Molecular Mechanism of activity dependent translocation of RGS8

G蛋白信号调节因子(RGS)蛋白由30多个成员组成，调节异源三聚体G蛋白信号转导。我们最初确定RGS8是大脑特异的RGS，然后发现RGS8在小脑浦肯野细胞中特异表达。由于RGS8的表观作用部位被认为与质膜上的G蛋白相邻，因此我们通过RGS8在非神经性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，并且与成分活性GaO(GaoQL)的共表达导致了RGS8向质膜的移位。关于RGS8蛋白的细节分布及其调节亚细胞分布的机制，我们研究了以下四点。不同Gα亚型与RGS8共表达。每个G-α亚基都有活性和非活性形式的表达，以及它们对…蛋白分布的影响更多的是调查。只有当GαI家族表达时，RGS8才发生膜转位。非活性或活性形式的GαI家族的表达也表现出类似的效果。通过产生和使用一个不与Gαo结合的点突变株RGS8(L153F)，我们确定了RGS8的膜募集是否是与GαI家族物理结合的直接结果。该RGS8(L153F)与GαI家族的非活性或活性形式共表达。只有活性G-αo引起RGS_8(L153F)的膜移位。这些结果表明，GO的激活可以在没有直接相互作用的情况下特异性地诱导RGS8的膜转位。利用培养的浦肯野细胞和小脑冰冻切片，对RGS8蛋白在小脑Pukinje细胞中的细胞分布进行了详细的研究。结果表明，该蛋白被排除在胞核之外，分布在浦肯野细胞的胞体和树突中，但轴突除外。我们研究了RGS8蛋白在神经元中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G-α依赖的膜转位，这两者都与在非神经细胞中观察到的非常相似。我们发现了RGS8的一种新的短亚型，RGS8S，它是通过选择性剪接产生的。RGS8S编码7个氨基酸的N末端，而不是RGS8的AA1-9和RGS8的10-180。我们研究了RGS8和RGS8S对GQ介导的信号转导的影响。RGS8可降低激活m1受体或P物质受体时的反应幅度，但不显著抑制m3受体发出的信号。相反，RGS8S对这两种GQ偶联受体的反应的抑制要小得多。因此，我们发现RGS8 N端的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，提示某种类型的GQ偶联受体可能通过直接作用将RGS8募集到质膜上。较少

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