由于RGS8的表观作用位点被认为与质膜上的G蛋白相邻，我们通过RGS8在非神经DDT1MF2细胞中的表达来检测RGS8蛋白的亚细胞分布。我们发现RGS8集中在细胞核中，并且组成活性高（GaoQL）的共表达导致RGS8向质膜易位。关于RGS8蛋白亚细胞分布的详细分布及其调控机制，本文主要从以下四个方面进行了研究。多种Gα亚型与RGS8共表达。研究了各Gα亚基的失活和活性形式对RGS8蛋白分布的影响。只有Gαi家族表达时，RGS8才会发生膜易位。Gαi家族的失活形式和活性形式的表达表现出相似的效果。通过产生和使用不与Gαo结合的点突变体RGS8(L153F)，我们确定了RGS8的膜募集是否可能是与Gαi家族物理关联的直接结果。该RGS8（L153F）与Gαi家族的非活性或活性形式共表达。只有活性Gαo引起RGS8（L153F）的膜移位。这些结果表明，Go的激活可以特异性诱导RGS8的膜易位，而不需要它们之间的直接相互作用。利用培养的浦肯野细胞和小脑冷冻切片，详细研究了RGS8蛋白在小脑浦肯野细胞中的细胞分布。结果表明，在浦肯野细胞中，除轴突外，该蛋白不存在于细胞核内，分布于细胞体和树突内。我们研究了RGS8蛋白在神经元中的分布受到不同调控的可能性。研究了RGS8蛋白在神经分化的P19细胞中的亚细胞分布。我们观察到RGS8的核分布和Gα依赖的膜易位，两者与非神经细胞的观察结果非常相似。我们发现了RGS8的一个新的短异构体RGS8S，它是通过选择性剪接产生的。RGS8S cDNA编码7个氨基酸的N-端，而不是RGS8的1-9和RGS8的10-180。我们研究了RGS8和RGS8S对gq介导的信号传导的影响。RGS8降低了ml毒蕈碱受体或P物质受体激活后的反应幅度，但没有显著抑制m3毒蕈碱受体的信号传导。相反，RGS8S对这两种gq偶联受体的抑制作用要小得多。因此，我们发现RGS8的n端有9个氨基酸以受体类型特异性的方式参与抑制gq偶联信号的功能，这表明某种类型的gq偶联受体可能通过直接相互作用将RGS8招募到质膜上。少