Gbeta5-R7 Complex in Signaling and Hormone Secretion

 描述(申请人提供)：G蛋白β亚单位Gβ5与R7蛋白家族的复合体是GI家族的缺口，参与许多生理功能。Gβ5和R7蛋白最初都在神经系统中发现，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，并推进了本实验室发展的一项原创性研究：Gβ5-R7对M3 M受体(M3R)的调节。最近的研究发现了一个新的途径，即Gβ5-R7可以增强M3R诱导的钙跨膜内流。这种对钙离子流量的影响与Gβ5-R7存在时胰岛素分泌水平较高是一致的。因为RGS蛋白被认为是G蛋白信号的负调节因子，所以这种积极的作用是新的和令人兴奋的。另一个令人费解的细节是，M3R是一种GQ偶联受体，Gβ5-R7成员不具有GQ的GAP活性。该项目将追求三个综合良好的目标。为了确定β细胞对Gβ5-R7敏感的特定信号机制，Aim 1将使用药物抑制剂或基因敲除和敲除来靶向M3R到胰岛素途径的成员。研究将在WT和G5基因敲除小鼠的胰岛和胰岛素瘤细胞系Min6中进行。实验将测量胰岛素分泌、[Cai]和其他对2+M3R激动剂的信号反应。AIM 2将建立和分析具有β细胞特异性Gβ5基因敲除的小鼠。这些小鼠的原代胰岛将在体外通过信号和分泌分析进行检测，并将研究这些胰岛在眼内移植到诱导性糖尿病小鼠体内后的长期功能。目的研究Gβ5-R7复合体对Gβ5 KO小鼠胰岛基因表达的影响。这些研究将对了解胰腺β细胞的信号转导、胰岛素分泌和应激适应产生重要影响。新的机械论见解将为GPCR信号的广泛领域、激素释放的调节以及糖尿病和肥胖症等代谢紊乱的病因学提供持续的思想流动。

 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.