Gbeta5-R7 Complex in Signaling and Hormone Secretion

 描述（由申请人提供）：G蛋白β亚基Gβ5与RGS蛋白的R7家族的复合物作为Gi家族的GAP，并参与许多生理功能。Gβ5和R7蛋白最初在神经系统中发现，但最近在其他一些组织中发现，特别是内分泌腺。目前的研究集中在Gβ5-R7在胰腺β细胞中的作用，并推进了本实验室开发的原始研究线：Gβ5-R7对M3毒蕈碱受体（M3 R）的调节。最近的研究确定了一种新的途径，其中Gβ5-R7可以增强M3 R诱导的Ca 2+跨质膜内流。这种对Ca 2+通量的影响与Gβ5-R7存在时较高水平的胰岛素分泌一致。这种积极的作用是新颖的和令人兴奋的，因为RGS蛋白被认为是G蛋白信号传导的负调节剂。另一个令人困惑的细节是，M3 R是一种Gq偶联受体，而Gβ5-R7成员对Gq没有GAP活性。该项目将追求三个综合目标。为了确定对Gβ5-R7敏感的特定β细胞信号传导机制，Aim 1将靶向M3 R-胰岛素途径的成员，使用药理学抑制剂或基因敲除和敲低。将在WT和G5敲除小鼠的胰岛和胰岛素瘤细胞系Min 6中进行研究。实验将测量胰岛素分泌、[Ca I]和对2+ M3 R激动剂的其它信号传导应答。目的2建立β细胞特异性Gβ5基因敲除小鼠模型并进行分析。将通过体外信号传导和分泌测定来检查来自这些小鼠的原代胰岛，并且将在这些胰岛眼内移植到具有诱导糖尿病的小鼠中后研究这些胰岛的长期功能。目的3采用RT-PCR、原位杂交和下一代测序相结合的方法，检测G β5基因敲除小鼠胰岛RNA中Gβ5-R7复合物对基因表达的影响。这些研究将对理解胰腺β细胞信号传导、胰岛素分泌和应激适应产生重大影响。新的机制见解将为GPCR信号传导，激素释放调节和糖尿病和肥胖等代谢疾病的病因学等广泛领域提供持续的想法。

 DESCRIPTION (provided by applicant): Complexes of the G protein βsubunit Gβ5 with the R7 family of RGS proteins serve as GAPs for Gi family and are involved in many physiological functions. Both Gβ5 and R7 proteins were originally found in the nervous system, but recently detected in some other tissues, notably endocrine glands. The current study concentrates on the role of Gβ5-R7 in pancreatic β cells and advances an original line of research developed in this laboratory: regulation of M3 muscarinic receptor (M3R) by Gβ5-R7. The most recent study identified a novel pathway where Gβ5-R7 can potentiate M3R-induced Ca2+ influx across the plasma membrane. This effect on Ca2+ flux is consistent with the higher level of insulin secretion in the presence of Gβ5-R7. The positive effect is novel and exciting because RGS proteins are thought to be negative regulators of G protein signaling. Another puzzling detail is that M3R is a Gq-coupled receptor, and Gβ5-R7 members do not have GAP activity for Gq. The project will pursue three well-integrated aims. To determine specific β cell signaling mechanisms sensitive to Gβ5-R7, Aim 1 will target members of the M3R-to-insulin pathway with pharmacologic inhibitors or gene knockout and knockdown. Studies will be performed in pancreatic islets from WT and G 5 knockout mice and insulinoma cell line Min6. Experiments will measure insulin secretion, [Ca I] and other signaling responses to 2+ M3R agonists. Aim 2 will generate and analyze mice with β cell-specific knockout of Gβ5. Primary islets from these mice will be examined by signaling and secretion assays in vitro and long-term functionality of these islets will be studied after their intraocular transplantation into mice with induced diabete. Aim 3 will determine the effect of Gβ5-R7 complex on gene expression using a combination of RT-PCR, in situ hybridization and next generation sequencing of pancreatic islet RNA from Gβ5 KO mice. These studies will have a strong impact on understanding pancreatic β cell signaling, insulin secretion and adaptation to stress. The new mechanistic insights will provide a sustained flow of ideas for the broad area of GPCR signaling, regulation of hormone release and etiology such of metabolic disorders as diabetes and obesity.