Inhibition Of G Protein-coupled Receptor Kinase 2 Research Articles

FMRP Acts as a Key Messenger for Dopamine Modulation in the Forebrain

The fragile X mental retardation protein (FMRP) is an RNA-binding protein that controls translational efficiency and regulates synaptic plasticity. Here, we report that FMRP is involved in dopamine (DA) modulation of synaptic potentiation. AMPA glutamate receptor subtype 1 (GluR1) surface expression and phosphorylation in response to D1 receptor stimulation were reduced in cultured Fmr1(-/-) prefrontal cortex (PFC) neurons. Furthermore, D1 receptor signaling was impaired, accompanied by D1 receptor hyperphosphorylation at serine sites and subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in both PFC and striatum of Fmr1(-/-) mice. FMRP interacted with GRK2, and pharmacological inhibition of GRK2 rescued D1 receptor signaling in Fmr1(-/-) neurons. Finally, D1 receptor agonist partially rescued hyperactivity and enhanced the motor function of Fmr1(-/-) mice. Our study has identified FMRP as a key messenger for DA modulation in the forebrain and may provide insights into the cellular and molecular mechanisms underlying fragile X syndrome.

Role of G‐protein coupled receptor kinase‐2 (GRK2) in the modulation of renal vascular response in SHR by PPAR

G‐protein coupled receptor (GPCR) kinase‐2 (GRK‐2) has been impllicated in hypertension and GRK‐2 inhibition reduced blood pressure (BP). Peroxisome proliferators activated receptor γ (PPARγ), a transcription factor regulates GPCRs but the connection with GRK‐2 is not known. Here, we examined GRK‐2 role in PPARγ‐mediated improvement in SHR. SHR/WKY rats were treated with GW1929 (GW), a PPARγ ligand (0.5mg/kg; 2 month) and BP was monitored. Whole kidney perfusion was determined (laser dopler scanner) and renal vascular response was measured with or without heparin, a GRK‐2 inhibitor. In SHR, GW reduced BP (25±1%) and increased renal perfusion (59±3%). Phenylephrine (PE) vasoconstriction was greater in SHRs (15±1%) then WKY rats and GW abolished this. Heparin attenuated PE response in untreated but not GW‐treated SHR. GW enhanced Ach vasodilatation in SHR (41±1%) and this was improved by heparin (79±18%). GW enhanced SNP vasodilatation in SHR (35±6%) but remained unchanged with heparin. In SHR, GW enhanced PPARγ mRNA but reduced GRK‐2 mRNA. These results suggest that down‐regulation of PPARγ and up‐regulation of GRK‐2 may responsible for increased renal vascular reactivity of SHR and that PPARγ transcriptionally attenuated GRK‐2 function in hypertension.

G‐protein coupled receptor kinase 2(GRK2) is involved in μ‐receptor signaling in the mouse ileum but not colon

Repeated administration of morphine results in tolerance to antinociception but not constipation. Tolerance develops to the effects of morphine in the ileum but not the colon. The objective of this study was to identify the intracellular signaling mechanisms responsible for these differences in these two organs. Morphine (100 nM) inhibited field‐stimulation evoked (EFS) cholinergic contractions in longitudinal muscle preparations and produced atropine‐insensitive contractions in the circular muscle from ileum and colon. In the presence of the GRK2 inhibitor, methyl 5‐[2‐(5‐nitro‐2‐furyl)vinyl]‐2‐furoate (100 μM), morphine‐mediated inhibition of EFS contractions in the longitudinal muscle and direct contractions of circular muscle were abolished in the ileum but not the colon. The concentration‐response to acetylcholine in both tissues was not affected by the GRK2 inhibitor. Cell lysates from morphine treated ileum and colon were immunoprecipitated with anti‐GRK2 antibody. Immunoblot with anti‐μ‐receptor (MOR) antibody showed a band of ~70 kDa in the ileum but not the colon demonstrating interaction of MOR with GRK2 in the ileum but not colon and may form the basis for the differential tolerance development between the ileum and colon. Supported by DK46367, DA01647, DA020836

Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of beta-adrenergic receptor (betaAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting alpha2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial alpha2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein-coupled receptor kinase 2 (GRK2). Adrenal gland-specific GRK2 inhibition reversed alpha2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac betaAR signaling and function, and increased sympatholytic efficacy of a alpha2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

Cross-regulation of VPAC2 receptor desensitization by M3 receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.

G Protein-coupled Receptor Kinase 2–mediated Phosphorylation of Ezrin Is Required for G Protein-coupled Receptor–dependent Reorganization of the Actin Cytoskeleton

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.

Killing Two Kinases with One Inhibitor

RKIP (Raf kinase inhibitor protein) associates with and inhibits the enzymatic activity of the protein kinase Raf, an upstream kinase in MAPK (mitogen-activated protein kinase) signaling cascades. RKIP appears to mediate crosstalk with other pathways, and phosphorylation of RKIP by protein kinase C (PKC) relieves inhibition of Raf-1. Now Lorenz et al. report a more extensive role of RKIP in coordinating signaling through G protein-coupled receptors (GPCRs). Their experiments show that RKIP is also an inhibitor of GRK-2 (G protein-coupled receptor kinase 2), a kinase that diminishes signaling through GPCRs by uncoupling them from G proteins and promoting their internalization. In transfected cells and various native tissues, the amount of RKIP associated with GRK-2 was increased after stimulation of appropriate GPCRs and correlated with phosphorylation of RKIP at a site phosphorylated by PKC. Such phosphorylation appeared to be necessary for inhibition of GRK-2 because mutants of RKIP lacking the phosphorylated serine residue were not effective inhibitors of GRK-2. The increased association of RKIP with GRK-2 coincided with reduced association of RKIP with Raf-1. Thus, in unstimulated cells, RKIP appears to hold Raf-1 in an inactive state. Depletion of RKIP from cardiomyocytes by electroporation in the presence of specific antibodies inhibited signaling and contraction in response to stimulation of β-adrenergic receptors. The authors propose that phosphorylation of RKIP by PKC after stimulation of GPCRs then contributes in two ways to GPCR signaling: Release of RKIP from Ras-1 promotes signaling through MAPK and the released protein then inhibits GRK-1, thereby preventing desensitization and internalization of GPCRs. K. Lorenz, M. J. Lohse, U. Quitterer, Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 426 , 574-579 (2003). [Online Journal]

Expression of GRK2 is increased in the left ventricles of cardiomyopathic hamsters.

Reduced beta-adrenergic responsiveness in the heart is a characteristic feature of heart failure. G protein-coupled receptor kinase 2 (GRK2) phosphorylates beta-adrenoceptors in an agonist-dependent manner, causing receptor uncoupling and desensitisation. Elevated levels of both GRK2 mRNA and activity have been shown to occur in the failing human heart (Ungerer et al. (1992) Circulation 87: 454-463). We have analysed levels of GRK2 protein in heart tissue from the cardiomyopathic Syrian hamster CHF 147 and compared these to GRK2 levels in age-matched, non-cardiomyopathic control hamsters (CHF 148). GRK2 protein levels were found to be significantly increased in the left ventricles of the cardiomyopathic hamsters compared to the controls. The relative amounts of GRK2 in the cardiomyopathic hamsters, as compared to normal controls, increased with age from 2-fold at 100 days to 5-fold at 350 days. These animals should provide a useful model for testing the effect of GRK2 inhibitors on the development of heart failure.

Molecular modeling of G-protein coupled receptor kinase 2: docking and biochemical evaluation of inhibitors.

G-protein coupled receptor kinase 2 (GRK2) regulates the activity of many receptors. Because potent inhibitors of GRK2 are thus far limited to polyanionic compounds like heparin, we searched for new inhibitors with the aid of a molecular model of GRK2. We used the available crystal structure of cAMP dependent protein kinase (cAPK) as a template to construct a 3D homology model of GRK2. Known cAPK and GRK2 inhibitors were docked into the active sites of GRK2 and cAPK using DOCK v3.5. H8 docked into the hydrophobic pocket of the adenosine 5'-triphosphate (ATP) binding site of cAPK, consistent with its known competitive cAPK inhibition relative to ATP. Similarly, 3 of 4 known GRK2 inhibitors docked into the ATP binding pocket of GRK2 with good scores. Screening the Fine Chemicals Directory (FCD, containing the 3D structures of 13,000 compounds) for docking into the active sites of GRK2 identified H8 and the known GRK2 inhibitor trifluoperazine as candidates. Whereas H8 indeed inhibited light-dependent phosphorylation of rhodopsin by GRK2, but with low potency, 3 additional FCD compounds with promising GRK2 scores failed to inhibit GRK2. This result demonstrates limitations of the GRK2 model in predicting activity among diverse chemical structures. Docking suramin, an inhibitor of protein kinase C (not present in FCD) yielded a good fit into the ATP binding site of GRK2 over cAPK. Suramin did inhibit GRK2 with IC50 32 microM (pA26.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 microM]) or representing half of the suramin molecule (NF520) also inhibited GRK2 as predicted by docking. In conclusion, suramin and analogues are lead compounds in the development of more potent and selective inhibitors of GRK2.