Beta adrenergic receptor kinase 1 and heart failure
Diseases

Author: Ylenia Marten Canavesio
Date: 31/01/2014

Description

We would like to analyze, in summary, the structure of Beta adrenergic kinase 1 and its importance in the heart failure as a target for new therapies.

DEFINITION

Beta adrenergic receptor kinase 1 (also referred to as βARK or BARK) is a serine/threonine intracellular kinase. It is activated by PKA and its target is the beta adrenergic receptor. It is one method by which the cell will desensitize itself from epinephrine overstimulation.
Now BARK1 is known as GRK2. GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance.

THE GENE

The gene spans approximately 23 kilobases and is composed of 21 exons interrupted by 20 introns. Exon sizes range from 52 bases (exon 7) to over 1200 bases (exon 21), intron sizes from 68 bases (intron L) to 10.8 kilobases (intron A).

DatabaseLink
WikigenesADRBK1
GeneCardsBARK1
Your Favorite Gene SigmaADRBK1
HGNCADRBK1

Gene expression pattern of the ADRBK1 gene (from: PBB GE ADRBK1 38447 at tn.png).

CHEMICAL STRUCTURE AND IMAGES

GRKs share a common structural architecture with a well-conserved, central catalytic domain (∼270 aa), similar to that of other serine-threonine kinases, flanked by an N-terminal domain (∼185 aa) and a variable-length carboxyl-terminal domain (∼105–230 aa). The N-terminal domain has been proposed to be important for receptor recognition, for intracellular membrane anchoring and also contains an RH domain (regulator of G protein signalling homology domain) of ∼120 aa. In the case of GRK2 and GRK3, the RH domain has been shown to specifically interact with Gαq family members, thus blocking its interaction with their effector, phospholipase C beta (PLCβ) (Selective Regulation of Gq Signaling by G Protein-Coupled Receptor Kinase 2: Direct Interaction of Kinase N Terminus with Activated Gαq, 1 April 2000). The C-terminal region of GRK2 contains a pleckstrin homology domain with binding sites for the membrane phospholipid PIP2 and free Gβγ subunits and therefore is involved in its agonist-dependent translocation to the plasma membrane.
The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets, 4 March 2010

Biological Assembly

Protein Aminoacids Percentage (Width 700 px)

CELLULAR FUNCTIONS

βARK1 activation consists of 8 steps:

  • Upon stimulation of the Beta adrenergic receptor by epinephrine, Gs will be activated.
  • Gs alpha will then stimulate adenyl cyclase to make cAMP.
  • cAMP will then activate cAMP dependent kinase (PKA), which among other proteins that it acts on, it will phosphorylate serine and threonine residues on βARK.
  • βARK, itself a serine/threonine kinase, will then phosphorylate serine and threonine resides on the β-adrenergic receptor itself.
  • This will facilitate Beta-arrestin's binding to the receptor. Additional stimulation by epinephrine will now be unable to activate Gs due to arrestin.
  • As a result of β-arrestin binding, phosphorylated receptors are also targeted for clathrin-mediated endocytosis, a process that classically serves to re-sensitize and recycle receptors back to the plasma membrane.

Therefore, βARK is a negative feedback enzyme which will prevent over stimulation of the β-adrenergic receptor.

βARK Activation

Transmembrane signaling systems for converting extracellular stimuli as divergent as hormones, drugs, or photons of light into intracellular metabolic changes have been remarkably conserved through evolution. Such systems generally consist of three major components: a receptor, such as the P-adrenergic receptor (J3AR) or the visual "light receptor" rhodopsin; a guanine nucleotide-binding regulatory protein, such as the stimulatory guanine nucleotide-binding regulatory protein (Gs) or transducin; and an effector, such as adenylate cyclase or cGMP phosphodiesterase (1-4). Not only are such systems analogous, but individual components are actually homologous proteins such as the ,BAR and rhodopsin or Gs and transducin. Moreover, two analogous enzymes that may function to regulate the activities of such systems have been described. Rhodopsin kinase is a cytosolic enzyme that phosphorylates only the light-bleached form of rhodopsin on multiple serine and threonine residues (up to 9 mol of phosphate per mol of receptor) and is thought to be involved in deactivating the illuminated form of rhodopsin so that it does not continue to activate transducin (5, 6). P3AR kinase is a ubiquitously distributed cytosolic enzyme that phosphorylates only the agonist-occupied form of the PAR
(up to 9 mol of phosphate per mol).
Functional desensitization of the isolated j3-adrenergic receptor by the ,3-adrenergic receptor kinase: Potential role of an analog of the retinal protein arrestin (48-kDa protein), December 1987

Recent data indicate that arrestins and GRKs can also regulate signalling mediated by other membrane receptor families, such as tyrosine kinase receptors for IGF-1, Insulin, PDGF or EGF. […]In addition, a growing number of non-GPCR, non-plasma membrane receptor substrates are being identified for GRKs, particularly GRK2. These include tubulin, synucleins, phosducin, ribosomal protein P2, the inhibitory g subunit of the type 6 retinal cyclic guanosine monophosphate (cGMP) phosphodiesterase, a subunit of the epithelial Na+ channel, the ERM family protein ezrin, the calcium-binding protein DREAM, IKappaBalpha or the p38 MAPK. GRK2 also inhibits TGF-beta-mediated cell growth arrest and apoptosis by inducing Smad. Besides such phosphorylation-dependent processes, GRK2 may also contribute to modulate cellular responses in a phosphorylation-independent manner thanks to its ability to interact with a plethora of proteins involved in signalling and trafficking.
The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets, 4 March 2010

GRK2 is linked to diverse regulatory networks acting at specific stages of the cell cycle. In response to both extrinsic and intrinsic cues, GRK2 protein plays a critical role in driving cell progression through G1/S and G2/M transitions in a kinase-dependent and independent manner. GRK2 is part of an intrinsic pathway that ensures timely progression of cell cycle at G2/M by means of its functional interaction with CDK2/cyclinA and Pin1 . Such pathway is disrupted upon DNA damage, when GRK2 appears to turn into a pro-arresting factor that promotes increased cell survival and to dampen p53-dependent responses by mechanisms that remain to be established (dotted lines/question mark). On the other hand, GRK2 contributes to the Hedgehog/Smoothened-triggered control of cell proliferation by promoting Smo activity and relieving the Patched-dependent inhibition of cyclin B (Jiang et al., 2009). CDK, cyclin-dependent kinase; GRK, G protein-coupled receptor kinase.

GRK2 in cardiovascular cells
If we focus on the heart, βARs are the most important GPCR class expressed in the human heart and represent the most powerful means to increase the pumping function of the heart. In particular, βARs are the prime modulators of heart rate and myocardial contractility in response to catecholamines originating from the SNS. Three βAR subtypes(β1, β2, and β3) have been identified in human cardiac tissue with the β1- and β2ARs representing the majority of βARs in the myocyte driving functional responses and these receptors are in a 3–4:ⁱ ratio (β1:β2) in the normal heart. The β3AR is relatively minor although it is present and may contribute to normal and diseased myocardial regulation. Following catecholamine stimulation, both β1- and β2ARs couple to adenylyl cyclase (AC) stimulatory Gprotein, Gs, leading to cAMP accumulation within the myocyte and activation of protein kinase A (PKA). This kinase phosphorylates many Ca2+ handling protein and some myofilament components leading to positive inotropic,lusitropic and chronotropic effects. [...] Known differences include myocyte cell death and survival as β1AR stimulation leads to apoptosis while β2AR signaling favors cell survival pathways. The later has been shown to be through a β2AR-Gi-Gβγ- PI3K-Akt cell survival signaling pathway and the inhibition of this pathway converts β2AR signaling from survival to apoptotic. β1- and β2ARs also manifest opposing effects on cardiac cell growth as stimulation of β1ARs, but not β2ARs, can cause hypertrophy in cultured neonatal and adult rat cardiac myocytes.
Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy, 26 September 2013

GRK2 plays a key role in the control of beta-adrenergic signalling in the heart. Hemizygous GRK2 mice expressing 50% less protein than control littermates are hyper-responsive to catecholamines and present increased cardiac contractility and function. The opposite happens with transgenic mice overexpressing different levels of this kinase (Koch et al., 1998) in which the adrenergic cardiac response is impaired. The relationship between increased GRK2 protein levels and end-stage heart failure (HF) has been established in animal models and in patients afflicted with different heart conditions. Increased left ventricular GRK2 mRNA and activity were reported in patients with ischemic or idiopathic dilated cardiomyopathy, cardiac ischemia, volume overload and left ventricular hypertrophy. Interestingly, in some animal models the development of overt HF is preceded by an elevation of GRK2 levels that correlates with progressive impaired myocardial contractility and β-adrenergic receptor (βAR) responsiveness. More interestingly, GRK2 levels are elevated post-infarction only in animals that develop heart failure. The levels and/or activity of GRK2 are also elevated in lymphocytes of patients with cardiac failure. This has lead to the possibility that lymphocyte GRK2 could be used as surrogate readout of myocardial levels of GRK2.

We crossed β1AR knockout (B1KO) mice with cardiac-specific transgenic mice expressing the βARKct, a known GRK2 inhibitor, and studied the offspring under normal conditions and in post-myocardial infarction (MI). βARKct expression in vivo proved essential for β2AR-dependent contractile function, as β2AR stimulation with isoproterenol fails to increase contractility in either healthy or post-MI B1KO mice and it only does so in the presence of βARKct. The main underlying mechanism for this is blockade of the interaction of phosphodiesterase (PDE) type 4D with the cardiac β2AR, which is normally mediated by the actions of GRK2 and βarrs on the receptor. The molecular “brake” that PDE4D poses on β2AR signaling to contractility stimulation is thus “released”. Regarding the other beneficial functions of cardiac β2AR, βARKct increased overall survival of the post-MI B1KO mice progressing to HF, via a decrease in cardiac apoptosis and an increase in wound healing-associated inflammation early (at 24 hrs) post-MI. However, these effects disappear by 4 weeks post-MI, and, in their place, upregulation of the other major GRK in the heart, GRK5, is observed.

GRK2 blockade with βARKct is essential for cardiac β2-adrenergic receptor signaling towards increased contractility, 28 August 2013

Data now supports GRK2 being a molecular link between the excessive neurohormonal activation that follows cardiac stress and initiation of defects in myocyte energy substrate use by negatively affecting glucose uptake in the myocyte through insulin-dependent phosphorylation of insulin receptor substrate-1 (IRS1) that causes a loss of signaling. [...] our group have also showed that elevated levels of GRK2 in cardiomyocytes cause excessive cell death after acute ischemic injury and targeted inhibition or genetic deletion significantly protects the heart. [...] Accordingly, GRK2 inhibition reduces IRS1-phosphorylation improving insulin signaling and as a result increase glucose uptake in the ischemic cardiomyocytes and its inhibition but also reduces the pro-apoptotic pathway activated by this kinase in response to ischemia.
Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy, 26 September 2013

REGULATION

The association of GRK2 with α-actinin, clathrin, calmodulin, caveolin or RKIP appear to participate in controlling GRK2 activity and in determining the complex subcellular distribution of the kinase.
Phosphorylation by different kinases has been shown to either enhance (PKA, PKC, Src) or decrease (ERK) membrane targeting and/or the catalytic activity of GRK2, thus opening the possibility of transmodulation by different signalling pathways. A recent work has put forward S-nitrosylation of GRK2 as a novel mechanism to inhibit its activity. Phosphorylation of GRK2 at given tyrosine or serine residues is also emerging as a key mechanism to dynamically modulate its interaction with cellular partners. Tyrosine phosphorylation by c-Src appears to enhance the interaction of GRK2 with Gαq and with the GIT1 scaffold protein. On the other hand, ERK1/2 phosphorylates GRK2 on S670, strongly impairing the GRK2/Gβγ interaction and inhibiting kinase translocation and catalytic activity towards receptor membrane substrates, while also modulating GRK2 interaction with GIT1.
Regarding the regulation of GRK2 expression little has been reported about the mechanisms governing GRK transcription. In aortic smooth muscle cells, we found that agents that induce physiological vasoconstriction and hypertrophy markedly enhance GRK2 promoter activity, whereas pro-inflammatory cytokines promote the opposite effect, suggesting that the expression of GRK2 is strongly controlled at the transcriptional level by the interplay between various signal transduction pathways. TGF-β induces GRK2 expression in hepatocarcinoma cells and in vascular smooth muscle cells. However, whether these mechanisms apply to other cell types awaits further investigation.
Regulation of GRK2 stability may provide an important mechanism for modulating its expression levels. We have shown that GRK2 is rapidly degraded by the proteasome pathway, and that GRK2 ubiquitination and turnover is enhanced by β2AR activation as a result of phosphorylation of GRK2 by c-Src and MAPK in a β-arrestin-dependent manner.

More recently, we have shown that Mdm2, an E3-ubiquitin ligase involved in the control of cell growth and apoptosis, plays a key role in GRK2 degradation. Mdm2 and GRK2 association and subsequent proteolysis are facilitated by the β-arrestin scaffold function upon β2-adrenergic receptor stimulation. On the contrary, activation of the PI3K/Akt pathway by agonists such as IGF-1 alters Mdm2 phosphorylation and triggers its nuclear localization thus hampering Mdm2-mediated GRK2 degradation and leading to enhanced GRK2 stability and increased kinase levels. It is tempting to suggest that deregulated activity of the PI3K/Akt pathway in pathophysiological contexts characterized by increased cell proliferation and survival would lead to GRK2 up-regulation in human tumour malignancies, and we are currently very actively investigating this possibility.
The presence of beta-agonists has been reported to up-regulate GRK2 mRNA, whereas ischemia might promote GRK2 degradation by the proteasome in some experimental models. However, there is limited knowledge of the mechanisms modulating GRK2 expression in cardiovascular cells in pathological settings.
Although the issue of whether elevated GRK2 expression is a precipitating factor for congestive heart failure or an initial adaptive process that eventually turns pathologic has not been solved, the available data underline the importance of GRK2 levels as a marker of predisposition to cardiac dysfunction and supports the idea that GRK2 offers a potential therapeutic target.
The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets, 4 March 2010

THERAPY

β-blockers are currently considered as the mainstay of HF therapy. Behind pharmacologic β-blockade action is the ability of these molecules to inhibit the excessive catecholamine stimulation of inotropic βARs that induce myocyte cell death so resulting in cardiac deterioration, and changes in ventricular mass and that promote ventricular dilation through HF. Importantly, although β-blockers only indirectly address key molecular signaling alterations within the cardiac myocyte, their clinical use substantially improves HF prognosis increasing survival rate of HF patients and reducing re-hospitalization. Interestingly, it has been observed that at molecular level, sustained therapy with β-blockers in HF is associated with resensitization of βARs, normalization of GRK2 levels and activity and as a consequence, β-blockers cause the upregulation of cardiac βARs (down-regulated in HF) increasing βAR signaling when they are activated. […]Of note, β-blocker therapy has limitations in the HF patient population and it is not tolerated by all patients and is certainly not an ideal therapeutic. For this reason the development of new potential molecules that minimize the unfavorable effects and potentially allow dose reduction is absolutely needed and we believe inhibiting GRK2 is an attractive target not only because it can also normalize βAR signaling but also it appears to have exciting and novel non-GPCR effects that has appeal for targeting its inhibition that can synergize with β-blocker use in HF patients.

Gene therapy for HF is now becoming a reality and AAV6-βARKct trials are in the planning stages, GRK2 inhibition by small pharmacological agents would offer many advantages to the HF patient. Interestingly, several recent molecules have been developed and described that have GRK2 inhibitory properties. Two decades ago, heparin and related compounds were shown to block GRK2 activity however, the direct access to GRK2, the high concentration and the intrinsic cytotoxicity made them not useful for either in cell-based assays or in in vivo scenarios.
Interestingly, molecules have recently emerged that target the GRK2-Gβγ protein-protein interaction and thus, have mechanisms identical to the βARKct. M119 is such a molecule and it has been shown to work in vitro and in vivo on cardiac cells and in the heart preventing ventricular dysfunction after chronic catecholamine exposure and also showing positive results similar to the βARKct in a genetic model of cardiomyopathy. Gallein is a related molecule that blocks GRK2-Gβγ and it has also shown positive results in vivo. These are promising results, however, these compounds are not true pharmacological agents in a “drugable” sense and have severe limitations that preclude human use.
Recently, we have found that an existing FDA-approved drug has significant GRK2 inhibitory properties and potentially this off-target effect may be seen in humans. The serotonin reuptake inhibitor (SSRI), paroxetine has affinity for GRK2 and has significant GRK2 inhibitory properties in vitro and in vivo. Paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Further, this drug causes increased isoproterenol-induced shortening and contraction amplitude in cardiomyocytes in vitro, and pretreatment in vivo of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve with no significant effect on heart rate. This agent used for clinical depression probably is not viable for use as a specific GRK2 inhibitor but is a great starting point for chemistry to develop novel GRK2 inhibitors that can be used
eventually for cardiovascular disorders.
Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy, 26 September 2013

Structure of GRK2 binding to paroxetine:

RNA aptamers can be identified by in vitro selection processes, also termed SELEX (systematic evolution of ligands by exponential enrichment), for various target molecules, including small molecules, peptides, proteins, and even living cells. Furthermore, aptamers have been shown to be potent and specific inhibitors of several protein classes both in the extracellular and intracellular environment. […]We applied in vitro selection to identify a highly specific RNA aptamer that binds to GRK2 and inhibits its kinase function.This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation. This RNA aptamer represents the most potent inhibitor of GRK2 activity reported so far. More importantly, the aptamer possesses a high specificity for GRK function, and thus it might allow the precise functional characterization of GRK2 both in cell culture and in vivo. Furthermore, this aptamer might represent a starting point for the development of small molecules that specifically target GRK2.
An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro, March 2008

Bibliography

Beta adrenergic receptor kinase, Wikipedia
The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets, 4 March 2010
Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy, 26 September 2013
An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro, March 2008
Selective Regulation of Gq Signaling by G Protein-Coupled Receptor Kinase 2: Direct Interaction of Kinase N Terminus with Activated Gαq, 1 April 2000
GRK2 blockade with βARKct is essential for cardiac β2-adrenergic receptor signaling towards increased contractility, 28 August 2013
Functional desensitization of the isolated j3-adrenergic receptor by the ,3-adrenergic receptor kinase: Potential role of an analog of the retinal protein arrestin (48-kDa protein), December 1987

Comments
2014-02-13T13:46:23 - Ylenia Marten Canavesio
2014-02-13T13:35:33 - Ylenia Marten Canavesio
2014-02-13T13:31:49 - Ylenia Marten Canavesio
2014-02-13T13:22:09 - Ylenia Marten Canavesio
2014-02-13T13:10:53 - Ylenia Marten Canavesio
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2014-02-12T18:52:43 - Ylenia Marten Canavesio
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2014-02-11T19:20:24 - Ylenia Marten Canavesio
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2014-02-11T12:20:24 - Ylenia Marten Canavesio
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