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G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson’s disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson’s disease.

Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca(2+)](i) release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein-coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca(2+)](i) response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca(2+)](i) response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca(2+)](i) response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin-mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.

The role of dopamine receptors in blood pressure regulation is well established. Genetic ablation of both dopamine D1-like receptor subtypes (D1, D5) and D2-like receptor subtypes (D2, D3, D4) results in a hypertensive phenotype in mice. This review focuses on the dopamine D1-like receptor subtypes D1 and D5 (especially D1 receptors), as they play a major role in regulating sodium homeostasis and blood pressure. Studies mostly describing the role of renal dopamine D1-like receptors are included, as the kidneys play a pivotal role in the maintenance of sodium homeostasis and the long-term regulation of blood pressure. We also attempt to describe the interaction between D1-like receptors and other proteins, especially angiotensin II type 1 and type 2 receptors, which are involved in the maintenance of sodium homeostasis and blood pressure. Finally, we discuss a new concept of renal D1 receptor regulation in hypertension that involves oxidative stress mechanisms.

The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.

G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.

A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 10(11)vg ml(-1) were used.

The dynorphin/kappa opioid receptor (KOR) system has been implicated as a critical component of the stress response. Stress-induced activation of dynorphin-KOR is well known to produce analgesia, and more recently, it has been implicated as a mediator of stress-induced responses including anxiety, depression, and reinstatement of drug seeking.Drugs selectively targeting specific KOR signaling pathways may prove potentially useful as therapeutic treatments for mood and addiction disorders.KOR is a member of the seven transmembrane spanning (7TM) G-protein coupled receptor (GPCR) superfamily. KOR activation of pertussis toxin-sensitive G proteins leads to Galphai/o inhibition of adenylyl cyclase production of cAMP and releases Gbetagamma, which modulates the conductances of Ca(+2) and K(+) channels. In addition, KOR agonists activate kinase cascades including G-protein coupled Receptor Kinases (GRK) and members of the mitogen-activated protein kinase (MAPK) family: ERK1/2, p38 and JNK. Recent pharmacological data suggests that GPCRs exist as dynamic, multi-conformational protein complexes that can be directed by specific ligands towards distinct signaling pathways. Ligand-induced conformations of KOR that evoke beta-arrestin-dependent p38 MAPK activation result in aversion; whereas ligand-induced conformations that activate JNK without activating arrestin produce long-lasting inactivation of KOR signaling.In this review, we discuss the current status of KOR signal transduction research and the data that support two novel hypotheses: (1) KOR selective partial agonists that do not efficiently activate p38 MAPK may be useful analgesics without producing the dysphoric or hallucinogenic effects of selective, highly efficacious KOR agonists and (2) KOR antagonists that do not activate JNK may be effective short-acting drugs that may promote stress-resilience.

Gタンパク質共役受容体キナーゼ（G Protein-coupled Receptor Kinase: GRK）の分子機能，分子構造，生理的役割を解説する．GRKは，Gタンパク質共役受容体（G Protein-Coupled Receptor: GPCR）をリン酸化するセリン・トレオニンキナーゼである．基本的な作用機構は，(1)アゴニスト結合した受容体（GPCR）がGRKを活性化，(2)活性化されたGRKが受容体をリン酸化，(3)リン酸化された受容体にアレスチン（arrestin）が結合，(4)その結果受容体の脱感受性（desensitization）が起こる，というものである．脱感受性は，受容体とGタンパク質との相互作用が損なわれる脱共役（uncoupling），受容体の細胞内への移行（internalization），細胞内移行した受容体の分解（down regulation）に区分される．Gqを介する反応をGRKがリン酸化非依存的に抑制する脱共役反応もある．受容体によるGタンパク質の活性化がonの反応であるのに対し，GRKの活性化による脱感受性はoffの反応である．一方，アレスチン上でMAPK（Mitogen Activated Protein Kinase）への情報伝達が起こることが報告されているが，この場合はGRKがonの反応を誘起していることになる．最近，GRKは多数の細胞内タンパク質や脂質と相互作用することが明らかになっている．細胞内シグナル伝達系の情報集積部位としての役割も推測されている．GRKは，網膜変性症，高血圧，心不全，リュウマチ性関節炎，アヘン耽溺などに関わると推測されている．

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is a major mechanism of desensitization of these receptors. GPCR activation of GRKs involves an allosteric site on GRKs distinct from the catalytic site. Although recent studies have suggested an important role of the N- and C-termini and domains surrounding the kinase active site in allosteric activation, the nature of that site and the relative roles of the RH domain in particular remain unknown. Based on evolutionary trace analysis of both the RH and kinase domains of the GRK family, we identified an important cluster encompassing helices 3, 9, and 10 in the RH domain in addition to sites in the kinase domain. To define its function, a panel of GRK5 and -6 mutants was generated and screened by intact-cell assay of constitutive GRK phosphorylation of the β2-adrenergic receptor (β2AR), in vitro GRK phosphorylation of light-activated rhodopsin, and basal catalytic activity measured by tubulin phosphorylation and autophosphorylation. A number of double mutations within helices 3, 9, and 10 reduced phosphorylation of the β2AR and rhodopsin by 50 to 90% relative to wild-type GRK, as well as autophosphorylation and tubulin phosphorylation. Based on these results, helix 9 peptide mimetics were designed, and several were found to inhibit rhodopsin phosphorylation by GRK5 with an IC50 of ∼30 μM. In summary, our studies have uncovered previously unrecognized functionally important sites in the regulator of G-protein signaling homology domain of GRK5 and -6 and identified a peptide inhibitor with potential for specific blockade of GRK-mediated phosphorylation of receptors.

Altered response to acute catecholamine increase in the synaptic cleft is considered to be the mechanism underlying transient left ventricular apical ballooning syndrome (LVABS).We postulated that polymorphisms associated with different bAR responsiveness might play a role in the risk of LVABS. We prospectively enrolled 22 consecutive LVABS patients (4 men, age63+13 years) admitted to the Intensive Coronary Care unit of the AOP Federico II, Naples, Italy between 2005 and 2008. Peripheral blood samples were collected from each patient for genetic analysis. The results were compared with those of 740 outpatients (control) aged 50+0.4 years, 57.8% male. The following polymorphisms were analysed: rs35230616 and rs1801253 for β1AR; rs1042713, rs1042714, and rs1800888 for β 2AR; rs11554276 for Gs-protein alpha subunit (GNAS);rs17098707 and rs34679178 for GRK5.The prevalence of polymorphisms of β1AR, β2AR, and GNAS were similar between patients and controls. Conversely, the percentage of LVABS patients who presented with the rs17098707 polymorphism of the GRK5 gene was significantly higher. This is the first study demonstrating a link between a polymorphism of one of the protein kinases most expressed in the heart and stress-induced acute ventricular dysfunction. The possibility of recurrence and case reports evaluating LVABS in relatives, support a genetic approach to the pathophysiology of this syndrome.

Differences in the mechanisms underlying tolerance and mu-opioid receptor desensitization resulting from exposure to opioid agonists of different efficacy have been suggested previously. The objective of this study was to determine the effects of protein kinase C (PKC) and G protein-coupled receptor kinase (GRK) inhibition on antinociceptive tolerance in vivo to opioid agonists of different efficacy. A rapid (8-h) tolerance-induction model was used where each opioid was repeatedly administered to naive mice. Animals were then challenged with the opioid after injection of a kinase inhibitor to determine its effects on the level of tolerance. Tolerance to meperidine, morphine, or fentanyl was fully reversed by the PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole (Gö6976). However, in vivo tolerance to [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors beta-adrenergic receptor kinase 1 inhibitor and 2-(8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide (Ro 32-0432) did not reverse the tolerance to meperidine, fentanyl, or morphine but did reverse the tolerance to DAMGO. To correlate GRK-dependent DAMGO-induced tolerance with mu-opioid receptor desensitization, we used in vitro whole-cell patch-clamp recording from mouse locus coeruleus neurons and observed that the GRK inhibitors reduced DAMGO-induced desensitization of mu-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy mu-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

Background The genetic expression of adrenergic receptors plays an important pathophysiologic role in heart failure. G protein–coupled receptor kinases (GRKs) desensitize the β-receptor to catecholaminergic stimulation. It has been suggested that their mRNA expression in peripheral lymphocytes could mirror the changes in their myocardial expression in the failing heart, but this relationship between the myocyte and lymphocyte has not been studied in heart transplantation (HT). The objective of this study was to analyze adrenergic receptor and GRK mRNA expression in myocardium and lymphocytes and their correlation. Methods Twenty-three HT patients without evidence of acute rejection or echocardiographic dysfunction were assessed. Myocardial biopsy samples and peripheral blood lymphocytes were obtained, and α 1 - and β-adrenoceptor subtype and GRK subtype mRNA was analyzed using reverse transcript–polymerase chain reaction (RT-PCR). Results Mean age was 45 ± 15 years, with a median of time since HT of 205 (351) days. In biopsies, the β 1 /β 2 -adrenoceptor ratio was 57%/42%, and GRK5 was the most commonly expressed, followed by GRK2. In lymphocytes, the β 1 /β 2 ratio was 3%/96%, whereas GRK2 mRNA expression was greater than that of other subtypes. There was no correlation between myocardial and lymphocyte parameters. There were no correlations with clinical variables, but lymphocyte β 2 and GRK2 were increased with time since HT. Conclusions In the transplanted heart, there is no correlation between mRNA expression of adrenoceptors and GRKs in myocardium and peripheral lymphocytes. With time since transplant, mRNA expression of lymphocyte but not myocardial β 2 -adrenoceptor and GRK2 increases. Therefore, this dissociation between myocardial and lymphocyte mRNA expression limits the potential use of peripheral blood samples for diagnosis of graft dysfunction.

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. beta(2)ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G(s) and G(i) proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the beta(2)AR coupling to G(i) proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the beta(2)AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G(i) inhibitor pertussis toxin (PTX). Further studies revealed that activated beta(2)ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the beta(2)AR blocked receptor/G(i) coupling. However, a sufficient beta(2)AR/G(i) coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (or = 100 nm). Disruption of the GRK phosphorylation sites on the beta(2)AR blocked receptor internalization and coupling to G(i) proteins, probably by preventing the receptor's transportation to access G(i) proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G(i)-specific inhibitor, G(i)CT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the beta(2)AR for agonist dose-dependent coupling to G(i) proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3beta, a cytosolic kinase for phosphorylation of a downstream effector beta-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of beta-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished beta-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Opiates, including morphine, are widely used drugs for antinociception in clinics. Prolonged treatments of opioids induce both tolerance and dependence, which are the major side effects of opioid therapy. One of the mechanisms for the development of tolerance and dependence is implicated to be opioid-receptor trafficking. Here we review the current understandings of opioid-receptor phosphorylation, endocytosis and desensitization after repeated agonist treatments. Also, the role of G-protein coupled receptor kinases in opioid-receptor phosphorylation is discussed. How to associate these observations to physiological and behavioral changes in animal models and clinics is still under investigation.

Phosphorylation by protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs) desensitize β 2 -adrenergic receptor (β 2 AR) signaling, and these are thought to be mechanisms involved with cell and organ homeostasis and tolerance to agonists. However, there is little direct evidence that these events are relevant to β 2 AR physiological function, such as airway smooth muscle (ASM) relaxation leading to bronchodilation. To maintain cell- and receptor-specificity without altering the natural complement of kinases/arrestins, transgenic mice were generated expressing the human WT and mutated β 2 ARs lacking PKA and/or GRK phosphorylation sites on ASM at ≈4-fold over background. Functional gains in response to β-agonist from the selective loss of these mechanisms were determined in mouse airways. Relaxation kinetics were altered in all mutant airways compared with β 2 WT. At low receptor occupancy, β 2 PKA(-) had enhanced agonist-promoted relaxation, while β 2 GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with β 2 GRK(-), with no evidence for additivity when PKA sites were also removed. For the full range of responses, the β 2 PKA(-)/GRK(-) airways had the greatest relaxation efficiency, indicating a graded effect of GRKs as agonist concentration increased. ASM cAMP levels paralleled relaxation phenotypes. No interaction between PKA phosphorylation of β 2 AR and GRK-promoted events was identified by β-arrestin-2 recruitment. Thus, these two mechanisms indeed impact a relevant β 2 AR physiologic function, acting as attenuators of the acute response, and represent specific interfaces where adjunct therapy or biased ligands may improve β-agonist treatment of obstructive lung disease.

Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [3H]inositol phosphate accumulation and intracellular Ca2+ concentration ([Ca2+]i); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific ≥75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

The majority of alpha-synuclein (alphaS) deposited in Lewy bodies, the pathological hallmark of Parkinson's disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of alphaS has been demonstrated to enhance the alphaS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of alphaS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of alphaS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of alphaS; however, knockdown of GRK2 or GRK5 did not decrease alphaS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of alphaS in HEK293 cells.

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs and initiate their desensitization. Many prior studies suggest that activated GPCRs dock to an allosteric site on the GRKs and thereby stimulate kinase activity. The extreme N-terminal region of GRKs is clearly involved in this process, but its role is not understood. Using our recent structure of bovine GRK1 as a guide, we generated mutants of solvent-exposed residues in the GRK1 kinase domain that are conserved among GRKs but not in the extended protein kinase A, G, and C family and evaluated their catalytic activity. Mutation of select residues in strands beta1 and beta3 of the kinase small lobe, alphaD of the kinase large lobe, and the protein kinase A, G, and C kinase C-tail greatly impaired receptor phosphorylation. The most dramatic effect was observed for mutation of an invariant arginine on the beta1-strand (approximately 1000-fold decrease in k(cat)/K(m)). These residues form a continuous surface that is uniquely available in GRKs for protein-protein interactions. Surprisingly, these mutants, as well as a 19-amino acid N-terminal truncation of GRK1, also show decreased catalytic efficiency for peptide substrates, although to a lesser extent than for receptor phosphorylation. Our data suggest that the N-terminal region and the newly identified surface interact and stabilize the closed, active conformation of the kinase domain. Receptor binding is proposed to promote this interaction, thereby enhancing GRK activity.

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.

G-protein receptor kinases (GRKs) are indispensable for terminating signaling of G-protein coupled receptors (GPCR) through receptor desensitization and downregulation. Increased neurohormone levels in heart failure and the adverse consequences of constant neurohormonal stimulation suggest an important protective role for mechanisms that desensitize neurohormone receptor responses. For that reason, GRK2, the first GRK identified in the heart, has been extensively studied in heart failure, cardiac hypertrophy, and myocardial infarction. However, our understanding of the roles of GRKs in general, and the differential effects of cardiac receptor phosphorylation by individual cardiac-expressed GRKs, have evolved considerably in the last few years. Here, recent developments are reviewed, with an emphasis on novel GRK functions and signaling pathways.

We examined the regulation of mGlu2 and mGlu3 metabotropic glutamate receptor signaling prompted by the emerging role of these receptor subtypes as therapeutic targets for psychiatric disorders, such as anxiety and schizophrenia. In transfected human embryonic kidney 293 cells, G-protein-coupled receptor kinase (GRK) 2 and GRK3 fully desensitized the agonist-dependent inhibition of cAMP formation mediated by mGlu3 receptors. In contrast, GRK2 or other GRKs did not desensitize the cAMP response to mGlu2 receptor activation. Desensitization of mGlu3 receptors by GRK2 required an intact kinase activity, as shown by the use of the kinase-dead mutant GRK2-K220R or the recombinant GRK2 C-terminal domain. Overexpression of beta-arrestin1 also desensitized mGlu3 receptors and did not affect the cAMP signaling mediated by mGlu2 receptors. The difference in the regulation of mGlu2 and mGlu3 receptors was signal-dependent because GRK2 desensitized the activation of the mitogen-activated protein kinase pathway mediated by both mGlu2 and mGlu3 receptors. In vivo studies confirmed the resistance of mGlu2 receptor-mediated cAMP signaling to homologous desensitization. Wild-type, mGlu2(-/-), or mGlu3(-/-) mice were treated intraperitoneally with saline or the mixed mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]-exhane-4,6-dicarboxylic acid (LY379268; 1 mg/kg) once daily for 7 days. Inhibition of forskolin-stimulated cAMP formation by LY379268 was measured in cortical slices prepared 24 h after the last injection. Agonist pretreatment fully desensitized the cAMP response in wild-type and mGlu2(-/-) mice but had no effect in mGlu3(-/-) mice, in which LY379268 could only activate the mGlu2 receptor. We predict the lack of tolerance when mixed mGlu2/3 receptor agonists or selective mGlu2 enhancers are used continually in patients.

Post-inflammatory pain is a poorly understood phenomenon. G protein-coupled receptors are involved in regulating pain signaling in the context of inflammation. G protein-coupled receptor kinases (GRK) modulate signaling through these receptors. We investigated whether GRK6 contributes to post-inflammatory visceral hyperalgesia. Colitis was induced in female mice by 1% dextran sodium sulphate in drinking water for 7 days. Disease score, colon length, and colonic cytokines were determined. On day 49, when animals had recovered from colitis, we induced visceral pain by intracolonic capsaicin instillation. Behavioral responses to capsaicin were monitored for 20 min. Referred hyperalgesia was measured using von Frey hairs. Spinal cord c-Fos was visualized by immunohistochemistry. In contrast to our earlier observations in male GRK6-/- and wild type (WT) mice, we did not detect differences in the course of colitis or in expression of colonic cytokines between female GRK6-/- and WT mice. After recovery from colitis, capsaicin-induced behavioral pain responses and spinal cord c-Fos expression were more pronounced in female GRK6-/- than WT mice. Naive GRK6-/- and WT animals did not differ in pain and c-Fos responses to capsaicin. Capsaicin-induced referred hyperalgesia post-colitis was increased in GRK6-/- compared to WT mice. However, referred hyperalgesia post-colitis was not affected by ablation of GRK6. Furthermore, in vitro IL-1beta sensitized the capsaicin receptor TRPV1 and this process was inhibited by over-expression of GRK6. We describe the novel concept that GRK6 inhibits post-inflammatory visceral hyperalgesia but does not contribute to visceral pain in naive animals. We propose that GRK6 regulates inflammation-induced sensitization of TRPV1.

The retina specific G protein-coupled receptor kinases, GRK1 and GRK7, have been implicated in the shutoff of the photoresponse and adaptation to changing light conditions via rod and cone opsin phosphorylation. Recently, we have defined sites of phosphorylation by cAMP-dependent protein kinase (PKA) in the amino termini of both GRK1 and GRK7 in vitro. To determine the conditions under which GRK7 is phosphorylated in vivo, we have generated an antibody that recognizes GRK7 phosphorylated on Ser-36, the PKA phosphorylation site. Using this phospho-specific antibody, we have shown that GRK7 is phosphorylated in vivo and is located in the cone inner and outer segments of mammalian, amphibian and fish retinas. Using Xenopus laevis as a model, GRK7 is phosphorylated under dark-adapted conditions, but becomes dephosphorylated when the animals are exposed to light. The conservation of phosphorylation at Ser-36 in GRK7 in these different species (which span a 400 million-year evolutionary period), and its light-dependent regulation, indicate that phosphorylation plays an important role in the function of GRK7. Our work demonstrates for the first time that cAMP can regulate proteins involved in the photoresponse in cones and introduces a novel mode of regulation for the retinal GRKs by PKA.

The European Society of Cardiology recommends that beta-blockers should be considered for treating all patients with stable, mild, moderate, or severe heart failure (HF) who are receiving standard treatment, unless there is a contraindication. Despite the significant benefit of the drug, there is widespread recognition of patient-to-patient variability in drug response. The genetic determinants of responses to drugs have important implications for the clinical course and management of HF. Pharmacogenetics (PGt) has drawn great attention for its potential to redirect personal care and public health paradigms. The aim of this review was to gather information on PGt of beta-blockers in HF treatment.We searched for articles related to PGt of beta-blockers in the PubMed database and attempted to cover all related articles.Several genetic polymorphisms affecting proteins in the beta-adrenergic receptor signaling pathway have been proposed as modifiers of HF risk. The most relevant of these to this review is the pharmacogenetic interactions between the genetic variants of catecholamine receptors or their effectors and beta-blockade for the treatment of HF.Interindividual variability of responsiveness to beta-blockers can be explained by PGt data of adrenaline-related genes. To demonstrate that pharmacogenetic intervention produces successful individualized beta-blocker treatment for HF patients, prospective, randomized, and pharmacogenomics (PGx)-based clinical trials are required. Our assessment is that we are already at a turning point in the history of clinical pharmacology.

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to Gq/11proteins upon PTH stimulation predominantly caused by elimination of Ser491/492/493, Ser501, or Ser504. Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to Gq/11proteins at least partially by direct binding to Gq/11proteins.

Hypertension is a prevalent condition in the developed world and disease severity is directly correlated with additional cardiovascular complications. It is estimated that 30% of the adult population in the United States has hypertension, which is classified as a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure ≥90 mmHg. A prolonged increase in afterload ultimately leads to congestive heart failure in the majority of cases. Currently, medication designed to treat hypertension is inadequate, thus new therapies need to be explored. Blood pressure is tightly regulated by blood vessel radius, which is established by hormones and/or peptides binding to GPCRs (G-protein-coupled receptors). Catecholamines and peptide hormones, such as AngII (angiotensin II), are elevated in hypertension and, therefore, signalling by these GPCRs is increased. Their signalling is tightly controlled by a class of proteins, the GRKs (GPCR kinases). Elevated levels of either GRK2 or GRK5 in both the lymphocytes and VSM (vascular smooth muscle) are associated with human hypertension and animal models of the disease. The focus of the present review is on the role GRKs, and their regulation of GPCRs, play in high blood pressure.

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Initially thought to play a role only in G-protein-coupled receptor desensitization, β-arrestins are ascribed with new roles such as scaffolding and signaling proteins by their own right. This review explores the many functions of β-arrestins, with an emphasis on their recently identified role as regulators of receptor signaling.

Dopamine and other G protein-coupled receptors (GPCRs) represent the major target of antipsychotic drugs. GPCRs undergo desensitization via activation-dependent phosphorylation by G protein-coupled receptor kinases (GRKs) followed by arrestin binding. Arrestins and GRKs are major regulators of GPCR signaling. We elucidated changes in expression of two arrestins and four GRKs following chronic (21 days) treatment with haloperidol (1 mg/kg i.p.) or clozapine (20 mg/kg i.p.) 2 or 24 h after the last injection in 11 brain regions. Haloperidol decreased GRK3 in ventrolateral caudate-putamen and transiently down-regulated GRK5 in globus pallidus and caudal caudate-putamen. Clozapine also caused a short-term suppression of the GRK5 expression in the caudal caudate-putamen and globus pallidus, but, unlike haloperidol, elevated GRK5 in the caudal caudate-putamen after 24 h. Unlike haloperidol, clozapine decreased arrestin2 and GRK3 in hippocampus and GRK3 in globus pallidus but increased arrestin2 in the core of nucleus accumbens and ventrolateral caudate-putamen and GRK2 in prefrontal cortex. Clozapine, but not haloperidol, induced long-term activation of extracellular signal-regulated kinase (ERK) 2 in ventrolateral caudate-putamen and transient in prefrontal cortex. The data demonstrate that haloperidol and clozapine differentially affect the expression of arrestins and GRKs and ERK activity, which may play a role in determining their clinical profile.

The recent, unfortunate death of Alfred G. ("Al") Gilman, M.D., Ph.D., represents a sad signpost for an era spanning over 40 years in molecular pharmacology. Gilman's discoveries, influence, and persona were dominant forces in research and training in pharmacology. Here, we review the progression of ideas and knowledge that spawned early work by Gilman and collaborators (among them, one of the authors) and later efforts (including those of the other author) that have recently yielded a comprehensive and precise structural understanding of fundamental topics in pharmacology: the binding of ligands to G protein-coupled receptors (GPCRs) and the interaction of GPCRs with heterotrimeric G proteins and effector molecules. Those data provide new and important insights into the molecular basis that underlies affinity and efficacy, two of the most important features of drug action, which represent the latest chapter in the saga that Al Gilman's work helped launch.

Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels., High aldosterone levels cause heart damage independently of its well-known effect on blood pressure. Here, Cannavo et al. show that aldosterone-mediated cardiac pathology involves G protein-coupled receptor (GPCR) kinase 2 (GRK2) and GRK5 that integrate signals from angiotensin II receptor (AT1R).

Oxytocin is a potent uterotonic agent and is used clinically for induction and augmentation of labor, as well as for prevention and treatment of postpartum hemorrhage. Oxytocin increases uterine contractility by activating the oxytocin receptor (OXTR), a member of the G protein-coupled receptor family, which is prone to molecular desensitization. After oxytocin binding, the OXTR is phosphorylated by a member of the G protein-coupled receptor kinase (GRK) family, which allows for recruitment of β-arrestin, receptor internalization, and desensitization. According to previous in vitro analyses, desensitization of calcium signaling by the OXTR is mediated by GRK6. The objective of this study was to determine the role of GRK6 in mediating uterine contractility. Here, we demonstrate that uterine GRK6 levels increase in pregnancy and using a telemetry device to measure changes in uterine contractility in live mice during labor, show that mice lacking GRK6 produce a phenotype of enhanced uterine contractility during both spontaneous and oxytocin-induced labor compared with wild-type or GRK5 knockout mice. In addition, the observed enhanced contractility was associated with high rates of term stillbirth. Lastly, using a heterologous in vitro model, we show that β-arrestin recruitment to the OXTR, which is necessary for homologous OXTR desensitization, is dependent on GRK6. Our findings suggest that GRK6-mediated OXTR desensitization in labor is necessary for normal uterine contractile patterns and optimal fetal outcome.

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCβ. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G proteincoupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal thatGαq/ PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling., Ministerio de Educación y Ciencia (SAF2011-23800, SAF2014-55511-R), Fundación Ramón Areces, The Cardiovascular Diseases Network of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid (S-2011/BMD-2332), and Instituto de Salud Carlos III (PI11/00126, PI14/00201) (to F. M. and C. R.). This work was also supported in part by the NIGMS, National Institutes of Health Grant R01-GM088242 (to G. T.), the Canadian Institutes of Health Research (CIHR) (MOP-GMX-231013) (to S. M.), an EMBO Short Fellowship (to G. S. F.), and Fondo Europeo de Desarrollo Regional (FEDER, European Union)

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.

G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

Receptors for hormones, neurotransmitters, drugs, sensory stimuli and many other agents represent the gateway to cellular metabolism and activity. They regulate virtually all physiological processes in mammals. Yet as recently as 40 years ago their very existence was still in question. One class of receptors, those coupled to G proteins (also known as GPCRs or seven transmembrane receptors) comprise by far the largest group (approx. 1000), and are the most important target of clinically used drugs. Here I provide a very personal retrospective of research over the past 35 years which ultimately led to the identification, purification, reconstitution and cloning of the adrenergic receptors; the discovery of their homology with the seven transmembrane spanning visual light receptor rhodopsin and the realization that there was a large gene family of G protein coupled receptors; the elucidation of the molecular mechanisms of receptor desensitization and signalling through G protein-coupled receptor kinases and beta-arrestins; and the appreciation that the structure, signalling, and regulatory mechanisms of the receptors are all highly conserved across the large receptor superfamily.

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck cancers with high invasiveness and poor prognosis. To identify targeted therapeutics against metastasis, a better understanding of the regulation of HSCC cell invasion is needed. In recent years, G protein-coupled receptor kinases (GRKs) have been implicated in cancer metastasis through phosphorylation of the activated form of G protein coupled receptors (GPCRs). However, there is little information regarding GRKs expression in HSCC. In the present study, we examined GRK6 expression in HSCC and also assessed the possible cause of its aberrant expression, as well as its clinical significance. Real-time quantitative PCR (qPCR) and western blotting were performed to analyze the expression of GRK6 in HSCC tissues and corresponding non-malignant tissues. Subsequently, paired HSCC and corresponding non-malignant tissues were evaluated for the methylation status of GRK6 gene promoter using methylation-specific PCR (MSP). Furthermore, we investigated the methylation status and the clinicopathological significance of GRK6 in 45 paired HSCC and corresponding non-malignant tissues. The suppression of GRK6 in hypopharyngeal cell line FaDu by GRK6-shRNA lentivirus transfection was utilized to detect the role of GRK6 in hypopharyngeal cancer progression. Our results showed that the expression of GRK6 mRNA and protein was significantly lower in HSCC than in corresponding adjacent non-tumor tissues, and this downregulation was found to be in accordance with aberrant methylation of the gene. Hypermethylation of the gene was observed in 77.8% (35/45) of the HSCC tissues, while it was found in only 42.2% (19/45) of the corresponding non-malignant tissues. GRK6 methylation was related to depth of tumor invasion and TNM stage. Upon treatment with 5-aza-2'-deoxycytidine, GRK6 expression was upregulated in FaDu cells, and cell invasion was signinficantly inhibited. Furthermore, the suppression of GRK6 by shRNA transfection enhanced FaDu cells invasion. Our results indicate that the aberrant methylation of GRK6 gene promoter may underlie its downregulation in HSCC, and may play an important role in the metastasis of HSCC.

Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors., G-protein-coupled receptors (GPCRs) signal via G proteins or arrestin-mediated pathways; the plasticity of arrestin proteins is thought to underlie their function. Here, the authors use NMR to examine how β-arrestin-1 recognizes different GPCR phospho-barcodes, and how this triggers structural rearrangements to fulfill selective functions.

The neuronal Ca 2+ -binding protein Recoverin has been shown to regulate phototransduction termination in mammalian rods. Here we identify four recoverin genes in the zebrafish genome, rcv1a , rcv1b , rcv2a and rcv2b , and investigate their role in modulating the cone phototransduction cascade. While Recoverin-1b is only found in the adult retina, the other Recoverins are expressed throughout development in all four cone types, except Recoverin-1a, which is expressed only in rods and UV cones. Applying a double flash electroretinogram (ERG) paradigm, downregulation of Recoverin-2a or 2b accelerates cone photoresponse recovery, albeit at different light intensities. Exclusive recording from UV cones via spectral ERG reveals that knockdown of Recoverin-1a alone has no effect, but Recoverin-1a/2a double-knockdowns showed an even shorter recovery time than Recoverin-2a-deficient larvae. We also showed that UV cone photoresponse kinetics depend on Recoverin-2a function via cone-specific kinase Grk7a. This is the first in vivo study demonstrating that cone opsin deactivation kinetics determine overall photoresponse shut off kinetics.

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.

Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the Vmax value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency.

The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.

We describe the 2.6-A crystal structure of human G protein-coupled receptor kinase (GRK)-6, a key regulator of dopaminergic signaling and lymphocyte chemotaxis. GRK6 is a member of the GRK4 subfamily of GRKs, which is represented in most, if not all, metazoans. Comparison of GRK6 with GRK2 confirms that the catalytic core of all GRKs consists of intimately associated kinase and regulator of G protein signaling (RGS) homology domains. Despite being in complex with an ATP analog, the kinase domain of GRK6 remains in an open, presumably inactive conformation, suggesting that G protein-coupled receptors activate GRKs by inducing kinase domain closure. The structure reveals a putative phospholipid-binding site near the N terminus of GRK6 and structural elements within the kinase substrate channel that likely influence G protein-coupled receptor access and specificity. The crystalline GRK6 RGS homology domain forms an extensive dimer interface using conserved hydrophobic residues distinct from those in GRK2 that bind Gαq, although dimerization does not appear to occur in solution and is not required for receptor phosphorylation.

SummaryPhosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of grk7 in zebrafish and knocked down gene expression of grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.

The deactivation of visual pigments involved in phototransduction is critical for recovering sensitivity after exposure to light in rods and cones of the vertebrate retina. In rods, phosphorylation of rhodopsin by rhodopsin kinase (GRK1) and the subsequent binding of visual arrestin completely terminates phototransduction. Although signal termination in cones is predicted to occur via a similar mechanism as in rods, there may be differences due to the expression of related but distinct gene products. While rods only express GRK1, cones in some species express only GRK1 or GRK7 and others express both GRKs. In the mouse, cone opsin is phosphorylated by GRK1, but this has not been demonstrated in mammals that express GRK7 in cones. We compared cone opsin phosphorylation in intact retinas from the 13-lined ground squirrel (GS) and pig, cone- and rod-dominant mammals, respectively, which both express GRK7. M opsin phosphorylation increased during continuous exposure to light, then declined between 3 and 6 min. In contrast, rhodopsin phosphorylation continued to increase during this time period. In GS retina homogenates, anti-GS GRK7 antibody blocked M opsin phosphorylation by 73%. In pig retina homogenates, only 20% inhibition was observed, possibly due to phosphorylation by GRK1 released from rods during homogenization. Our results suggest that GRK7 phosphorylates M opsin in both of these mammals. Using an in vitro GTPgammaS binding assay, we also found that the ability of recombinant M opsin to activate G(t) was greatly reduced by phosphorylation. Therefore, phosphorylation may participate directly in the termination of phototransduction in cones by decreasing the activity of M opsin.

The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

Dysregulation of dopamine receptors (DARs) is believed to contribute to Parkinson disease (PD) pathology. G protein-coupled receptors (GPCR) undergo desensitization via activation-dependent phosphorylation by G protein-coupled receptor kinases (GRKs) followed by arrestin binding. Using quantitative Western blotting, we detected profound differences in the expression of arrestin2 and GRKs among four experimental groups of nonhuman primates: (1) normal, (2) parkinsonian, (3) parkinsonian treated with levodopa without or (4) with dyskinesia. Arrestin2 and GRK6 expression was significantly elevated in the MPTP-lesioned group in most brain regions; GRK2 was increased in caudal caudate and internal globus pallidus. Neither levodopa-treated group differed significantly from control. The only dyskinesia-specific change was an elevation of GRK3 in the ventral striatum of the dyskinetic group. Changes in arrestin and GRK expression in the MPTP group were accompanied by enhanced ERK activation and elevated total ERK expression, which were also reversed by L-DOPA. The data suggest the involvement of arrestins and GRKs in Parkinson disease pathology and the effects of levodopa treatment.