Islet GPCR regulation by GRKs and RGS proteins

目前全球有超过4亿人患有2型糖尿病(T2D)，外周细胞对胰岛素的敏感性降低，胰岛β细胞不能分泌足够的胰岛素来维持低血糖水平。β细胞数量减少是T2D的根本问题，但目前的药物治疗并不能有效地增加功能性β细胞质量。G蛋白偶联受体(GPCRs)是许多临床治疗药物的靶标，我们的GPC罗马图谱表明，人的胰岛表达近300个GPCRs，其中至少有一些可以用于治疗T2D(1-3)。现在清楚的是，GPCRs的调节超越了激动剂结合、相关G蛋白的激活和效应器介导的下游信号的修饰。因此，GPCRs在被激动剂激活后被GPCRK(GRKs)磷酸化，并招募arrestin蛋白，然后与磷酸化的GPCRs结合来抑制它们与蛋白质的相互作用，这反过来又减少了活性GPCRs的下游信号(综述4)。到目前为止，已经克隆和鉴定了七个人类GRK，并确定了两个非视觉拦阻(4)。除了GRK和阻滞素的减敏作用外，蛋白质信号转导调节蛋白(RGS)蛋白还提供了另一种水平的GPCR调节，其中RGS结构域中的保守氨基酸与亚单位内开关区的保守氨基酸结合，增加GTP的水解率并终止信号(5)。虽然我们对b-arrestins在b细胞功能中的作用的理解已经取得了进展(6)对胰岛中GRK和RGS的表达和功能知之甚少，这一点将在本博士项目中进行研究，以更好地了解靶向胰岛GPCRs治疗T2D的可能性。此外，如果在这个项目中产生的数据表明抑制GRKs和/或RGS蛋白可以改善胰岛素分泌并保护β细胞免受凋亡，这将对优化T1D移植前的胰岛功能质量具有重要意义。目前必要的技术正在监督实验室(1，2，7-11)中使用，并将向Jessica提供全面的培训。研究目的-本博士项目有4个主要目标：1.量化来自低BMI和高BMI捐赠者的人胰岛中编码GRKs和RGS蛋白的mRNAs，并确定在肥胖条件下发生的变化。2.确定GRKs和RGS蛋白在瘦人和肥胖者和小鼠胰腺样本中的细胞定位。3.确定选择性GRK和RGS抑制剂对GPCR激动剂调节的胰岛功能的影响。4.确定删除β细胞中关键的GRKs和RGS蛋白的功能效应。

Over 400 million people worldwide currently have type 2 diabetes (T2D), in which peripheral cells show reduced sensitivity toinsulin and islet beta-cells do not secrete sufficient insulin to maintain low blood glucose levels. Declining beta-cell number isthe fundamental problem in T2D but current pharmacotherapies do not effectively increase functional beta-cell mass. Gprotein-coupled receptors (GPCRs) are the targets for many clinically used therapeutics, and our "GPCRome" mapping has indicated thathuman islets express nearly 300 GPCRs, at least some of which could be harnessed therapeutically to treat T2D(1-3).It is now clear that GPCR regulation extends beyond agonist binding, activation of associated G proteins and modifications in effector-mediated downstream signalling. Thus,GPCRs are phosphorylated by GPCR kinases (GRKs)after their activation by agonists andrecruited arrestin proteins then bind to phosphorylated GPCRs to inhibit their interactions with proteins which, in turn, reduces downstream signallingof active GPCRs(reviewed in 4). To date seven human GRKs have been cloned and characterised, and two non-visual arrestins have been identified(4). In addition the desensitising effects of GRKs and arrestins, regulator of protein signalling (RGS) proteins provide another level of GPCR regulation in which conserved amino acids within the RGS domainbind toconserved amino acids in the switch regionswithin subunitstoincrease GTP hydrolysis and terminate signalling(5). While progress has been made in our understanding of the roles of b-arrestins in b-cell function (6) very little is known about GRK and RGS expression and function in islets,and this will be examined in this PhD project to better understand the potential of targeting islet GPCRs to treat T2D. In addition, if data generated during this project demonstrate that inhibition of GRKs and/or RGS proteins improves insulin secretion and protects beta-cells from apoptosis this will be of importance in optimising islet functional mass prior to transplantation in T1D. The necessary techniques are in current use in the supervisors' labs (1,2, 7-11)and full training will be providedto Jessica.Aim of the investigation-This PhD project has 4 main objectives:1.Quantify mRNAs encoding GRKs and RGS proteins in human islets from low and high BMI donors and determine changes that occur under conditions of obesity.2.Identify cellular localisation of GRKs and RGS proteins in pancreas samples from lean and obese humans and mice.3.Determine the effects of selective GRK and RGS inhibitors on GPCR agonist-regulated islet function.4.Determine functional effects of deleting key GRKs and RGS proteinsin beta-cells.