Molecular Mechanism of activity dependent translocation of RGS8

RGS（regulators of G protein signaling，G蛋白信号调节子）蛋白包括一个由30多个成员组成的大家族，其调节异源三聚体G蛋白信号。我们最初将RGS 8鉴定为脑特异性RGS，然后发现RGS 8在小脑浦肯野细胞中特异性表达。由于RGS 8的表观作用位点被认为与质膜上的G蛋白相邻，因此我们通过在非神经DDT 1 MF 2细胞中表达RGS 8来检查RGS 8蛋白的亚细胞分布。我们发现RGS 8集中在细胞核中，并且组成型活性Gao（GaoQL）的共表达导致RGS 8易位到质膜。关于RGS 8蛋白在细胞内的分布及其调控机制，本文主要研究了以下四个方面：1.各种Gα亚型与RGS 8共表达。每个Gα亚基都表达有活性和非活性两种形式，它们对RGS 8蛋白分布的影响与Gα亚基的表达有关。 ...更多信息 在调查。只有当Gαi家族表达时，RGS 8才发生膜转位。Gαi家族的非活性或活性形式的表达显示出类似的效果。通过产生和使用不与Gαo结合的点突变体RGS 8（L153 F），我们确定RGS 8的膜募集是否可能是与Gαi家族物理关联的直接结果。该RGS 8（L153 F）与Gαi家族的无活性或活性形式共表达。RGS 8（L153 F）只有活性Gαo引起膜移位。这些结果表明，Go的激活可以特异性地诱导RGS 8的膜转位，而不需要它们之间的直接相互作用. RGS 8蛋白在小脑浦肯野细胞中的细胞分布使用培养的浦肯野细胞和小脑的冷冻切片进行了详细研究。结果表明，该蛋白质被排除在细胞核之外，分布于浦肯野细胞的胞体和树突中，轴突除外.我们研究了RGS 8蛋白在神经元中的分布受到不同调节的可能性。研究了RGS 8蛋白在神经元分化的P19细胞中的亚细胞分布。我们观察到RGS 8的核分布和Gα依赖性膜转位，这两个现象与在非神经细胞中观察到的非常相似.我们鉴定了RGS 8的一种新的短同种型RGS 8 S，其通过选择性剪接产生。RGS 8 S cDNA编码7个氨基酸的N-末端，而不是RGS 8的aa 1-9和RGS 8的aa 10-180。我们研究了RGS 8和RGS 8 S对Gq介导的信号传导的影响。RGS 8降低ml毒蕈碱或P物质受体激活后的响应幅度，但不显著抑制来自m3毒蕈碱受体的信号传导。相比之下，RGS 8 S对这些Gq偶联受体中的任一种的反应的抑制要小得多。因此，我们发现RGS 8的N-末端的9个氨基酸以受体类型特异性的方式对抑制Gq偶联信号传导的功能有贡献，这表明某种类型的Gq偶联受体可以通过直接相互作用将RGS 8募集到质膜。少

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