The Experts below are selected from a list of 8811 Experts worldwide ranked by ideXlab platform
Jeffrey L. Benovic - One of the best experts on this subject based on the ideXlab platform.
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M 3 Muscarinic Acetylcholine Receptor-Mediated Signaling Is Regulated by Distinct Mechanisms
2020Co-Authors: Jiansong Luo, John M. Busillo, Jeffrey L. BenovicAbstract:ABSTRACT We have used RNA interference previously to demonstrate that G protein-coupled receptor kinase 2 (GRK2) regulates endogenously expressed H1 histamine receptor in human embryonic kidney 293 cells. In this report, we investigate the regulation of endogenously expressed M 3 muscarinic acetylcholine receptor (M 3 mAChR). We show that knockdown of GRK2, GRK3, or GRK6, but not GRK5, significantly increased carbachol-mediated calcium mobilization. Stable expression of wild-type GRK2 or a kinase-dead mutant (GRK2-K220R) reduced calcium mobilization after receptor activation, whereas GRK2 mutants defective in G␣ q binding (GRK2-D110A, GRK2-R106A, and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates G q after M 3 mAChR activation. The knockdown of arrestin-2 or arrestin-3 also significantly increased carbachol-mediated calcium mobilization. Knockdown of GRK2 and the arrestins also significantly enhanced carbachol-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), whereas prolonged ERK1/2 activation was only observed with GRK2 or arrestin-3 knockdown. We also investigated the role of casein kinase-1␣ (CK1␣) and found that knockdown of CK1␣ increased calcium mobilization but not ERK activation. In summary, our data suggest that multiple proteins dynamically regulate M 3 mAChR-mediated calcium signaling, whereas GRK2 and arrestin-3 play the primary role in regulating ERK activation
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g protein coupled receptor kinase 5 phosphorylates nucleophosmin and regulates cell sensitivity to polo like kinase 1 inhibition
Journal of Biological Chemistry, 2012Co-Authors: Allison M Michal, Rouzbeh Mashayekhi, Jeffrey L. BenovicAbstract:G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, leading to their desensitization and endocytosis. GRKs have also been implicated in phosphorylating other classes of proteins and can localize in a variety of cellular compartments, including the nucleus. Here, we attempted to identify potential nuclear substrates for GRK5. Our studies reveal that GRK5 is able to interact with and phosphorylate nucleophosmin (NPM1) both in vitro and in intact cells. NPM1 is a nuclear protein that regulates a variety of cell functions including centrosomal duplication, cell cycle control, and apoptosis. GRK5 interaction with NPM1 is mediated by the N-terminal domain of each protein, and GRK5 primarily phosphorylates NPM1 at Ser-4, a site shared with polo-like kinase 1 (PLK1). NPM1 phosphorylation by GRK5 and PLK1 correlates with the sensitivity of cells to undergo apoptosis with cells having higher GRK5 levels being less sensitive and cells with lower GRK5 being more sensitive to PLK1 inhibitor-induced apoptosis. Taken together, our results demonstrate that GRK5 phosphorylates Ser-4 in nucleophosmin and regulates the sensitivity of cells to PLK1 inhibition.
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g protein coupled receptor kinase 5 is localized to centrosomes and regulates cell cycle progression
Journal of Biological Chemistry, 2012Co-Authors: Allison M Michal, Christopher H So, Neil Beeharry, Haripriya Shankar, Rouzbeh Mashayekhi, Jeffrey L. BenovicAbstract:Abstract G protein-coupled receptor kinases (GRKs) are important regulators of G protein-coupled receptor function and mediate receptor desensitization, internalization, and signaling. While GRKs also interact with and/or phosphorylate many other proteins and modify their function, relatively little is known about the cellular localization of endogenous GRKs. Here we report that GRK5 co-localizes with γ-tubulin, centrin, and pericentrin in centrosomes. The centrosomal localization of GRK5 is observed predominantly at interphase and although its localization is not dependent on microtubules, it can mediate microtubule nucleation of centrosomes. Knockdown of GRK5 expression leads to G2/M arrest, characterized by a prolonged G2 phase, which can be rescued by expression of wild type but not catalytically inactive GRK5. This G2/M arrest appears to be due to increased expression of p53, reduced activity of aurora A kinase and a subsequent delay in the activation of polo-like kinase 1. Overall, these studies demonstrate that GRK5 is localized in the centrosome and regulates microtubule nucleation and normal cell cycle progression.
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Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling
The Journal of biological chemistry, 2010Co-Authors: John M. Busillo, Sylvain Armando, Rajarshi Sengupta, Olimpia Meucci, Michel Bouvier, Jeffrey L. BenovicAbstract:The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.
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m3 muscarinic acetylcholine receptor mediated signaling is regulated by distinct mechanisms
Molecular Pharmacology, 2008Co-Authors: Jiansong Luo, John M. Busillo, Jeffrey L. BenovicAbstract:We have used RNA interference previously to demonstrate that G protein-coupled receptor kinase 2 (GRK2) regulates endogenously expressed H1 histamine receptor in human embryonic kidney 293 cells. In this report, we investigate the regulation of endogenously expressed M3 muscarinic acetylcholine receptor (M3 mAChR). We show that knockdown of GRK2, GRK3, or GRK6, but not GRK5, significantly increased carbachol-mediated calcium mobilization. Stable expression of wild-type GRK2 or a kinase-dead mutant (GRK2-K220R) reduced calcium mobilization after receptor activation, whereas GRK2 mutants defective in Gαq binding (GRK2-D110A, GRK2-R106A, and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates Gq after M3 mAChR activation. The knockdown of arrestin-2 or arrestin-3 also significantly increased carbachol-mediated calcium mobilization. Knockdown of GRK2 and the arrestins also significantly enhanced carbachol-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), whereas prolonged ERK1/2 activation was only observed with GRK2 or arrestin-3 knockdown. We also investigated the role of casein kinase-1α (CK1α) and found that knockdown of CK1α increased calcium mobilization but not ERK activation. In summary, our data suggest that multiple proteins dynamically regulate M3 mAChR-mediated calcium signaling, whereas GRK2 and arrestin-3 play the primary role in regulating ERK activation.
Antonio De Blasi - One of the best experts on this subject based on the ideXlab platform.
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analysis of differential modulatory activities of grk2 and grk4 on galphaq coupled receptor signaling
Methods in Enzymology, 2004Co-Authors: Antonietta Picascia, Luisa Iacovelli, Loredana Capobianco, Antonio De BlasiAbstract:Abstract G-protein-coupled receptor kinases (GRK) contain a regulator of G-protein signaling (RGS)-like domain located at the N terminus (GRK-Nter) of their sequence. This domain is present in all the GRK subtypes, but the RGS-like domain of GRK2 was documented to be functionally active, as it is able to interact selectively with Gαq (both in vitro and in cells) and to inhibit Gαq–dependent signaling. In contrast GRK4, GRK5, and GRK6 are unable to interact with Gαq. This article describes the methodology to investigate the modulatory activity of GRK2 and GRK4 on GPCR-stimulated Gαq signaling. This analysis is essentially based on three types of experiments: (a) study of the effect of the GRK-Nter on GPCR-dependent signaling; (b) analysis of the binding of GRK-Nter to Gαq in vitro; and (c) analysis of the interaction of GRK with Gαq in cells.
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the g protein coupled receptor kinase grk4 mediates homologous desensitization of metabotropic glutamate receptor 1
The FASEB Journal, 2000Co-Authors: Michele Sallese, Marianna Storto, E Durbano, Thomas Launey, Gessica Sala, Lorena Salvatore, Ferdinando Nicoletti, Thomas Knopfel, Antonio De BlasiAbstract:G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or β-adrenergic receptor ...
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chromosome mapping of the human arrestin sag β arrestin 2 arrb2 and β adrenergic receptor kinase 2 adrbk2 genes
Genomics, 1994Co-Authors: G. Calabrese, Anna Stornaiuolo, Michele Sallese, Giandomenico Palka, Liborio Stuppia, Antonio De BlasiAbstract:Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.
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chromosome mapping of the human arrestin sag beta arrestin 2 arrb2 and beta adrenergic receptor kinase 2 adrbk2 genes
Genomics, 1994Co-Authors: G. Calabrese, Anna Stornaiuolo, Michele Sallese, Giandomenico Palka, Liborio Stuppia, Antonio De BlasiAbstract:Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.
John J. G. Tesmer - One of the best experts on this subject based on the ideXlab platform.
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structure based design of selective covalent g protein coupled receptor kinase 5 inhibitors
ACS Medicinal Chemistry Letters, 2019Co-Authors: Rachel A Rowlands, John J. G. Tesmer, Scott D Larsen, Qiuyan Chen, Helen V Waldschmidt, Claire M Cato, Renee A Bouley, Larisa Avramova, Andrew D WhiteAbstract:The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating heart failure and other diseases such as cancer. Although advances have been made toward developing inhibitors that are selective for GRK2, there have been far fewer reports of GRK5 selective compounds. Herein, we describe the development of GRK5 subfamily selective inhibitors, 5 and 16d that covalently interact with a nonconserved cysteine (Cys474) unique to this subfamily. Compounds 5 and 16d feature a highly amenable pyrrolopyrimidine scaffold that affords high nanomolar to low micromolar activity that can be easily modified with Michael acceptors with various reactivities and geometries. Our work thereby establishes a new pathway toward further development of subfamily selective GRK inhibitors and establishes Cys474 as a new and useful covalent handle in GRK5 drug discovery.
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Identification and structure-function analysis of sub-family selective G protein-coupled receptor kinase inhibitors
2016Co-Authors: Kristoff T Homan, Kelly M. Larimore, Jonathan M. Elkins, Marta Szklarz, John J. G. TesmerAbstract:*S Supporting Information ABSTRACT: Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85
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crystal structure of g protein coupled receptor kinase 5 in complex with a rationally designed inhibitor
Journal of Biological Chemistry, 2015Co-Authors: Kristoff T Homan, Walter J Koch, Scott D Larsen, Alisa Glukhova, Alessandro Cannavo, Helen V Waldschmidt, Jianliang Song, Joseph Y Cheung, John J. G. TesmerAbstract:G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 A crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.
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g protein coupled receptor kinase 2 grk2 and 5 GRK5 exhibit selective phosphorylation of the neurotensin receptor in vitro
Biochemistry, 2015Co-Authors: Sayaka Inagaki, Sergey A Vishnivetskiy, Kristoff T Homan, John J. G. Tesmer, Jim F. White, Rodolfo Ghirlando, Vsevolod V Gurevich, Reinhard GrisshammerAbstract:G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activ...
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g protein coupled receptor kinase 2 grk2 and 5 GRK5 exhibit selective phosphorylation of the neurotensin receptor in vitro
Biochemistry, 2015Co-Authors: Sayaka Inagaki, Sergey A Vishnivetskiy, Kristoff T Homan, John J. G. Tesmer, Rodolfo Ghirlando, Vsevolod V Gurevich, Jim White, Reinhard GrisshammerAbstract:G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β2-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our in vitro study revealed NTSR1 differential phosphorylation by GRK2 and GRK5.
Kristoff T Homan - One of the best experts on this subject based on the ideXlab platform.
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structure based design of highly selective and potent g protein coupled receptor kinase 2 inhibitors based on paroxetine
Journal of Medicinal Chemistry, 2017Co-Authors: Helen V Waldschmidt, Kristoff T Homan, Alessandro Cannavo, Marilyn C Cato, Osvaldo Cruzrodriguez, Michael W WilsonAbstract:In heart failure, the β-adrenergic receptors (βARs) become desensitized and uncoupled from heterotrimeric G proteins. This process is initiated by G protein-coupled receptor kinases (GRKs), some of which are upregulated in the failing heart, making them desirable therapeutic targets. The selective serotonin reuptake inhibitor, paroxetine, was previously identified as a GRK2 inhibitor. Utilizing a structure-based drug design approach, we modified paroxetine to generate a small compound library. Included in this series is a highly potent and selective GRK2 inhibitor, 14as, with an IC50 of 30 nM against GRK2 and greater than 230-fold selectivity over other GRKs and kinases. Furthermore, 14as showed a 100-fold improvement in cardiomyocyte contractility assays over paroxetine and a plasma concentration higher than its IC50 for over 7 h. Three of these inhibitors, including 14as, were additionally crystallized in complex with GRK2 to give insights into the structural determinants of potency and selectivity of t...
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Identification and structure-function analysis of sub-family selective G protein-coupled receptor kinase inhibitors
2016Co-Authors: Kristoff T Homan, Kelly M. Larimore, Jonathan M. Elkins, Marta Szklarz, John J. G. TesmerAbstract:*S Supporting Information ABSTRACT: Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85
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crystal structure of g protein coupled receptor kinase 5 in complex with a rationally designed inhibitor
Journal of Biological Chemistry, 2015Co-Authors: Kristoff T Homan, Walter J Koch, Scott D Larsen, Alisa Glukhova, Alessandro Cannavo, Helen V Waldschmidt, Jianliang Song, Joseph Y Cheung, John J. G. TesmerAbstract:G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 A crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.
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g protein coupled receptor kinase 2 grk2 and 5 GRK5 exhibit selective phosphorylation of the neurotensin receptor in vitro
Biochemistry, 2015Co-Authors: Sayaka Inagaki, Sergey A Vishnivetskiy, Kristoff T Homan, John J. G. Tesmer, Jim F. White, Rodolfo Ghirlando, Vsevolod V Gurevich, Reinhard GrisshammerAbstract:G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activ...
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g protein coupled receptor kinase 2 grk2 and 5 GRK5 exhibit selective phosphorylation of the neurotensin receptor in vitro
Biochemistry, 2015Co-Authors: Sayaka Inagaki, Sergey A Vishnivetskiy, Kristoff T Homan, John J. G. Tesmer, Rodolfo Ghirlando, Vsevolod V Gurevich, Jim White, Reinhard GrisshammerAbstract:G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β2-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our in vitro study revealed NTSR1 differential phosphorylation by GRK2 and GRK5.
Michele Sallese - One of the best experts on this subject based on the ideXlab platform.
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Role of G Protein-coupled Receptor Kinase 4 and β-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases
Journal of Biological Chemistry, 2003Co-Authors: Luisa Iacovelli, Antonietta Picascia, Marianna Storto, Loredana Capobianco, Emanuela Barletta, Lorena Salvatore, Antonio Porcellini, Stefania Mariggio, Michele Sallese, Ferdinando NicolettiAbstract:Abstract The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, β-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of β-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70–80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and β-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of β-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing β-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1receptor by different mechanisms and that β-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.
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the g protein coupled receptor kinase grk4 mediates homologous desensitization of metabotropic glutamate receptor 1
The FASEB Journal, 2000Co-Authors: Michele Sallese, Marianna Storto, E Durbano, Thomas Launey, Gessica Sala, Lorena Salvatore, Ferdinando Nicoletti, Thomas Knopfel, Antonio De BlasiAbstract:G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or β-adrenergic receptor ...
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chromosome mapping of the human arrestin sag β arrestin 2 arrb2 and β adrenergic receptor kinase 2 adrbk2 genes
Genomics, 1994Co-Authors: G. Calabrese, Anna Stornaiuolo, Michele Sallese, Giandomenico Palka, Liborio Stuppia, Antonio De BlasiAbstract:Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.
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chromosome mapping of the human arrestin sag beta arrestin 2 arrb2 and beta adrenergic receptor kinase 2 adrbk2 genes
Genomics, 1994Co-Authors: G. Calabrese, Anna Stornaiuolo, Michele Sallese, Giandomenico Palka, Liborio Stuppia, Antonio De BlasiAbstract:Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.
