G-protein-coupled receptor kinases (GRKs) comprise a family of seven mammalian serine/threonine protein kinases that phosphorylate and regulate agonist-occupied or constitutively active G-protein-coupled receptors (GPCRs). Studies of the details and consequences of these mechanisms have focused heavily on the original b-adrenoceptor kinase (b-ARK) family (GRK2 and GRK3) and, in particular, on phosphorylation-dependent recruitment of adaptor proteins such as the b-arrestins. However, recent work has indicated roles for the other, non-visual GRKs (GRK4, GRK5 and GRK6) and has revealed potential phosphorylation-independent regulation of GPCRs by GRK2 and GRK3. In this article, we review this newer information and attempt to put it into context with GRKs as physiological regulators that could be appropriate targets for future pharmacological intervention.G-protein-coupled receptors (GPCRs) constitute a very large family of heptahelical, integral membrane proteins that mediate a wide variety of physiological processes ranging from the transmission of light and odorant signals to the mediation of neurotransmission and hormonal actions [1,2]. GPCRs represent a major target for therapeutic agents, and the continuing identification of orphan GPCRs offers opportunity for future pharmacological and therapeutic development [3].Most GPCRs display a rapid loss of responsiveness in the continuing (or recurring) presence of an agonist or stimulus, and there is now substantial evidence that this process of desensitization, at least in part, is a consequence of ligand-induced phosphorylation of serine and threonine residues located within the C-terminal domain and/or the third intracellular loop of GPCRs. Two families of protein kinases appear to be predominantly involved: the G-protein-coupled receptor kinases (GRKs), which phosphorylate agonist-occupied GPCRs to mediate homologous receptor phosphorylation, and the second messengeractivated kinases, such as cAMP-dependent kinase (PKA) and protein kinase C (PKC), which can phosphorylate both ligand-bound and other inactive GPCRs in a heterologous manner [4,5]. Phosphorylation by GRKs enhances receptor affinity for non-visual b-arrestins 1 and 2 (arrestin2 and arrestin3), which not only sterically suppresses further interaction between the receptor and G proteins, but also initiates clathrin-mediated endocytosis of phosphorylated receptors and can promote the activation of additional signalling pathways by acting as agonist-regulated adaptor scaffolds [6].Other protein kinases have also been implicated in GPCR regulation. For example, evidence has accumulated that casein kinase 1a might bring about agonistmediated phosphorylation of acetylcholine muscarinic M 3 receptors and light-activated rhodopsin [7]. Casein kinase 1a-mediated phosphorylation does not appear to be associated with reduced responsiveness of the M 3 receptor but probably contributes to the activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) by this receptor [8,9].Although research in this area h...

AimsProlonged endothelin (ET) receptor signalling causes vasoconstriction and can lead to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. Usually, G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs), preventing prolonged or inappropriate signalling. This study investigated whether GRKs regulate ET receptor contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).Methods and resultsET-1-stimulated phospholipase C (PLC) activity and changes in [Ca2+]i were assessed using confocal microscopy in rat MSMCs transfected with the pleckstrin-homology domain of PLCδ1 (eGFP-PH) and loaded with Fura-Red. ET-1 applications (30 s) stimulated transient concentration-dependent eGFP-PH translocations from plasma membrane to cytoplasm and graded [Ca2+]i increases. ET-1-mediated PLC signalling was blocked by the type A endothelin receptor (ETAR) antagonist, BQ123. To characterize ETAR desensitization, cells were stimulated with a maximally effective concentration of ET-1 (50 nM, 30 s) followed by a variable washout period and a second identical application of ET-1. This brief exposure to ET-1 markedly decreased ETAR responsiveness to re-challenge, and reversal was incomplete even after increasing the time period between agonist challenges to 60 min. To assess GRK involvement in ETAR desensitization, MSMCs were co-transfected with eGFP-PH and catalytically inactive D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 constructs. D110A,K220RGRK2 expression significantly attenuated ETAR desensitization, whereas other constructs were ineffective. Small interfering RNA-targeted GRK2 depletion equally attenuated ETAR desensitization. Finally, immunocyotchemical data showed that ETAR activation recruited endogenous GRK2 from cytoplasm to membrane.ConclusionThese studies identify GRK2 as a key regulator of ETAR responsiveness in resistance arteries, highlighting the potential importance of this GRK isoenzyme in regulating vasoconstrictor signalling pathways implicated in vascular disease.

AimsProlonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5′-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).Methods and resultsMesenteric arteries contracted in response to UTP challenge: When an EC50 UTP concentration (30 µM, 5 min) was added 5 min before (R1) and after (R2) the addition of a maximal UTP concentration (Rmax: 100 µM, 5 min), R2 responses were decreased relative to R1, indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca2+-sensitive dyes. A similar protocol (R1/R2 = 10 µM; Rmax = 100 µM, applied for 30 s) revealed markedly reduced R2 when compared with R1 responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y2-receptor almost completely abolished UTP-stimulated IP3/Ca2+ signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype.ConclusionThese new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y2-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.