G Protein-coupled Receptor Kinase 2 Activity Research Articles

The selective serotonin reuptake inhibitor paroxetine improves right ventricular systolic function in experimental pulmonary hypertension

BackgroundPulmonary hypertension (PH) often leads to right ventricle (RV) failure, a significant cause of morbidity and mortality. Despite advancements in PH management, progression to RV maladaptation and subsequent failure remain a clinical challenge. This study explored the effect of paroxetine, a selective serotonin reuptake inhibitor (SSRI), on RV function in a rat model of PH, hypothesizing that it improves RV function by inhibiting G protein-coupled receptor kinase 2 (GRK2) and altering myofilament protein phosphorylation. MethodsThe Su5416/hypoxia (SuHx) rat model was used to induce PH. Rats were treated with paroxetine and compared to vehicle-treated and control groups. Parameters measured included RV morphology, systolic and diastolic function, myofilament protein phosphorylation, GRK2 activity, and sympathetic nervous system (SNS) markers. ResultsParoxetine treatment significantly improved RV systolic function, evidenced by increased stroke volume, cardiac output, and ejection fraction, without significantly affecting RV hypertrophy, myosin heavy chain/titin isoform switching, or fibrosis. Enhanced phosphorylation of titin and myosin light chain-2 was observed, correlating positively with improved systolic function. Contrary to the hypothesis, improvements occurred independently of GRK2 inhibition or SNS modulation, suggesting an alternate mechanism, potentially involving antioxidant properties of paroxetine. ConclusionParoxetine improves RV systolic function in PH rats, likely through mechanisms beyond GRK2 inhibition, possibly related to its antioxidant effects. This highlights the potential of paroxetine in managing RV dysfunction in PH, warranting further investigation into its detailed mechanisms of action and clinical applicability.

GRK2 inhibits Flt-1+ macrophage infiltration and its proangiogenic properties in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/− mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/− mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.

GRK2 Mediates Macrophage Polarization by Regulating EP4-cAMP-pCREB Signaling in Ulcerative Colitis and the Therapeutic Effect of Paroxetine on Mice with DSS-Induced Colitis.

G protein-coupled receptor kinase 2 (GRK2) is one of the cytosolic enzymes, and GRK2 translocation induces prostaglandin E2 receptor 4 (EP4) over-desensitization and reduces the level of cyclic adenosine monophosphate (cAMP) to regulate macrophage polarization. However, the role of GRK2 in the pathophysiology of ulcerative colitis (UC) remains unclear. In this study, we investigated the role of GRK2 in macrophage polarization in UC, using biopsies from patients, a GRK2 heterozygous mouse model with dextran sulfate sodium (DSS)-induced colitis, and THP-1 cells. The results showed that a high level of prostaglandin E2 (PGE2) stimulated the receptor EP4 and enhanced the transmembrane activity of GRK2 in colonic lamina propria mononuclear cells (LPMCs), resulting in a down-regulation of membrane EP4 expression. Then, the suppression of cAMP-cyclic AMP responsive element-binding (CREB) signal inhibited M2 polarization in UC. Paroxetine is acknowledged as one of the selective serotonin reuptake inhibitors (SSRI), which is also considered as a potent GRK2 inhibitor with a high selectivity for GRK2. We found that paroxetine could alleviate symptoms of DSS-induced colitis in mice by regulating GPCR signaling to affect macrophage polarization. Taken together, the current results show that GRK2 may act as a novel therapeutic target in UC by regulating macrophage polarization, and paroxetine as a GRK2 inhibitor may have therapeutic effect on mice with DSS-induced colitis.

Mitochondrial GRK2 is a Novel Regulator of Cardiac Energetics

G protein‐coupled receptor (GPCR) kinase 2 (GRK2) is highly expressed in the heart, where during injury or heart failure (HF), both its levels and activity increase. GRK2 is canonically studied in the context of GPCR phosphorylation; however, noncanonical activities of GRK2 have emerged and it is now appreciated that GRK2 has a large non‐GPCR interactome. For example, in cardiac myocytes, GRK2 translocates from the cytosol to mitochondria (mtGRK2) following oxidative stress or ischemia injury, and this pool of mtGRK2 is associated with negative effects on metabolism and also induces myocyte cell death. However, the mechanisms by which mtGRK2 contributes to cardiac dysfunction and HF are not fully understood. We hypothesized that mtGRK2 could have novel substrates and phosphorylate proteins involved in mitochondrial bioenergetics, thus contributing to our previously established post‐injury phenotype. Stress‐induced mitochondrial translocation of cytosolic GRK2 was validated in cell and animal models and the mtGRK2 interactome was identified using liquid chromatography‐mass spectroscopy (LCMS). Proteomics analysis identified mtGRK2 interacting proteins which were involved in mitochondrial dysfunction, bioenergetics, and OXPHOS, particularly complexes I, II, IV and V of the electron transport chain (ETC). Specifically, mtGRK2 interactions with Complex V (ATP synthase) subunits were particularly increased following stress. We established that mtGRK2 phosphorylates ATP synthase on the F1 catalytic barrel, which is critical for oxidative phosphorylation and ATP production. We have also determined that alterations in either the levels or activity of GRK2 appear to alter ATP synthase enzymatic activity in vitro. Excitingly, in vivo data suggest that reducing levels of GRK2 in a mouse model of myocardial infarction prevents the post‐injury reduction in ATP synthesis. We are currently assessing the ability of the SSRI drug paroxetine, a GRK2 inhibitor, to preserve mitochondrial bioenergetics in a transgenic GRK2 mouse model. Thus, phosphorylation of the ATP synthesis machinery by mtGRK2 may contribute to the impaired mitochondrial phenotype observed in injured or failing heartssuch as reduced fatty acid metabolism and substrate utilization. These data uncover a druggable, novel target for rescuing cardiac function in patients with injured and/or failing hearts.

GRK2 regulates group 2 innate lymphoid cell mobilization in sepsis

BackgroundSepsis induces group 2 innate lymphoid cell (ILC2) expansion in the lung. However, the origin of these lung-recruited ILC2 and the mechanism of ILC2 expansion are unclear. This study aims to determine the origin of lung-recruited ILC2 and its underlying mechanism in sepsis.MethodsSepsis was induced by cecal ligation and puncture (CLP) model in wild-type, IL-33-deficient and ST2-deficient mice. The frequency, cell number and C-X-C chemokine receptor 4 (CXCR4) expression of ILC2 in bone marrow (BM), blood and lung were measured by flow cytometry. In the in vitro studies, purified ILC2 progenitor (ILC2p) were challenged with IL-33 or G protein-coupled receptor kinase 2 (GRK2) inhibitor, the CXCR4 expression and GRK2 activity were detected by confocal microscopy or flow cytometry.ResultsWe show that IL-33 acts through its receptor, ST2, on BM ILC2p to induce GRK2 expression and subsequent downregulation of cell surface expression of CXCR4, which results in decreasing retention of ILC2p in the BM and promoting expansion of ILC2 in the lung. Importantly, we demonstrate that reduced IL-33 level in aging mice contributes to impaired ILC2 mobilization from BM and accumulation in the lung following sepsis.ConclusionThis study identifies a novel pathway in regulating ILC2p mobilization and expansion during sepsis and indicates BM as the main source of ILC2 in the lung following sepsis.

GRK2 promotes activation of lung fibroblast cells and contributes to pathogenesis of pulmonary fibrosis through increasing Smad3 expression.

Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor-β1 (TGF-β1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-β1-induced signal transduction in different cell types; however, the role of GRK2 in TGF-β1-induced activation of lung fibroblast cells and development of pulmonary fibrosis has not been revealed. In this study, we found that GRK2 levels were increased in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-β1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Posttreatment with GRK2 inhibitor reduced extracellular matrix (ECM) accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-β1-induced lung fibroblast cells or myofibroblast cells isolated from patients with pulmonary fibrosis. Furthermore, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-β1 signal pathway and ECM accumulation.

CP-25 exerts therapeutic effects in mice with dextran sodium sulfate-induced colitis by inhibiting GRK2 translocation to downregulate the TLR4-NF-κB-NLRP3 inflammasome signaling pathway in macrophages.

Deficiency of G protein-coupled receptor kinase 2 (GRK2) was found to protect mice from dextran sulfate sodium (DSS)-induced colitis. Paeoniflorin-6'-O-benzene sulfonate (CP-25) has been shown to exert anti-inflammatory immune regulatory effects in animal models of inflammatory autoimmune disease. This study aimed to investigate the of GRK2 in the pathogenesis of ulcerative colitis (UC) and its effects on macrophage polarization, macrophage subtype regulation of intestinal barrier function, and therapeutic effects of CP-25 in mice with DSS-induced colitis. We found imbalanced macrophage polarization, intestinal barrier dysfunction, and abnormal activation of GRK2 and TLR4-NF-κB-NLRP3 inflammasome signaling pathway in the colonic mucosa of patients with UC. CP-25, restored the damaged intestinal barrier function by inhibiting the transmembrane region of GRK2 in macrophages stimulated by lipopolysaccharides. CP-25 exerted therapeutic effects by ameliorating clinical manifestation, regulating macrophage polarization, and restoring abnormally activated TLR4-NF-κB-NLRP3 inflammasome signaling pathway by inhibiting GRK2. These data suggest the pathogenesis of UC may be related to the imbalance of macrophage polarization, which leads to abnormal activation of TLR4-NF-κB-NLRP3 inflammasome signaling pathway mediated by GRK2 and destruction of the intestinal mucosal barrier. CP-25 confers therapeutic effects on colitis by inhibiting GRK2 translocation to induce the downregulation of TLR4-NF-κB-NLRP3 inflammasome signaling in macrophages.

Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury.

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.

Apelin‐Induced Relaxation is Impaired in Coronary Arteries from Spontaneously Hypertensive Rats

Hypertension is a major modifiable risk factor for coronary artery disease. The pathophysiological mechanisms underlying hypertension as a risk factor for coronary artery disease are not fully understood. Apelin is a vasoactive peptide that binds to APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. APJ receptors signal via G-protein-dependent and -independent pathways, including activation of G-protein-coupled-receptor kinase 2 (GRK2), which can potentially lead to inhibition of eNOS and nitric oxide (NO) production. We have previously shown that apelin causes endothelium-dependent, NO-mediated relaxation of coronary arteries from normotensive animals (JPET 366:265, 2018), but its effects in hypertensive coronary arteries are unknown. Here, we tested the hypothesis that apelin-induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Western blot analysis demonstrated increased expression of GRK2 in cultured coronary endothelial cells from SHR in comparison with normotensive WKY (control) rats. Isolated coronary arterial rings were mounted in wire myographs for isometric tension recording and contracted with 5-HT (10-7 M). Apelin (10-9-10-6 M) caused endothelium-dependent relaxation of WKY coronary arteries (pD2 = 7.00 ± 0.11; Emax = 54 ± 4% relaxation; n=6), but had no effect in rings from SHR. Conversely, endothelium-dependent relaxations to an APJ receptor biased agonist, CMF-019 (10-9-10-5 M), which preferentially activates the G-protein-dependent pathway with minimal effect on GRK2, did not differ between SHR and WKY. In the presence of apelin (10-7 M), endothelium-dependent relaxation to acetylcholine (ACh) (10-9-10-5 M) was impaired in SHR coronary arteries (pD2 = 6.82 ± 0.10 vs 6.52 ± 0.18, and Emax = 81 ± 8 vs 60 ± 10% relaxation, in the absence and presence of apelin, respectively; n=5; p<0.05); however, apelin had no effect on ACh-induced relaxation of WKY coronary arteries. By contrast, ACh-induced relaxation of SHR and WKY coronary arterial rings was unaffected by CMF-019 (10-7 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 × 10-5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2 = 6.24 ± 0.30; Emax = 42 ± 13% relaxation; n=3). Relaxations in response to the NO-donor, DEA NONOate (10-9-10-4 M), were not altered by apelin (10-7 M) treatment in either WKY or SHR coronary arteries. Taken together, the data indicate that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired production or release of NO from endothelial cells rather than interfering with the action of NO on coronary smooth muscle cells. In SHR coronary arteries, apelin instead actively inhibited relaxation to another endothelium-dependent vasodilator; i.e. ACh. That the effects of the G-protein biased agonist, CMF-019, did not differ between WKY and SHR coronary arteries is consistent with a role for GRK2 activation in the altered response to apelin in hypertensive arteries. APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself, as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in coronary arteries from SHR.

Dynamics of adrenergic signaling in cardiac myocytes and implications for pharmacological treatment

Dense innervation of the heart by the sympathetic nervous system (SNS) allows cardiac output to respond appropriately to the needs of the body under varying conditions, but occasionally the abrupt onset of SNS activity can trigger cardiac arrhythmias. Sympathetic activity leads to the release of norepinephrine (NE) onto cardiomyocytes, activating β1-adrenergic receptors (β1-ARs) and leading to the production of the second messenger cyclic AMP (cAMP). Upon sudden activation of β1-ARs in experiments, intracellular cAMP can transiently rise to a high concentration before converging to a steady state level. Although changes to cellular cAMP concentration are important in modulating the overall cardiovascular response to sympathetic tone, the underlying mechanisms of the cAMP transients and the parameters that control their magnitude are unclear.We reduce a detailed computational model of the β1-adrenergic signaling cascade to a system of two differential equations by eliminating extraneous variables and applying quasi-steady state approximation. The structure of the reduced model reveals that the large cAMP transients associated with abrupt β1-AR activation are generated by the interplay of production/degradation of cAMP and desensitization/resensitization of β1-ARs. The reduced model is used to predict how the dynamics of intracellular cAMP depend on the concentrations of norepinephrine (NE), phosphodiesterases 3 and 4 (PDE3,4), G-protein coupled receptor kinase 2 (GRK2), and β1-AR, in healthy conditions and a simple model of early stages of heart failure.The key findings of the study are as follows: 1) Applying a reduced model of the dynamics of cardiac sympathetic signaling we show that the concentrations of two variables, cAMP and non-desensitized β1-AR, capture the overall dynamics of sympathetic signaling; 2) The key factors influencing cAMP production are AC activity and PDE3,4 activity, while those that directly impact β1-AR phosphorylation are GRK2 and PKA1. Thus, disease states that affect sympathetic control of the heart can be thoroughly assessed by studying AC activity, PDE3,4, GRK2 and PKA activity, as these factors directly impact cAMP production/degradation and β1-AR (de) phosphorylation and are therefore predicted to comprise the most effective pharmaceutical targets in diseases affecting cardiac β1-adrenergic signaling.

Hit-to-lead optimization and discovery of a potent, and orally bioavailable G protein coupled receptor kinase 2 (GRK2) inhibitor

G-protein coupled receptor kinase 2 (GRK2), which is upregulated in the failing heart, appears to play a critical role in heart failure (HF) progression in part because enhanced GRK2 activity promotes dysfunction of β-adrenergic signaling and myocyte death. An orally bioavailable GRK2 inhibitor could offer unique therapeutic outcomes that cannot be attained by current heart failure treatments that directly target GPCRs or angiotensin-converting enzyme. Herein, we describe the discovery of a potent, selective, and orally bioavailable GRK2 inhibitor, 8h, through high-throughput screening, hit-to-lead optimization, structure-based design, molecular modelling, synthesis, and biological evaluation. In the cellular target engagement assays, 8h enhances isoproterenol-mediated cyclic adenosine 3′,5′-monophosphate (cAMP) production in HEK293 cells overexpressing GRK2. Compound 8h was further evaluated in a human stem cell-derived cardiomyocyte (HSC-CM) contractility assay and potentiated isoproterenol-induced beating rate in HSC-CMs.

Role of Endothelial G Protein-Coupled Receptor Kinase 2 in Angioedema.

Excessive BK (bradykinin) stimulation is responsible for the exaggerated permeabilization of the endothelium in angioedema. However, the molecular mechanisms underlying these responses have not been investigated. BK receptors are Gq-protein-coupled receptors phosphorylated by GRK2 (G protein-coupled receptor kinase 2) with a hitherto unknown biological and pathophysiological significance. In the present study, we sought to identify the functional role of GRK2 in angioedema through the regulation of BK signaling. We found that the accumulation of cytosolic Ca2+ in endothelial cells induced by BK was sensitive to GRK2 activity, as it was significantly augmented by inhibiting the kinase. Accordingly, permeabilization and NO production induced by BK were enhanced, as well. In vivo, mice with reduced GRK2 levels in the endothelium (Tie2-CRE/GRK2fl+/fl-) exhibited an increased response to BK in terms of vascular permeability and extravasation. Finally, patients with reduced GRK2 levels displayed a severe phenotype of angioedema. Taken together, these findings establish GRK2 as a novel pivotal regulator of BK signaling with an essential role in the pathophysiology of vascular permeability and angioedema.

Abstract MP122: Effects of Cardiac Beta3-Adrenoceptors on the Inhibition of G Protein-coupled Receptor Kinase 2 via S-nitrosylation

G Protein-Coupled Receptor Kinase 2 (GRK2) is a culprit in the loss of cardiac contractile function in heart failure due to β-Adrenoceptor (AR) desensitization after its upregulation. Indeed, its inhibition has been demonstrated to improve cardiac function and increased GRK2 in the heart leads to larger injury after an ischemic insult. Nitric oxide (NO) via S-nitrosothiol (SNO) at residue Cys340 is a reported endogenous inhibitor of GRK2 activity. ß3ARs, on the other hand, are known to be resistant to desensitization by GRK2 and they are upregulated in cardiac pathologies. Activation of ß3ARs can be cardioprotective via NO signalling. Thus, in the present study, we aimed to investigate the interaction between ß3ARs and GRK2 through NO signaling to determine if ß3AR cardioprotection can occur via NO-mediated GRK2 inhibition. We used wild type C57BL/6 mice (WT), global β3AR knockout (KO) mice and GRK2-C340S knockin mice, which harbor a point mutation that changes Cys340 with a serine, meaning all endogenous GRK2 globally cannot be inhibited via NO-mediated S-nitrosylation. We exposed WT, GRK2-C340s KI and β3AR KO mice to ischemia/reperfusion (I/R) injury (40 min ischemia followed by 24 hrs reperfusion). We found that WT mice had significantly diminished cardiac function evaluated by echocardiography and Millar Catheterization, and this was rescued by treating these mice with CL316,243 (a selective β3AR agonist) at the time of reperfusion. On the other hand, GRK2-C340S KI mice did worse after I/R injury compared to WT mice and CL316,243 did not rescue this dysfunction as it did in WT mice. As expected, β3AR KO mice had worsened cardiac function compared to WT mice and CL316,243 had no functional benefit. Infarct size measurements after I/R revealed that β3AR KO mice had larger infarcts than WT mice supporting β3ARs as being protective. Indeed, CL316,243 induced robust cardioprotection in WT mice, reducing infarct size. GRK2-C340S mice had larger infarcts than WT mice and CL316,243 failed to offer any cardioprotection. Thus, β3AR-mediated cardioprotection clearly involves inhibition of GRK2 as part of its therapeutic mechanism and this appears to involve NO-mediated S-nitrosylation.

Nintedanib Reduces Neutrophil Chemotaxis via Activating GRK2 in Bleomycin-Induced Pulmonary Fibrosis.

Neutrophils are involved in the alveolitis of idiopathic pulmonary fibrosis (IPF). However, their pathogenic mechanisms are still poorly understood. Nintedanib has antifibrotic and anti-inflammatory activity in IPF. This study aimed to investigate the regulatory mechanism of nintedanib on neutrophil chemotaxis in bleomycin (BLM)-induced pulmonary fibrosis. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h after a bleomycin intratracheal injection (1.5 U/kg). Lung histopathological findings, the expression of cytokines, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed. The effect of nintedanib was also investigated in a mouse model with adoptive neutrophil transfer in vivo. Nintedanib significantly decreased the histopathological changes and neutrophil recruitment in BLM-induced pulmonary fibrosis. Nintedanib mediated a downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and very late antigen 4 (VLA-4) expression, as well as an upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in BLM-induced pulmonary fibrosis. Nintedanib also decreased the activation of endothelial cells by the decreased expression of vascular cell adhesion molecule 1 (VCAM-1). The effect of nintedanib on regulating neutrophil chemotaxis was also confirmed by a mouse model with adoptive neutrophil transfer in vivo. In conclusion, nintedanib reduces neutrophil chemotaxis and endothelial cell activation to regulate the severity of BLM-induced pulmonary fibrosis. These effects are associated with an enhancement of GRK2 activity and a reduction in CXCR2 and VLA-4 expression on neutrophils and a decrease in VCAM-1 expression on endothelial cells.

Kinetic control of signaling: role of RGS proteins and GRKs

G protein‐coupled receptors interact with the GDP‐bound heterotrimeric G‐protein, G protein‐coupled receptor kinases (GRKs), and arrestins through their cytosolic surface. Previous studies suggest that the interaction of GRKs and the heterotrimer with the receptor occur in the same space; therefore, there is likely steric hindrance allowing only one pathway to operate at a time. Supporting this claim, experimentally induced competition for the intracellular heterotrimeric G‐protein binding site results in a reduction in GRK and Gα function. We created a Gαs chimera, called sis, containing most of the helical domain of Gαi1. In initial studies, sis prevented GRK2, 4, 5, and 6‐mediated reductions in isoproterenol‐mediated cAMP accumulation. Therefore, we tested the hypothesis that sis disrupts GRK/arrestin function due to an alteration in the turnover of the sis protein. To exclude any background effects of endogenous Gαs, we used GnasE2−/E2− MEFs re‐expressing Gαs (control) or sis (experimental) as well as the β2‐adrenergic receptor. Experimentally, cAMP accumulation in the presence and absence of receptor and GRK inhibitors, receptor‐Gs kinetics, and receptor internalization were measured. The introduction of the Gαi1 helical domain into Gαs did not significantly affect the biochemistry of the chimeric sis protein; however, GRK function and cAMP production altered. GRK2 failed to attenuate isoproterenol‐mediated cAMP accumulation in sis‐expressing cells, but attenuated cAMP accumulation in Gs‐expressing cells. Accordingly, the rate of endocytosis decreased in sis‐expressing cells compared to Gαs‐expressing cells at 10 μM, but not 0.1 and 1 μM, isoproterenol (0.77 ± 0.10 versus 1.00 ± 0.14 min−1). The kinetics of 10 μM isoproterenol‐mediated cAMP production demonstrates that the rate of accumulation of cAMP is similar, yet sis‐expressing cells have an increase in maximal response, and the rate of degradation of cAMP fits two different models. Degradation of cAMP in Gαs‐expressing cells is fit to a classical first‐order equation with a rate constant of 0.068 ± 0.012 luminescence/min, whereas, in sis‐expressing cells, the degradation of cAMP fit a linear zero‐order model with a slope of −0.012 ± 0.0012 luminescence/min. As there is 10 μM of isoproterenol present, we hypothesized that the receptor is nearly always active, and sis has altered kinetics and GRK activity due to rapid cycling between the cyclase and receptor. To test this second hypothesis, exploratory experiments are being conducted to determine if the alterations observed can be explained through a chimerically‐derived interaction with RGS proteins. The collected preliminary data suggest that altered kinetics of signaling alter GRK function and receptor internalization, and consequently, RGS proteins may indirectly inhibit GRK function.Support or Funding InformationWesternU MSPS program funds and NIH grants DK073911 (MB), DK079307 (EKJ), and HL109002 (EKJ) supported this work.

Investigation of G protein‐coupled receptor kinase 2 (GRK2) regulation of D2 dopamine receptor signaling: effects of GRK2 functional domains and pharmacologic and genetic inhibition of GRK2 activity

The D2 dopamine (DA) receptor (D2R) signals through two major pathways, namely activation of G proteins and recruitment of β‐arrestin. Agonist binding stabilizes a receptor state that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family, and this phosphorylation is known to enhance β‐arrestin recruitment and initiate the process of internalization for many GPCRs. Previously, we found that only GRK2 and GRK3 were involved in D2R phosphorylation in response to agonist stimulation (Namkung et al., JBC: 284: 15038, 2009). In this study, we further investigated the influence of GRK2 on receptor signaling, β‐arrestin recruitment, and internalization of the D2R. Overexpression of GRK2 was found to increase DA‐stimulated β‐arrestin recruitment and receptor internalization, whereas it suppressed D2R‐mediated inhibition of cAMP accumulation. In contrast, a catalytically inactive GRK2 mutant (GRK2‐K220R) did not affect any of these functional outputs suggesting that GRK2 kinase activity is required to influence D2R signaling. A disabling mutation of the GRK2 RGS homology (RH) domain (GRK2‐D110A) behaved similarly as WT GRK2, suggesting that this domain is not required for GRK2’s regulatory effects on the D2R. Finally, overexpression of a GRK2 construct containing a mutation in the pleckstrin homology (PH) domain (GRK2‐R587Q), which negates GRK2 interaction with Gβγ subunits, increased DA‐stimulated β‐arrestin recruitment and receptor internalization (more than observed with WT GRK2), but did not affect D2R regulation of cAMP accumulation, indicating that this GRK2 domain is involved in regulating some, but not all, aspects of D2R signaling. To study D2R regulation in the complete absence of GRK2, we utilized a cell line in which GRK2 expression was knocked out using CRISPR‐Cas9 technology. DA‐stimulated β‐arrestin recruitment and receptor internalization were decreased, but not eliminated, in the GRK2 KO cells suggesting that, while GRK2 may be facilitatory, it is not essential for these processes. In parallel studies we characterized the effects of Compound 101, a dual GRK2/3 inhibitor. Compound 101 was found to negate GRK2 interactions with the D2R and attenuate, but not eliminate, β‐arrestin recruitment and receptor internalization. In contrast, in the GRK2 KO cells, Compound 101 had no effect on the residual DA‐stimulated β‐arrestin recruitment. In conclusion, our data suggest that different GRK2 domains affect specific regulatory pathways of the D2R and that both agonist‐induced β‐arrestin recruitment and receptor internalization can occur in the absence of GRK2 interactions with the D2R.Support or Funding InformationNINDS Intramural Research Program

LDK378 inhibits the recruitment of myeloid-derived suppressor cells to spleen via the p38-GRK2-CCR2 pathway in mice with sepsis.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that are persistently increased in abundance and associated with adverse clinical outcomes in sepsis. Here, we investigated the therapeutic potential of an anaplastic lymphoma kinase inhibitor, LDK378, in cecal ligation and puncture (CLP)-induced polymicrobial sepsis and examined its effects on the recruitment of MDSCs. LDK378 significantly improved the survival of CLP-induced polymicrobial septic mice, which was paralleled by reduced organ injury, decreased release of inflammatory cytokines and decreased recruitment of MDSCs to the spleen. Importantly, LDK378 inhibited the migration of MDSCs to the spleen by blocking the CLP-mediated upregulation of CC chemokine receptor 2 (CCR2), a chemokine receptor critical for the recruitment of MDSCs. Mechanistically, LDK378 treatment blocked the CLP-induced CCR2 upregulation of MDSCs via partially inhibiting the phosphorylation of p38 and G-protein-coupled receptor kinase-2 (GRK2) in bone marrow MDSCs of septic mice. Furthermore, invitro experiments also showed that lipopolysaccharide (LPS)-induced migration of MDSCs was similarly owing to the activation of GRK2 and upregulation of CCR2 by LPS, whereas the treatment with LDK378 partially blocked the LPS-induced phosphorylation of p38 and GRK2 and decreased the expression of CCR2 on the cell surface, therefore leading to the suppression of MDSC migration. Together, these findings unravel a novel function of LDK378 in the host response to infection and suggest that LDK378 could be a potential therapeutic agent for sepsis.

Aldosterone Jeopardizes Myocardial Insulin and β-Adrenergic Receptor Signaling via G Protein-Coupled Receptor Kinase 2.

Hyperaldosteronism alters cardiac function, inducing adverse left ventricle (LV) remodeling either via increased fibrosis deposition, mitochondrial dysfunction, or both. These harmful effects are due, at least in part, to the activation of the G protein-coupled receptor kinase 2 (GRK2). In this context, we have previously reported that this kinase dysregulates both β-adrenergic receptor (βAR) and insulin (Ins) signaling. Yet, whether aldosterone modulates cardiac Ins sensitivity and βAR function remains untested. Nor is it clear whether GRK2 has a role in this modulation, downstream of aldosterone. Here, we show in vitro, in 3T3 cells, that aldosterone impaired insulin signaling, increasing the negative phosphorylation of insulin receptor substrate 1 (ser307pIRS1) and reducing the activity of Akt. Similarly, aldosterone prevented the activation of extracellular signal-regulated kinase (ERK) and the production of cyclic adenosine 3′,5′-monophosphate (cAMP) in response to the β1/β2AR agonist, isoproterenol. Of note, all of these effects were sizably reduced in the presence of GRK2-inhibitor CMPD101. Next, in wild-type (WT) mice undergoing chronic infusion of aldosterone, we observed a marked GRK2 upregulation that was paralleled by a substantial β1AR downregulation and augmented ser307pIRS1 levels. Importantly, in keeping with the current in vitro data, we found that aldosterone effects were wholly abolished in cardiac-specific GRK2-knockout mice. Finally, in WT mice that underwent 4-week myocardial infarction (MI), we observed a substantial deterioration of cardiac function and increased LV dilation and fibrosis deposition. At the molecular level, these effects were associated with a significant upregulation of cardiac GRK2 protein expression, along with a marked β1AR downregulation and increased ser307pIRS1 levels. Treating MI mice with spironolactone prevented adverse aldosterone effects, blocking GRK2 upregulation, and thus leading to a marked reduction in cardiac ser307pIRS1 levels while rescuing β1AR expression. Our study reveals that GRK2 activity is a critical player downstream of the aldosterone signaling pathway; therefore, inhibiting this kinase is an attractive strategy to prevent the cardiac structural disarray and dysfunction that accompany any clinical condition accompanied by hyperaldosteronism.

G Protein-Coupled Receptor Kinase 2 (GRK2) as a Potential Therapeutic Target in Cardiovascular and Metabolic Diseases.

G protein-coupled receptor kinase 2 (GRK2) is a central signaling node involved in the modulation of many G protein-coupled receptors (GPCRs) and also displaying regulatory functions in other cell signaling routes. GRK2 levels and activity have been reported to be enhanced in patients or in preclinical models of several relevant pathological situations, such as heart failure, cardiac hypertrophy, hypertension, obesity and insulin resistance conditions, or non-alcoholic fatty liver disease (NAFLD), and to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Therefore, targeting GRK2 by different strategies emerges as a potentially relevant approach to treat cardiovascular disease, obesity, type 2 diabetes, or NAFLD, pathological conditions which are frequently interconnected and present as co-morbidities.

Abstract 318: MicroRNA Regulation of G Protein-Coupled Receptor Kinase 2 After Cardiac Injury

The increase in myocardial expression and activity of G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2) during the development and progression of heart failure (HF) has been documented to be pathological. Upregulation of GRK2 that is involved with deactivating agonist-activated β-adrenergic receptors (βARs) results in weakened contractile responsiveness through decreases in βAR density, coupling, and sensitivity. Non-GPCR activity of GRK2 has also been shown to be involved in cardiac dysfunction. Studies with knockout or blockade of GRK2 are able to significantly reduce and/or prevent HF phenotypes. Conversely, models with over-expressed GRK2 produce increased injury and accelerated HF development. Being such an important kinase in cardiac disease, regulation of GRK2 expression and activity is critical. Of interest, there is not much known about how GRK2 is regulated (mRNA or protein) in the heart and mechanisms for its up-regulation after cardiac injury are not well understood. There is evidence of GRK2 being regulated by microRNAs (miRNAs) in endothelial and blood cells. We believe cardiac GRK2 may also be regulated in a similar miRNA-mediated fashion. MiRNA profiles become dysregulated with the onset of stress/injury and contribute to orchestrating many classical HF phenotypes, including βAR aberrations. We hypothesize that the expression of a GRK2-regulating miRNA is altered in HF, which contributes to changes in GRK2 regulation resulting in its pathological increase. MiRNA microarray analysis was done on 2-week post-myocardial infarction (MI) murine hearts to examine miRNA profiles compared to sham-operated. Cardiac miRNA species were examined for their expression levels, seed sequence, and TargetScan prediction and alignment to mouse and human GRK2 3’UTR. Candidate miRNAs underwent 3’UTR analysis against GRK2 3’UTR and transfection into AC16 cardiac cells. Two miRNAs were identified to significantly reduce GRK2 protein and/or RNA levels as well directly bind to the 3’UTR of GRK2. Further studies will be done to elucidate the mechanism these miRNAs are able to regulate GRK2 levels. Results from these studies can be applied towards GRK2 inhibition via miRNA-based therapeutics.