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 μM (pA₂ 6.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 μM]) 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.     

Therapeutic potential of G-protein coupled receptor kinases in the heart

The actions of G-protein coupled receptor kinases (GRKs) critically regulate β-adrenergic receptor (βAR) signalling. In the cardiovascular system, the βAR signalling pathway controls important responses of the heart such as the ability to contract (inotropy), the ability to contract faster (chronotropy) and the ability to relax (lusotropy). The observation that the βAR kinase (βARK1, also known as GRK2), the most abundant GRK in the heart, is increased in cardiovascular disease associated with impaired cardiac function, suggests that this molecule could have pathophysiological relevance in the setting of heart failure. Technological advances in the genetic engineering of mice have provided a powerful tool to study the physiological implications of altering GRK activity and expression in the heart. Recent studies have demonstrated that βARK1 plays a key role in not only the regulation of myocardial signalling, but also in cardiac function and development. Importantly, targeting the activity of GRKs, and βARK1 in particular, appears to represent a novel therapeutic strategy for the treatment of the failing heart. At present, gene therapy modalities are being tested which inhibit the activity of βARK1 in the heart. This technology makes it possible to test directly whether βARK1 inhibition in the setting of heart disease will improve the function of the compromised heart. Thus, these genetic approaches or the development of small molecule inhibitors of GRK activity, may lead to novel therapeutic approaches for cardiovascular disease.     

G蛋白偶联受体激酶和arrestins在受体调节中的作用

Ｇ 蛋白偶联受体（ Ｇｐｒｏｔｅｉｎ ｃｏｕｐｌｅｄ ｒｅｃｅｐｔｏｒ ， Ｇ Ｐ Ｃ Ｒｓ）是一大家族，介导许多激素的信号转导。在激动剂的持续作用下， Ｇ Ｐ Ｃ Ｒｓ 可发生对激动剂的敏感性下降，即受体减敏，现认为这一过程主要由 Ｇ 蛋白偶联受体激酶（ Ｇｐｒｏｔｅｉｎ ｃｏｕ ｐｌｅｄ ｒｅｃｅｐｔｏｒ ｋｉｎａｓｅｓ ， Ｇ Ｒ Ｋｓ） 和ａｒｒｅｓｔｉｎｓ 两大蛋白家族介导： Ｇ Ｒ Ｋｓ 先结合并磷酸化被激动剂占领的受体，然后ａｒｒｅｓｔｉｎｓ与磷酸化的受体结合，阻止受体与 Ｇ 蛋白发生作用，导致受体功能减退。近来发现， Ｇ Ｒ Ｋｓ 和ａｒｒｅｓｔｉｎｓ 还参与受体的内陷机制，而受体的复敏又与内陷密切相关     

Molecular mechanism of selectivity among G protein-coupled receptor kinase 2 inhibitors

G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-Gβγ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-Gβγ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.     

Phosphorylation-independent attenuation of GPCR signalling

The uncoupling of G-protein-coupled receptors (GPCRs) from their cognate heterotrimeric G proteins provides an essential physiological 'feedback' mechanism that protects against both acute and chronic overstimulation of receptors. The primary mechanism by which GPCR activity is regulated is the feedback phosphorylation of activated GPCRs by kinases that are dependent on second messengers, GPCR kinases (GRKs) and arrestins. It has recently become apparent, however, that GRK2-mediated regulation of GPCR responsiveness also involves a phosphorylation-independent component that requires both heterotrimeric G-protein alpha-subunit interactions and GPCR binding. Moreover, in addition to GRK2, a growing number of GPCR-interacting proteins might contribute to the phosphorylation-independent G-protein uncoupling of GPCRs. Here, new information about the mechanisms underlying this phosphorylation-independent regulation of receptor and G-protein coupling is reviewed.     

Exploiting Fluorescence Lifetime Plasticity in FLIM: Target Molecule Localization in Cells and Tissues

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Therapeutic potential of G-protein coupled receptor kinases in the heart

The actions of G-protein coupled receptor kinases (GRKs) critically regulate beta-adrenergic receptor (betaAR) signalling. In the cardiovascular system, the betaAR signalling pathway controls important responses of the heart such as the ability to contract (inotropy), the ability to contract faster (chronotropy) and the ability to relax (lusotropy). The observation that the betaAR kinase (betaARK1, also known as GRK2), the most abundant GRK in the heart, is increased in cardiovascular disease associated with impaired cardiac function, suggests that this molecule could have pathophysiological relevance in the setting of heart failure. Technological advances in the genetic engineering of mice have provided a powerful tool to study the physiological implications of altering GRK activity and expression in the heart. Recent studies have demonstrated that betaARK1 plays a key role in not only the regulation of myocardial signalling, but also in cardiac function and development. Importantly, targeting the activity of GRKs, and betaARK1 in particular, appears to represent a novel therapeutic strategy for the treatment of the failing heart. At present, gene therapy modalities are being tested which inhibit the activity of betaARK1 in the heart. This technology makes it possible to test directly whether betaARK1 inhibition in the setting of heart disease will improve the function of the compromised heart. Thus, these genetic approaches or the development of small molecule inhibitors of GRK activity, may lead to novel therapeutic approaches for cardiovascular disease.     

Tissue dependent differences in G-protein coupled receptor kinases associated with 5-HT4 receptor desensitization in the rat gastro-intestinal tract

Desensitization of 5-HT₄ receptors is regulated by G-protein coupled receptor kinases (GRKs). However, the specific GRK(s) that regulates the desensitization of 5-HT₄ receptors in the in vivo setting is unknown. We investigated the in situ expression of 5-HT₄ receptors and the GRKs in the rat gastrointestinal tract using immunohistochemistry and their interaction using coimmunoprecipitation. 5-HT₄ receptors were expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus and muscularis mucosae of both the proximal and distal colon. GRK2 was expressed in longitudinal muscle and occasionally in myenteric plexus whilst GRK5 showed limited expression in the nerve endings of the myenteric plexus and submucosal plexus of the colon. GRK3 was expressed in the tunica muscularis mucosae of the oesophagus, circular muscle, submucosal plexus and muscularis mucosae of the colon. GRK6 was expressed in the tunica muscularis mucosae of the oesophagus, longitudinal muscle, circular muscle, and muscularis mucosae of the colon. Stimulation of tunica muscularis mucosae of the oesophagus and distal colon using the 5-HT₄ receptor agonist, tegaserod, followed by analysis of the 5-HT₄ receptor antibody immunoprecipitate, revealed the coimmunoprecipitation of GRK6 with 5-HT₄ receptors in the tunica muscularis mucosae of oesophagus while GRK2 and GRK6 were coimmunoprecipitated with 5-HT₄ receptors in the distal colon. This study indicates that GRK6 may be involved in the regulation of the desensitization of 5-HT₄ receptors in the rat oesophagus whilst GRK2 and GRK6 may be involved in regulation of the desensitization of 5-HT₄ receptors in the distal colon.     

Non-visual GRKs: are we seeing the whole picture?

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 β-adrenoceptor kinase (β-ARK) family (GRK2 and GRK3) and, in particular, on phosphorylation-dependent recruitment of adaptor proteins such as the β-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.     

Morphine-induced physiological and behavioral responses in mice lacking G protein-coupled receptor kinase 6

G protein-coupled receptor kinases (GRKs) are a family of intracellular proteins that desensitize and regulate the responsiveness of G protein-coupled receptors (GPCRs). In the present study, we assessed the contribution of GRK6 to the regulation and responsiveness of the G protein-coupled mu-opioid receptor (μOR) in response to morphine in vitro and in vivo using mice lacking GRK6. In cell culture, overexpression of GRK6 facilitates morphine-induced beta-arrestin2 (βarrestin2) recruitment and receptor internalization, suggesting that this kinase may play a role in regulating the μOR. In vivo, we find that acute morphine treatment induces greater locomotor activation but less constipation in GRK6 knockout (GRK6-KO) mice compared to their wild-type (WT) littermates. The GRK6-KO mice also appear to be “presensitized” to the locomotor stimulating effects induced by chronic morphine treatment, yet these animals do not display more conditioned place preference than WT mice do. Furthermore, several other morphine-mediated responses which were evaluated, including thermal antinociception, analgesic tolerance, and physical dependence, were not affected by ablation of the GRK6 gene. Collectively, these results suggest that GRK6 may play a role in regulating some, but not all morphine-mediated responses. In addition, these findings underscore that the contribution of a particular regulatory factor to receptor function can differ based upon the specific cell composition and physiology assessed, and illustrate the need for using caution when interpreting the importance of interactions observed in cell culture.     

G protein-coupled receptor kinase 2--a feedback regulator of Gq pathway signalling

G protein coupled receptors or serpentine receptors work as signalling switches that turn extracellular signals into activation of multiple molecules at the intracellular face of the plasma membrane. Serpentine receptors are the targets of around 70% of all current drugs in clinical medicine. We suggest that these receptors can be pharmacologically targeted by modification of their unique internal inhibitors the G protein coupled receptor kinases (GRKs). The GRKs constitute a family of serine/threonine kinases that specifically bind to and phosphorylate agonist-activated serpentine receptors. The phosphorylated receptors are recognized by arrestins that bind to the receptor and uncouple them from attached G proteins thereby terminating G protein signalling. This review focuses on a ubiquitously expressed GRK family member dubbed GRK2 (previously called beta-adrenergic receptor kinase 1) that regulates cellular signalling at multiple levels. In Gq-coupled signalling modules GRK2 may function as a feedback inhibitor molecule that monitors, inhibits and re-directs the information flow. GRK2 acts as a negative feedback protein by interacting with at least six key signalling molecules in the Gq pathway including; receptors, free G beta gamma subunits, activated G alpha q subunits, phosphatidylinositol-4, 5-bisphosphate (PIP2), protein kinase C (PKC) and calmodulin (CaM). GRK signalling is important for immune, endocrine and cardiovascular function manifesting itself in disorders such as heart failure and lymphocyte activation especially in chronic inflammation. This review summarizes the advances made in understanding the many actions of GRKs and addresses their potential as novel therapeutic targets.     

Enhanced expression of G protein-coupled receptor kinase 2 selectively increases the sensitivity of A2A adenosine receptors to agonist-induced desensitization

1. G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor (GPCR) responses. We have previously shown that stable expression of a dominant negative mutant G protein- coupled receptor kinase 2 (GRK2) construct in NG108-15 mouse neuroblastoma x rat glioma cells suppresses the agonist-induced desensitization of A_2A and A_2B adenosine receptor-stimulated adenylyl cyclase activity (Mundell et al., 1997). To further determine the role of GRK2 in agonist-induced desensitization of these adenosine receptors, we stably overexpressed wild type GRK2 in NG108-15 cells.
2. In homogenates prepared from cells overexpressing GRK2, the acute stimulation of adenylyl cyclase by activation of A_2A and A_2B adenosine receptors was markedly reduced, but could be reversed by pretreating the cells with AD (adenosine deaminase), to remove extracellular adenosine from the medium. On the other hand, acute stimulation of adenylyl cyclase by secretin, iloprost, NaF and forskolin was the same in GRK2 overexpressing cells and plasmid-transfected control cells.
3. Cells overexpressing GRK2 were more sensitive to adenosine receptor agonist-induced desensitization than plasmid-transfected control cells. This effect was selective since the agonist sensitivity of desensitization for secretin and IP-prostanoid receptor-stimulated adenylyl cyclase activity was not affected by GRK2 overexpression.
4. These results further implicate GRK2 as the likely mechanism by which A₂ adenosine receptors undergo short-term desensitization in NG108-15 cells, and indicate that even when overexpressed, GRK2 retains its substrate specificity for native receptors in intact cells. Furthermore, the susceptibility of GPCRs to desensitization appears to depend on the level of GRK expression, such that in cells that express high levels of GRK2, low agonist concentrations may be sufficient to trigger GRK-mediated desensitization.

     

The identification of G-protein coupled receptors in sequence databases

In the race to identify high quality drug targets from the enormous amount of information generated by the Human Genome Project, many companies are focussing their efforts on proteins already known to be good therapeutic targets. The G-protein coupled receptors (GPCRs) represent the most common class of therapeutic target. In this review we explore the sequence attributes of GPCRs and outline how these might be applied to focused database searches for novel GPCRs and to efficiently evaluate these new gene products pharmacologically.     

A polymorphism of G-protein coupled receptor kinase5 alters agonist-promoted desensitization of beta2-adrenergic receptors

Beta-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of beta2-adrenergic (beta2AR) signaling genes. beta2AR function undergoes desensitization during persistent agonist exposure because of receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for beta-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared with GRK5-Gln41. Consistent with this functional effect, agonist-promoted beta2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated beta2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of beta2AR during persistent agonist exposure, and thus may contribute to beta-agonist variability in asthma treatment of African-Americans.     

Regulatory mechanisms underlying GKR2 levels in U937 cells: evidence for GRK3 involvement

G protein-coupled receptors represent the most diverse group of proteins involved in transmembrane signalling, that participate in the regulation of a wide range of physicochemical messengers through the interaction with heterotrimeric G proteins. In addition, GPCRs stimulation also triggers a negative feedback mechanism, known as desensitization that prevents the potentially harmful effects caused by persistent receptor stimulation. In this adaptative response, G protein-coupled receptor kinases (GRKs) play a key role and alterations in their function are related to diverse pathophysiological situations. Based on the scarce knowledge about the regulation of GRK2 by other kinases of the same family, the aim of the present work was to investigate the regulation of GRK2 levels in systems where other GRKs are diminished by antisense technique. Present findings show that in U937 cells GRK2 levels are regulated by GRK3 and not by GRK6 through a mechanism involving InsP upregulation. This work reports a novel GRK3-mediated GRK2 regulatory mechanism and further suggests that GRK2 may also act as a compensatory kinase tending to counterbalance the reduction in GRK3 levels. This study provides the first evidence for the existence of GRKs cross-regulation.     

Beta-arrestin2 as a competitor for GRK2 interaction with the GLP-1 receptor upon receptor activation

The signaling of seven transmembrane receptors/G-protein- coupled receptors (GPCRs) is regulated by a number of receptor interacting proteins, including βarrestins (βarrs) and GPCR kinases (GRKs). In the present report, we have analyzed the interaction pattern between the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), βarr2, and GRK2 using bioluminescence resonance energy transfer assays. We found that βarr2 interacts with the GLP-1R in a biphasic manner with a phosphorylation-independent and a phosphorylation-dependent component. In competition experiments, we observed βarr2 competing with GRK2 for interaction with GLP-1R. We propose a model were βarr2 competes with GRK2 for interaction with the activated and GRK phosphorylated GLP-1R, suggesting a new role of βarr2 in regulating the orchestration of GRK2 functionality.     

基于G-蛋白偶联受体激酶2抑制剂治疗心力衰竭的合理药物设计（英文）

G protein-coupled receptors(GPCRs)convert extracellular stimuli in the form of hormones,odorants and light into profound changes in cell homeostasis.Their timely desensitization is critical for cells to rapidly respond to changes in their environment and to avoid damage from sustained signaling.Seven GPCR kinases(GRKs)phosphorylate and regulate the activity of most of the~800 GPCRs in the human genome.Although GRKs normally play an adaptive role,in conditions such as chronic heart failure they are overexpressed and linked to disease progression.GRK2 and GRK5 have thus become important targets for the treatment of heart failure and pathological cardiac hypertrophy,respectively.Our lab has determined atomic structures representing all three vertebrate GRK subfamilies,and is now in the midst of a campaign to develop selective inhibitors of these enzymes using structure-based rational design.We have identified the FDA approved drug paroxetine as a selective GRK2 inhibitor,determined the crystal structure of the GRK2·paroxetine complex and,in collaboration with the Koch lab,showed that the drug improves contractility in myocytes and,most impressively,recovery in postmyocardial infarcted mice.Since then,we have identified additional chemical scaffolds that exhibit even higher potency and/or selectivity for GRK5.Using a"hybrid"inhibitor design approach we have generated GRK selective chemical probes that exhibit improved potency and stability and are able to increase inotropy and dampen the hypertrophic response in cardiomyocytes and small animal models.Structural analysis has revealed the molecular basis for selectivity and potency in many of these compounds,allowing for the design of future generations of GRK chemical probes.     

Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

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.     

Pharmacogenomics of the heptahelical receptor regulators G-protein-coupled receptor kinases and arrestins: the known and the unknown

Heptahelical G-protein-coupled receptors are the most diverse and therapeutically important family of receptors, playing major roles in the physiology of various organs and tissues. They couple their ligand binding to G-protein activation, which then transmits intracellular signals. G-protein signaling is terminated by phosphorylation of the receptor by the family of G-protein-coupled receptor kinases (GRKs), followed by arrestin (Arr) binding, which uncouples the phosphorylated receptor from the G-protein and subsequently targets the receptor for internalization. Moreover, Arrs can transmit signals in their own right during receptor internalization. Genetic polymorphisms in receptors, as well as in GRK and Arr family members per se, which affect regulation of receptor signaling and function, have just started being identified and characterized. The present review will discuss what is known so far in this evolving field of GRK/Arr pharmacogenomics, as well as highlight important areas likely to produce invaluable information in the future.