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Robert J Lefkowitz - One of the best experts on this subject based on the ideXlab platform.
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Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: David A. Zidar, Jonathan D Violin, Erin J Whalen, Robert J LefkowitzAbstract:CCL19 and CCL21 are endogenous agonists for the seven-transmembrane receptor CCR7. They are equally active in promoting G protein stimulation and chemotaxis. Yet, we find that they result in striking differences in activation of the G protein-coupled receptor kinase (GRK)/ss-arrestin system. CCL19 leads to robust CCR7 phosphorylation and beta-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Although each ligand leads to beta-arrestin2 recruitment, only CCL19 leads to redistribution of beta-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, these agonists are both capable of signaling through GRK6 and beta-arrestin2 to ERK kinases. Thus, this mechanism for "ligand bias" whereby endogenous agonists activate different GRK isoforms leads to functionally distinct pools of beta-arrestin.
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Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN).
Clinical immunology (Orlando Fla.), 2008Co-Authors: Teresa K. Tarrant, Richard T. Premont, Robert J Lefkowitz, Rishi R. Rampersad, Denise A. Esserman, Lisa R. Rothlein, Peng Liu, David M. Lee, Dhavalkumar D. PatelAbstract:Abstract Objective Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model. Methods Clinical, histologic, and cytokine responses in GRK6−/−, GRK5−/−, GRK2+/−, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays. Results Both GRK6−/− and GRK2+/− mice had increased arthritis disease severity (p 10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro. Conclusions GRK6 and -2, but not GRK5, are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.
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β arrestin2 mediated inotropic effects of the angiotensin ii type 1a receptor in isolated cardiac myocytes
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Keshava Rajagopal, Richard T. Premont, Erin J Whalen, Jonathan D Violin, Jonathan A Stiber, Paul B Rosenberg, Thomas M Coffman, Howard A Rockman, Robert J LefkowitzAbstract:The G protein-coupled receptor kinases (GRKs) and β-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/β-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/β-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT1AR) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the “biased” AT1AR agonist [Sar1, Ile4, Ile8]-angiotensin II (SII), which is unable to stimulate Gαq-mediated signaling, but which has previously been shown to promote β-arrestin interaction with the AT1AR. Cardiomyocytes from WT, but not AT1AR-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from β-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2+/−) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent Gαq/PKC- and GRK6/β-arrestin2-dependent mechanisms by which stimulation of the AT1AR can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.
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different g protein coupled receptor kinases govern g protein and β arrestin mediated signaling of v2 vasopressin receptor
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Xiurong Ren, Eric Reiter, Seungkirl Ahn, Jihee Kim, Wei Chen, Robert J LefkowitzAbstract:Signaling through β-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally a prerequisite for β-arrestin binding, we studied the roles of different GRKs in promoting β-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gs- and β-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and β-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of β-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and β-arrestin recruitment, but yet appeared exclusively to support β-arrestin 2-mediated ERK activation. GRK2 suppression actually increased β-arrestin-stimulated ERK activation. These results suggest that β-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials.
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functional antagonism of different g protein coupled receptor kinases for β arrestin mediated angiotensin ii receptor signaling
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Jihee Kim, Erin J Whalen, Xiurong Ren, Eric Reiter, Seungkirl Ahn, Huijun Wei, Robert J LefkowitzAbstract:β-arrestins bind to G protein-coupled receptor kinase (GRK)-phosphorylated seven transmembrane receptors, desensitizing their activation of G proteins, while concurrently mediating receptor endocytosis, and some aspects of receptor signaling. We have used RNA interference to assess the roles of the four widely expressed isoforms of GRKs (GRK 2, 3, 5, and 6) in regulating β-arrestin-mediated signaling to the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2 by the angiotensin II type 1A receptor. Angiotensin II-stimulated receptor phosphorylation, β-arrestin recruitment, and receptor endocytosis are all mediated primarily by GRK2/3. In contrast, inhibiting GRK 5 or 6 expression abolishes β-arrestin-mediated ERK activation, whereas lowering GRK 2 or 3 leads to an increase in this signaling. Consistent with these findings, β-arrestin-mediated ERK activation is enhanced by overexpression of GRK 5 and 6, and reciprocally diminished by GRK 2 and 3. These findings indicate distinct functional capabilities of β-arrestins bound to receptors phosphorylated by different classes of GRKs.
Jeffrey L. Benovic - One of the best experts on this subject based on the ideXlab platform.
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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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arrestin 2 and g protein coupled receptor kinase 5 interact with nfκb1 p105 and negatively regulate lipopolysaccharide stimulated erk1 2 activation in macrophages
Journal of Biological Chemistry, 2006Co-Authors: Narayanan Parameswaran, Christina S Pao, Kristi S Leonhard, Dong Soo Kang, Michelle Kratz, Steven C Ley, Jeffrey L. BenovicAbstract:Abstract Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IκB kinase promotes phosphorylation and degradation of NFκB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IκB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.
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The Structure of G Protein-coupled Receptor Kinase (GRK)-6 Defines a Second Lineage of GRKs.
Journal of Biological Chemistry, 2006Co-Authors: D.t. Lodowski, Valerie M. Tesmer, Jeffrey L. Benovic, John J. G. TesmerAbstract:Abstract 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.
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L-DOPA reverses the MPTP-induced elevation of the arrestin2 and GRK6 expression and enhanced ERK activation in monkey brain.
Neurobiology of disease, 2005Co-Authors: Erwan Bezard, Vsevolod V. Gurevich, Jeffrey L. Benovic, C.e. Gross, Li Qin, Eugenia V GurevichAbstract: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.
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Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors.
European Journal of Pharmacology, 2000Co-Authors: Nambi Aiyar, Jeffrey L. Benovic, Alexey Pronin, Jyoti Disa, Khoa Dang, Ponnal NambiAbstract:Abstract This investigation was undertaken to study the mechanisms of calcitonin gene-related peptide (CGRP)-mediated desensitization using recombinant porcine CGRP receptors stably expressed in human embryonic kidney (HEK-293) cells. Pretreatment of these cells with human αCGRP resulted in an ∼60% decrease in CGRP-stimulated adenylyl cyclase activity and an ∼10-fold rightward shift in the dose–response curve of CGRP. This effect was rapid ( t 1/2 ∼5 min) and was accompanied by a significant decrease in [ 125 I]CGRP binding to membrane preparations from CGRP-pretreated cells. In contrast, CGRP pretreatment had no effect on isoproterenol- or forskolin-stimulated adenylyl cyclase activity in these cells. The potential involvement of protein kinase A or protein kinase C in CGRP-mediated desensitization was studied using selective inhibitors or activators of these kinases. Pretreatment of the cells with forskolin (adenylyl cyclase activator) or phorbol dibutyrate (protein kinase C activator) had no effect on CGRP-mediated adenylyl cyclase activity and did not influence CGRP-mediated desensitization. However, pretreatment of the cells with 2-(8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methylindol-3-yl)maleimide hydrochloride (Ro 32-0432) (a potent inhibitor of protein kinase C) resulted in significant attenuation of CGRP-mediated desensitization with an IC 50 ∼3 μM. To establish whether this effect might be due to inhibition of other protein kinases by Ro 32-0432, its effect was tested against several G protein-coupled receptor kinases (GRKs). Ro 32-0432 was found to inhibit GRK2, GRK5, and GRK6 with IC 50 values of 29, 3.6, and 16 μM, respectively, suggesting that its effect on CGRP-mediated desensitization might be a result of GRK inhibition. To further test this hypothesis, as well as the potential GRK specificity, the cells were treated with antisense oligonucleotides to GRK2, GRK5, and GRK6. While GRK2 and GRK5 antisense nucleotides had no effect on CGRP-mediated desensitization, the GRK6 antisense nucleotide treatment significantly reversed CGRP-mediated desensitization. These results suggest the involvement of GRK6 in CGRP-mediated desensitization in HEK-293 cells.
Richard T. Premont - One of the best experts on this subject based on the ideXlab platform.
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Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN).
Clinical immunology (Orlando Fla.), 2008Co-Authors: Teresa K. Tarrant, Richard T. Premont, Robert J Lefkowitz, Rishi R. Rampersad, Denise A. Esserman, Lisa R. Rothlein, Peng Liu, David M. Lee, Dhavalkumar D. PatelAbstract:Abstract Objective Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model. Methods Clinical, histologic, and cytokine responses in GRK6−/−, GRK5−/−, GRK2+/−, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays. Results Both GRK6−/− and GRK2+/− mice had increased arthritis disease severity (p 10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro. Conclusions GRK6 and -2, but not GRK5, are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.
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β arrestin2 mediated inotropic effects of the angiotensin ii type 1a receptor in isolated cardiac myocytes
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Keshava Rajagopal, Richard T. Premont, Erin J Whalen, Jonathan D Violin, Jonathan A Stiber, Paul B Rosenberg, Thomas M Coffman, Howard A Rockman, Robert J LefkowitzAbstract:The G protein-coupled receptor kinases (GRKs) and β-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/β-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/β-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT1AR) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the “biased” AT1AR agonist [Sar1, Ile4, Ile8]-angiotensin II (SII), which is unable to stimulate Gαq-mediated signaling, but which has previously been shown to promote β-arrestin interaction with the AT1AR. Cardiomyocytes from WT, but not AT1AR-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from β-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2+/−) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent Gαq/PKC- and GRK6/β-arrestin2-dependent mechanisms by which stimulation of the AT1AR can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.
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Defective lymphocyte chemotaxis in β-arrestin2- and GRK6-deficient mice
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Alan M. Fong, Richard T. Premont, Robert J Lefkowitz, Ricardo M Richardson, Dhavalkumar D. PatelAbstract:Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated (“desensitized”) by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and β-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various β-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of β-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the β-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from β-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that β-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.
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the grk4 subfamily of g protein coupled receptor kinases alternative splicing gene organization and sequence conservation
Journal of Biological Chemistry, 1999Co-Authors: Richard T. Premont, Samuel A J R Aparicio, Humphrey E Kendall, Jeffrey E Welch, Alexander D. Macrae, Robert J LefkowitzAbstract:Abstract G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.
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Phosphatidylinositol 4,5-Bisphosphate (PIP2)-enhanced G Protein-coupled Receptor Kinase (GRK) Activity: LOCATION, STRUCTURE, AND REGULATION OF THE PIP2 BINDING SITE DISTINGUISHES THE GRK SUBFAMILIES
Journal of Biological Chemistry, 1996Co-Authors: Julie A. Pitcher, Zl Fredericks, Robert H. Stoffel, Richard T. Premont, Walter J Koch, W C Stone, Robert J LefkowitzAbstract:Abstract The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) enhances GRK5-mediated β-adrenergic receptor (βAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of βARK (β-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the βγ subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem.270, 11707-11710). Thus, five members of the GRK family bind PIP2, βARK (GRK2), βARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the βARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
Walter J Koch - One of the best experts on this subject based on the ideXlab platform.
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G protein-coupled receptor kinases as therapeutic targets in the heart
Nature Reviews Cardiology, 2019Co-Authors: Jessica Pfleger, Kenneth Gresham, Walter J KochAbstract:G protein-coupled receptors (GPCRs) mediate a range of physiological responses in various cardiovascular cell types. β-Adrenergic receptors (β-ARs) regulate cardiomyocyte contractility in response to sympathetic nervous system stimulation. In the failing heart, increased levels of GPCR kinases (GRKs) phosphorylate, and thereby desensitize and downregulate, β-ARs, resulting in a loss of cardiomyocyte contractile reserve. GRK2 and GRK5 can be therapeutically targeted to protect the heart against injury and failure using novel small-molecule inhibitors. G protein-coupled receptor (GPCR) kinases (GRKs) can desensitize and downregulate GPCRs. In this Review, Pfleger and colleagues describe the changes in GPCR and GRK signalling in the heart under disease conditions and how GRKs can be targeted to treat heart failure. G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as β-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.
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G protein-coupled receptor kinases as therapeutic targets in the heart
Nature Reviews Cardiology, 2019Co-Authors: Jessica Pfleger, Kenneth Gresham, Walter J KochAbstract:G protein-coupled receptors (GPCRs) are critical cellular sensors that mediate numerous physiological processes. In the heart, multiple GPCRs are expressed on various cell types, where they coordinate to regulate cardiac function by modulating critical processes such as contractility and blood flow. Under pathological settings, these receptors undergo aberrant changes in expression levels, localization and capacity to couple to downstream signalling pathways. Conventional therapies for heart failure work by targeting GPCRs, such as β-adrenergic receptor and angiotensin II receptor antagonists. Although these treatments have improved patient survival, heart failure remains one of the leading causes of mortality worldwide. GPCR kinases (GRKs) are responsible for GPCR phosphorylation and, therefore, desensitization and downregulation of GPCRs. In this Review, we discuss the GPCR signalling pathways and the GRKs involved in the pathophysiology of heart disease. Given that increased expression and activity of GRK2 and GRK5 contribute to the loss of contractile reserve in the stressed and failing heart, inhibition of overactive GRKs has been proposed as a novel therapeutic approach to treat heart failure.G protein-coupled receptor (GPCR) kinases (GRKs) can desensitize and downregulate GPCRs. In this Review, Pfleger and colleagues describe the changes in GPCR and GRK signalling in the heart under disease conditions and how GRKs can be targeted to treat heart failure.Key pointsG protein-coupled receptors (GPCRs) mediate a range of physiological responses in various cardiovascular cell types.β-Adrenergic receptors (β-ARs) regulate cardiomyocyte contractility in response to sympathetic nervous system stimulation.In the failing heart, increased levels of GPCR kinases (GRKs) phosphorylate, and thereby desensitize and downregulate, β-ARs, resulting in a loss of cardiomyocyte contractile reserve.GRK2 and GRK5 can be therapeutically targeted to protect the heart against injury and failure using novel small-molecule inhibitors.
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lymphocyte levels of grk2 βark1 mirror changes in the lvad supported failing human heart lower grk2 associated with improved β adrenergic signaling after mechanical unloading
Journal of Cardiac Failure, 2006Co-Authors: Jonathan A Hata, Matthew L Williams, Jacob N Schroder, Brian Lima, Janelle R Keys, Burns C Blaxall, Jason A Petrofski, Andre Jakoi, Carmelo A Milano, Walter J KochAbstract:Abstract Background In human heart failure, increased expression of G protein–coupled receptor kinases (GRKs) causes the loss of β-adrenergic receptor (βAR) signaling and function. Mechanical unloading with a left ventricular assist device (LVAD) promotes reverse remodeling, which includes restoration of βAR responsiveness. We tested the hypothesis that LVAD support of the failing human heart alters the expression and activity of GRKs and we sought to determine whether changes in myocardial GRKs could be tracked in lymphocytes. Methods and Results Paired samples of human LV tissue (n = 12) and blood were obtained at the time of LVAD implantation (heart failure) and subsequent cardiac transplantation (LVAD). βAR signaling was quantified by receptor density and adenylyl cyclase activity. Immunoblotting and real-time reverse transcription polymerase chain reaction were used to measure GRK2 and GRK5 protein and mRNA levels. Rhodopsin phosphorylation was used to assess total GRK activity. Consistent with reverse remodeling, βAR density and signaling were restored to nonfailing levels after LVAD support. GRK2 protein levels were significantly reduced 55% after LVAD support and GRK2 mRNA was similarly reduced. In contrast, GRK5 protein and mRNA levels were unchanged. Total myocardial GRK activity was reduced similar to the drop in GRK2 expression. In lymphocytes, GRK2 protein levels were decreased after LVAD support and there was a significant positive correlation between myocardial and lymphocyte GRK2 levels in both heart failure and LVAD samples. Conclusion The changes in myocardial GRK2 expression and activity that are mirrored in lymphocytes provide a possible mechanism for the restoration of βAR signaling and reverse remodeling after mechanical unloading in the failing heart. Moreover, lymphocytes may provide a surrogate marker of myocardial GRK2 in these patients.
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Phosphatidylinositol 4,5-Bisphosphate (PIP2)-enhanced G Protein-coupled Receptor Kinase (GRK) Activity: LOCATION, STRUCTURE, AND REGULATION OF THE PIP2 BINDING SITE DISTINGUISHES THE GRK SUBFAMILIES
Journal of Biological Chemistry, 1996Co-Authors: Julie A. Pitcher, Zl Fredericks, Robert H. Stoffel, Richard T. Premont, Walter J Koch, W C Stone, Robert J LefkowitzAbstract:Abstract The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) enhances GRK5-mediated β-adrenergic receptor (βAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of βARK (β-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the βγ subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem.270, 11707-11710). Thus, five members of the GRK family bind PIP2, βARK (GRK2), βARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the βARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
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Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies.
The Journal of biological chemistry, 1996Co-Authors: Julie A. Pitcher, Zl Fredericks, Robert H. Stoffel, Richard T. Premont, Walter J Koch, W C Stone, Robert J LefkowitzAbstract:The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 11707-11710). Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
Julie A. Pitcher - One of the best experts on this subject based on the ideXlab platform.
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Distinct structural features of G protein-coupled receptor kinase 5 (GRK5) regulate its nuclear localization and DNA-binding ability.
PLOS ONE, 2013Co-Authors: Laura Ruth Johnson, James D. Robinson, Katrina N. Lester, Julie A. PitcherAbstract:G protein-coupled receptor kinases (GRKs) act to desensitize G protein-coupled receptors (GPCRs). In addition to this role at the plasma membrane, a nuclear function for GRK5, a member of the GRK4 subfamily of GRKs, has been reported. GRK5 phosphorylates and promotes the nuclear export of the histone deacetylase, HDAC5. Here we demonstrate that the possession of a nuclear localization sequence (NLS) is a common feature of GRK4 subfamily members (GRKs 4, 5 and 6). However, the location of the NLS and the ability of these GRKs to bind DNA in vitro are different. The NLSs of GRK5 and 6 bind DNA in vitro, whilst the NLS of GRK4 does not. Using mutants of GRK5 we identify the regions of GRK5 required for DNA-binding in vitro and nuclear localization in cells. The DNA-binding ability of GRK5 requires both the NLS and an N-terminal calmodulin (CaM)-binding site. A functional nuclear export sequence (NES), required for CaM-dependent nuclear export of the kinase, is also identified. Based on our observations we propose a model to explain how nuclear localization of GRK5 may be regulated. Notably, the nuclear localization of GRK5 and 6 is differentially regulated. These results suggest subfamily specific nuclear functions for the GRK4 subfamily members. Identification of GRK specific small molecule inhibitors of nuclear localization and/or function for the GRK4 subfamily may thus be an achievable goal.
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G Protein-Coupled Receptor Kinase 5 Contains a DNA-Binding Nuclear Localization Sequence
Molecular and Cellular Biology, 2004Co-Authors: Laura Ruth Johnson, Mark G. H. Scott, Julie A. PitcherAbstract:G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5(DeltaNLS)), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca2+/calmodulin (Ca2+/CaM) -dependent fashion. Ca2+/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5(DeltaNLS) or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.
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Phosphatidylinositol 4,5-Bisphosphate (PIP2)-enhanced G Protein-coupled Receptor Kinase (GRK) Activity: LOCATION, STRUCTURE, AND REGULATION OF THE PIP2 BINDING SITE DISTINGUISHES THE GRK SUBFAMILIES
Journal of Biological Chemistry, 1996Co-Authors: Julie A. Pitcher, Zl Fredericks, Robert H. Stoffel, Richard T. Premont, Walter J Koch, W C Stone, Robert J LefkowitzAbstract:Abstract The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) enhances GRK5-mediated β-adrenergic receptor (βAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of βARK (β-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the βγ subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem.270, 11707-11710). Thus, five members of the GRK family bind PIP2, βARK (GRK2), βARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the βARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
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Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies.
The Journal of biological chemistry, 1996Co-Authors: Julie A. Pitcher, Zl Fredericks, Robert H. Stoffel, Richard T. Premont, Walter J Koch, W C Stone, Robert J LefkowitzAbstract:The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 11707-11710). Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.
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characterization of the g protein coupled receptor kinase grk4 identification of four splice variants
Journal of Biological Chemistry, 1996Co-Authors: Richard T. Premont, Robert H. Stoffel, Julie A. Pitcher, Namjin Chung, Christine Ambrose, Alexander D. Macrae, Marcy E Macdonald, James Inglese, Robert J LefkowitzAbstract:A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
