Islet GPCR regulation by GRKs and RGS proteins

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.