M ₃ -muscarinic receptor promotes insulin release via receptor phosphorylation/arrestin-dependent activation of protein kinase D1

Abstract

The activity of G protein-coupled receptors is regulated via hyperphosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M 3 -muscarinic receptor, a prototypical G q/11 -coupled receptor. This mutant mouse strain was used here to investigate the role of M 3 -muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G q/11 -signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and β-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M 3 -muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of β-arrestin to the phosphorylated M 3 -muscarinic receptor. In conclusion, our findings support the unique concept that M 3 -muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/ arrestin-dependent coupling of the receptor to protein kinase D1. G-protein coupled receptor | ligand biasT he vast majority of G protein-coupled receptors (GPCRs) respond to agonist occupation by becoming rapidly hyperphosphorylated within intracellular domains (1-3). This process not only leads to the uncoupling of the receptor from its cognate G proteins, but also allows for the activation of G proteinindependent signaling, a process that is driven largely by the recruitment of β-arrestin adaptor proteins (4-7). As a consequence, GPCRs regulate an extensive array of signaling pathways and biological responses (3). G protein-independent signaling pathways have mostly been studied in recombinant systems. However, the current challenge is to understand to what extent these processes are involved in the regulation of key physiological responses.In the present study, we examined the in vivo role of GPCR phosphorylation by generating a knock-in mouse strain expressing a phosphorylation-deficient GPCR. Specifically, we used the M 3 -muscarinic acetylcholine receptor, a prototypic G q/11 -coupled GPCR, as a model system (8, 9). We and others have previously demonstrated that the M 3 -muscarinic receptor is rapidly phosphorylated on agonist occupation by a range of protein kinases that include members of the GPCR kinase (GRK) family, as well as casein kinase 1α and protein kinase CK2 (10-13). To define the potential physiological role of M 3 -muscarinic receptor phosphorylation, we generated a mouse strain where the wild-type M 3 -muscarinic receptor gene had been replaced by a phosphorylationdeficient mutan...

G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1

“…Oleate phosphorylates PKD via GPR40 PKD is a DAGsensitive kinase involved in the regulation of insulin secretion in response to the M3-muscarinic receptor [28,29]. There are three isoforms of PKD (PKD1-3) encoded by three different genes.…”

“…The role of PKD in beta cell function was first demonstrated by Sumara et al [29], who showed that PKD1 was negatively regulated by the δ isoform of p38 mitogen-activated protein kinase and promoted both insulin secretion and beta cell survival. Kong et al [28] further showed that PKD1 mediates insulin secretion in response to muscarinic stimulation. Here, we provide unique evidence that PKD is also activated rapidly, in a GPR40-dependent manner, in response to oleate.…”

“…In beta cells, however, our [43,44] and other [45] previous observations that glucose and phorbol esterstimulated insulin secretion are unaltered by deletion or overexpression of a kinase-negative isoform of PKCδ, as well as the current results showing normal fatty-acid potentiation of GSIS in Prkcd −/− islets, suggest that PKCδ is unlikely involved, in this context, in trans-phosphorylation of PKD. Alternatively, it is possible that GPR40 signalling activates PKD via G αq -dependent autophosphorylation [31,46] or, as shown for M3-muscarinic receptor stimulation of insulin release, by G-protein-independent, β-arrestindependent activation [28]. However, since exogenous DAG mimics the effect of oleate on PKD activation and insulin secretion, and since inhibition of G αq/11 inhibits fatty-acid potentiation of GSIS [7], it is likely that GPR40 signalling involves a G αq/11 -dependent pathway rather than a G-protein independent mechanism.…”

A stress response pathway in mice upregulates somatostatin level and transcription in pancreatic delta cells through Gs and β-arrestin 1

“…New studies have revealed that β-arrestin 1 is a major downstream mediator of GPCRs, such as β2AR, GLP-1R, M3R and A1TaR, and is involved in genome stability, pancreatic beta cell function, podocyte activation and renal injury [5,19,35,36]. However, the functional importance of β-arrestin 1 in pancreatic delta cells had not been investigated.…”

β-Arrestin-Mediated Signaling Improves the Efficacy of Therapeutics

“…This may lead to the discovery of more efficient therapies against Alzheimer disease. In the pancreas, M 3 -muscarinic receptor activation augments sustained insulin secretion by a G protein-independent but β-arrestin-dependent pathway through M 3 -muscarinic receptor and protein kinase D1 activation (29). Selective activation of β-arrestinmediated signaling will be a novel way to improve the control of blood glucose levels in diabetic patients.…”