The Ubiquitination of Arrestin3 within the Nucleus Triggers the Nuclear Export of Mdm2, Which, in Turn, Mediates the Ubiquitination of GRK2 in the Cytosol.

GRK2 and arrestin3, key players in the functional regulation of G protein-coupled receptors (GPCRs), are ubiquitinated by Mdm2, a nuclear protein. The agonist-induced increase in arrestin3 ubiquitination occurs in the nucleus, underscoring the crucial role of its nuclear translocation in this process. The ubiquitination of arrestin3 occurs in the nucleus, highlighting the pivotal role of its nuclear translocation in this process. In contrast, GRK2 cannot translocate into the nucleus; thus, facilitation of the cytosolic translocation of nuclear Mdm2 is required to ubiquitinate GRK2 in the cytosol. Among the explored cellular components and processes, arrestin, Gßγ, clathrin, and receptor phosphorylation were found to be required for the nuclear import of arrestin3, the ubiquitination of arrestin3 in the nucleus, nuclear export of Mdm2, and the ubiquitination of GRK2 in the cytosol. In conclusion, our findings demonstrate that agonist-induced ubiquitination of arrestin3 in the nucleus is interconnected with cytosolic GRK2 ubiquitination.

G-Protein-Coupled Receptor Kinase 2 Inhibition Induces Meiotic Arrest by Disturbing Ca2+ Release in Porcine Oocytes.

G-protein-coupled receptor kinase 2 (GRK2) interacts with Gßγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (ßi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; ßi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; ßi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of ßi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.

GRK2 kinases in the primary cilium initiate SMOOTHENED-PKA signaling in the Hedgehog cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Unveiling the intracellular dynamics of α4ß2 nAChR-mediated ERK activation through the interplay of arrestin, Gßγ, and PKCßII.

AIMS: In contrast to G protein-coupled receptors or receptor tyrosine kinases, the mechanism underlying ERK activation through nicotine acetylcholine receptors (nAChRs), members of the ligand-gated ion channel family, remains poorly elucidated. This study aimed to delineate the signaling pathway responsible for ERK activation by the α4ß2 nAChR subtype, which is implicated in nicotine addiction and various mental disorders. MATERIALS AND METHODS: Loss-of-function strategies and mutants of arrestin2/PKCßII with distinct functional characteristics were employed to identify the cellular components and processes involved in ERK activation. KEY FINDINGS: ERK activation via α4ß2 nAChR was observed within the nucleus and necessitated the nuclear translocation of arrestin2 and PKCßII, which exhibited mutual augmentation. Activation of PKCßII by α4ß2 nAChR stimulation facilitated the nuclear translocation of arrestin2 by enhancing its interaction with importin ß1. Apart from scaffolding ERK activation in the nucleus, arrestin2, in cooperation with GRK2, facilitated the activation of the Src/Syk/PKCßII signaling cascade, leading to the nuclear entry of PKCßII in a Gßγ-dependent manner. Upon nuclear localization, PKCßII underwent ubiquitination by Mdm2 and interacted with MEK1, resulting in ERK activation. In summary, α4ß2 nAChR-mediated ERK activation in the nucleus involves the nuclear translocation of arrestin2 and PKCßII, which is reciprocally facilitated via positive feedback augmentation. SIGNIFICANCE: As α4ß2 nAChRs play a pivotal role in various cellular processes including drug addiction and mental disorders, our findings will offer insights into understanding the pathogenesis of α4ß2 nAChR-related disorders and may facilitate the development of targeted therapeutic interventions.

Cinnamaldehyde activates AMPK/PGC-1α pathway via targeting GRK2 to ameliorate heart failure.

BACKGROUND: According to recent research, treating heart failure (HF) by inhibiting G protein-coupled receptor kinase 2 (GRK2) to improve myocardial energy metabolism has been identified as a potential approach. Cinnamaldehyde (CIN), a phenylpropyl aldehyde compound, has been demonstrated to exhibit beneficial effects in cardiovascular diseases. However, whether CIN inhibits GRK2 to ameliorate myocardial energy metabolism in HF is still unclear. PURPOSE: This study examines the effects of CIN on GRK2 and myocardial energy metabolism to elucidate its underlying mechanism to treat HF. METHODS: The isoproterenol (ISO) induced HF model in vivo and in vitro were constructed using Sprague-Dawley (SD) rats and primary neonatal rat cardiomyocytes (NRCMs). Based on this, the effects of CIN on myocardial energy metabolism and GRK2 were investigated. Additionally, validation experiments were conducted after interfering and over-expressing GRK2 in ISO-induced NRCMs to verify the regulatory effect of CIN on GRK2. Furthermore, binding capacity between GRK2 and CIN was explored by Cellular Thermal Shift Assay (CETSA) and Microscale Thermophoresis (MST). RESULTS: In vivo and in vitro, CIN significantly improved HF as demonstrated by reversing abnormal changes in myocardial injury markers, inhibiting myocardial hypertrophy and decreasing myocardial fibrosis. Additionally, CIN promoted myocardial fatty acid metabolism to ameliorate myocardial energy metabolism disorder by activating AMPK/PGC-1α signaling pathway. Moreover, CIN reversed the inhibition of myocardial fatty acid metabolism and AMPK/PGC-1α signaling pathway by GRK2 over-expression in ISO-induced NRCMs. Meanwhile, CIN had no better impact on the stimulation of cardiac fatty acid metabolism and the AMPK/PGC-1α signaling pathway in ISO-induced NRCMs when GRK2 was disrupted. Noticeably, CETSA and MST confirmed that CIN binds to GRK2 directly. The binding of CIN and GRK2 promoted the ubiquitination degradation of GRK2 mediated by murine double mimute 2. CONCLUSION: This study demonstrates that CIN exerts a protective intervention in HF by targeting GRK2 and promoting its ubiquitination degradation to activate AMPK/PGC-1α signaling pathway, ultimately improving myocardial fatty acid metabolism.

Chronic Partial Sleep Deprivation Increased the Incidence of Atrial Fibrillation by Promoting Pulmonary Vein and Atrial Arrhythmogenesis in a Rodent Model.

Sleep deprivation (SD) is a recognized risk factor for atrial fibrillation (AF), yet the precise molecular and electrophysiological mechanisms behind SD-induced AF are unclear. This study explores the electrical and structural changes that contribute to AF in chronic partial SD. We induced chronic partial SD in Wistar rats using a modified multiple-platform method. Echocardiography demonstrated impaired systolic and diastolic function in the left ventricle (LV) of the SD rats. The SD rats exhibited an elevated heart rate and a higher low-frequency to high-frequency ratio in a heart-rate variability analysis. Rapid transesophageal atrial pacing led to a higher incidence of AF and longer mean AF durations in the SD rats. Conventional microelectrode recordings showed accelerated pulmonary vein (PV) spontaneous activity in SD rats, along with a heightened occurrence of delayed after-depolarizations in the PV and left atrium (LA) induced by tachypacing and isoproterenol. A Western blot analysis showed reduced expression of G protein-coupled receptor kinase 2 (GRK2) in the LA of the SD rats. Chronic partial SD impairs LV function, promotes AF genesis, and increases PV and LA arrhythmogenesis, potentially attributed to sympathetic overactivity and reduced GRK2 expression. Targeting GRK2 signaling may offer promising therapeutic avenues for managing chronic partial SD-induced AF. Future investigations are mandatory to investigate the dose-response relationship between SD and AF genesis.

GRK2 is critical for the cleavage of the porcine embryo by regulating HSP90 and the AKT pathway.

In brief: GRK2 deficiency disrupts the early embryonic development in pigs. The regulation of GRK2 on HSP90 and AKT may also play an important role during embryo development and tumor formation. Abstract: Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, G-protein-coupled receptor kinase 2 (GRK2) binds to HSP90 in response to hypoxia or other stresses. In this study, we investigated the effects of GRK2 knockdown and inhibition on porcine embryonic development from the zygote stage. Immunofluorescence and western blotting were used to determine the localization and expression, respectively, of GRK2 and related proteins. First, GRK2 and p-GRK2 were expressed in both the cytoplasm and membrane and co-localized with HSP90 on the membrane. The mRNA level of GRK2 increased until the 8C-morula stage, suggesting that GRK2 may play an essential role during the early development of the porcine embryos. GRK2 knockdown reduced porcine embryo development capacity and led to significantly decreased blastocyst quality. In addition, inhibition of GRK2 also induced poor ability of embryo development at an early stage, indicating that GRK2 is critical for embryonic cleavage in pigs. Knockdown and inhibition of GRK2 reduced HSP90 expression, AKT activation, and cAMP levels. Additionally, GRK2 deficiency increased LC3 expression, suggesting enhanced autophagy during embryo development. In summary, we showed that GRK2 binds to HSP90 on the membrane to regulate embryonic cleavage and AKT activation during embryonic development in pigs.

Inhibition of GRK2 ameliorates the pristane-induced mouse SLE model by suppressing plasma cells differentiation.

Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder characterized by diverse clinical manifestations and organ damage. Despite its elusive etiology, dysregulated subsets and functions of B cells are pivotal in SLE pathogenesis. Peoniflorin-6'-O-benzene sulfonate (CP-25), an esterification modification of Paeoniflorin, exhibits potent anti-inflammatory and immunomodulatory properties in autoimmune diseases (AID). However, the involvement of CP-25 and its target, GRK2, in SLE development has not been explored. In this study, we demonstrate that both genetic deficiency and pharmacological inhibition of GRK2 attenuate autoantibodies production, reduce systemic inflammation, and mitigate histopathological alterations in the spleen and kidney in the pristane-induced mouse SLE model. Importantly, our findings highlight that both genetic deficiency and pharmacological inhibition of GRK2 suppress plasma cells generation and restore dysregulated B-cell subsets by modulating two crucial transcription factors, Blimp1 and IRF4. Collectively, targeting GRK2 with CP-25 emerges as a promising therapeutic approach for SLE.

Cardioprotective Effects of the GRK2 Inhibitor Paroxetine on Isoproterenol-Induced Cardiac Remodeling by Modulating NF-κB Mediated Prohypertrophic and Profibrotic Gene Expression.

Pathological cardiac remodeling is associated with cardiovascular disease and can lead to heart failure. Nuclear factor-kappa B (NF-κB) is upregulated in the hypertrophic heart. Moreover, the expression of the G-protein-coupled receptor kinase 2 (GRK2) is increased and linked to the progression of heart failure. The inhibitory effects of paroxetine on GRK2 have been established. However, its protective effect on IκBα/NFκB signaling has not been elucidated. This study investigated the cardioprotective effect of paroxetine in an animal model of cardiac hypertrophy (CH), focusing on its effect on GRK2-mediated NF-κB-regulated expression of prohypertrophic and profibrotic genes. Wistar albino rats were administered normal saline, paroxetine, or fluoxetine, followed by isoproterenol to induce CH. The cardioprotective effects of the treatments were determined by assessing cardiac injury, inflammatory biomarker levels, histopathological changes, and hypertrophic and fibrotic genes in cardiomyocytes. Paroxetine pre-treatment significantly decreased the HW/BW ratio (p < 0.001), and the expression of prohypertrophic and profibrotic genes Troponin-I (p < 0.001), BNP (p < 0.01), ANP (p < 0.001), hydroxyproline (p < 0.05), TGF-ß1 (p < 0.05), and αSMA (p < 0.01) as well as inflammatory markers. It also markedly decreased pIκBα, NFκB(p105) subunit expression (p < 0.05) and phosphorylation. The findings suggest that paroxetine prevents pathological cardiac remodeling by inhibiting the GRK2-mediated IκBα/NF-κB signaling pathway.

GRK2 expression and catalytic activity are essential for vasoconstrictor/ERK-stimulated arterial smooth muscle proliferation.

Prolonged vasoconstrictor signalling found in hypertension, increases arterial contraction, and alters vessel architecture by stimulating arterial smooth muscle cell (ASMC) growth, underpinning the development of re-stenosis lesions and vascular remodelling. Vasoconstrictors interact with their cognate G protein coupled receptors activating a variety of signalling pathways to promote smooth muscle proliferation. Here, angiotensin II (AngII) and endothelin 1 (ET1), but not UTP stimulates ASMC proliferation. Moreover, siRNA-mediated depletion of endogenous GRK2 expression, or GRK2 inhibitors, compound 101 or paroxetine, prevented AngII and ET1-promoted ASMC growth. Depletion of GRK2 expression or inhibition of GRK2 activity ablated the prolonged phase of AngII and ET-stimulated ERK signalling, while enhancing and prolonging UTP-stimulated ERK signalling. Increased GRK2 expression enhanced and prolonged AngII and ET1-stimulated ERK signalling, but suppressed UTP-stimulated ERK signalling. In ASMC prepared from 6-week-old WKY and SHR, AngII and ET1-stimulated proliferation rates were similar, however, in cultures prepared from 12-week-old rats AngII and ET1-stimulated growth was enhanced in SHR-derived ASMC, which was reversed following depletion of GRK2 expression. Furthermore, in ASMC cultures isolated from 6-week-old WKY and SHR rats, AngII and ET1-stimulated ERK signals were similar, while in cultures from 12-week-old rats ERK signals were both enhanced and prolonged in SHR-derived ASMC, and were reversed to those seen in age-matched WKY-derived ASMC following pre-treatment of SHR-derived ASMC with compound 101. These data indicate that the presence of GRK2 and its catalytic activity are essential to enable pro-proliferative vasoconstrictors to promote growth via recruitment and activation of the ERK signalling pathway in ASMC.

GPCR activation and GRK2 assembly by a biased intracellular agonist.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1-6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.

Ubiquitination of GRK2 Is Required for the ß-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and ß-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying ß-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R ß-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R ß-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Overexpression of GRK2 in vascular smooth muscle leads to inappropriate hypertension and acute heart failure as in clinical scenario 1.

Clinical scenario 1 (CS1) is acute heart failure (HF) characterized by transient systolic blood pressure (SBP) elevation and pulmonary congestion. Although it is managed by vasodilators, the molecular mechanism remains unclear. The sympathetic nervous system plays a key role in HF, and desensitization of cardiac ß-adrenergic receptor (AR) signaling due to G protein-coupled receptor kinase 2 (GRK2) upregulation is known. However, vascular ß-AR signaling that regulates cardiac afterload remains unelucidated in HF. We hypothesized that upregulation of vascular GRK2 leads to pathological conditions similar to CS1. GRK2 was overexpressed in vascular smooth muscle (VSM) of normal adult male mice by peritoneally injected adeno-associated viral vectors driven by the myosin heavy chain 11 promoter. Upregulation of GRK2 in VSM of GRK2 overexpressing mice augmented the absolute increase in SBP (+ 22.5 ± 4.3 mmHg vs. + 36.0 ± 4.0 mmHg, P < 0.01) and lung wet weight (4.28 ± 0.05 mg/g vs. 4.76 ± 0.15 mg/g, P < 0.01) by epinephrine as compared to those in control mice. Additionally, the expression of brain natriuretic peptide mRNA was doubled in GRK2 overexpressing mice as compared to that in control mice (P < 0.05). These findings were similar to CS1. GRK2 overexpression in VSM may cause inappropriate hypertension and HF, as in CS1.

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation.

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6'-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-ß-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

Selectivity mechanism of GRK2/5 inhibition through in silico investigation.

As two representative isoforms of G protein-coupled receptor kinases family, the largest known membrane receptor family, GRK2 and GRK5 are ubiquitously distributed in human heart, brain, lung, kidney, skeletal muscle and other tissues. GRK2 and GRK5 have common functions implicated in the regulation of heart failure, though GRK5 has also been involved in diseases like hypertension, cancer, diabetes and Alzheimer's disease. Therefore, to clarify the selectivity mechanism towards GRK2 and GRK5 will be of great significance for the discovery of effective and selective inhibitors. To this end, the structures and chemical properties of key residues were analyzed among GRK2 and GRK5 derived from their respective protein crystal structures. Furthermore, a combination of multiple computational strategies, including sequence superposition, receptor-ligand docking, molecular dynamics, MM-GBSA calculation, QM/MM approach and pharmacological modeling, were integrated to validated and elucidate their unique binding modes towards highly selective inhibitors. In addition, the specific amino acid distribution within the GRK2/5 target site is also analyzed in this paper, which can guide future research and development of selective inhibitors in a more targeted manner. Overall, our study comprehensively clarifies the selectivity mechanism of GRK2/5 inhibition, thereby providing guidance for further rational design of selective inhibitors targeting GRK2/5.

The tyrosine phosphorylation of GRK2 is responsible for activated D2R-mediated insulin resistance.

Dopamine D2 receptor (D2R) plays a key role in the regulation of glucose homeostasis by stimulating the secretion of many glucoregulatory hormones. Insulin resistance (IR) is associated with the pathogenesis of metabolic disorders which occurs when PI3K/Akt signaling pathway is downregulated. However, the potential involvement of D2R in insulin resistance remains unclear. In the present study, we investigated the regulation of glucose transport by D2-like receptors and discovered that activation of D2R, but not D3R or D4R, suppressed insulin-induced 2-DOG uptake and Glut4 membrane translocation in a GRK2- and Src-dependent manner. Further study revealed that activation of D2R inhibits insulin-induced phosphorylation of Akt at Thr308 and Ser473, which are hallmarks of its kinase activity, by increasing the interaction of tyrosine phosphorylated GRK2 with Akt and then preventing Akt from interacting with PDK1. Thus, this study demonstrates that Src mediated GRK2 tyrosine phosphorylation is an essential physiological event that mediates the roles of D2R in insulin resistance.

Targeting GRK2 and GRK5 for treating chronic degenerative diseases: Advances and future perspectives.

The development of small-molecule inhibitors targeting G protein-coupled receptor kinase 2 (GRK2) and G protein-coupled receptor kinase 5 (GRK5) for the treatment of chronic degenerative diseases has attracted wide attention. GRK2 and GRK5 can regulate essential physiological processes by phosphorylating G protein-coupled receptor (GPCR). Alterations in the functional levels of GRK2 and GRK5 have been found in a variety of chronic degenerative diseases, such as cardiovascular diseases, neurodegenerative diseases, cancers, type 2 diabetes, and rheumatoid arthritis (RA). Abnormal GRK2 and GRK5 expression contribute to the development of chronic degenerative diseases through environmental molecular mechanisms, making them promising molecular targets for treating chronic degenerative diseases. To date, many novel GRK2 and GRK5 inhibitors have been reported for the treatment of chronic degenerative diseases. We focus on the recent progress of single and dual-target inhibitors of GRK2/GRK5. This review summarizes the structural optimization rationale, structure-activity relationships (SARs), and the latest application in the treatment of chronic degenerative diseases. We believe it will shed light on the future development of small molecule inhibitors of GRK2 and GRK5, as well as the clinical applications in chronic degenerative diseases.

GRK2 in cardiovascular disease and its potential as a therapeutic target.

Cardiovascular diseases (CVDs) represent the leading cause of death globally. Despite major advances in the field of pharmacological CVD treatments, particularly in the field of heart failure (HF) research, case numbers and overall mortality remain high and have trended upwards over the last few years. Thus, identifying novel molecular targets for developing HF therapeutics remains a key research focus. G protein-coupled receptors (GPCRs) are critical myocardial signal transducers which regulate cardiac contractility, growth, adaptation and metabolism. Additionally, GPCR dysregulation underlies multiple models of cardiac pathology, and most pharmacological therapeutics currently used in HF target these receptors. Currently-approved treatments have improved patient outcomes, but therapies to stop or reverse HF are lacking. A recent focus on GPCR intracellular-regulating proteins such as GPCR kinases (GRKs) has uncovered GRK2 as a promising target for combating HF. Current literature strongly establishes increased levels and activity of GRK2 in multiple models of CVD. Additionally, the GRK2 interactome includes numerous proteins which interact with differential domains of GRK2 to modulate both beneficial and deleterious signaling pathways in the heart, indicating that these domains can be targeted with a high level of specificity unique to various cardiac pathologies. These data support the premise that GRK2 should be at the forefront of a novel investigative drug search. This perspective reviews cardiac GPCRs, describes the structure and functions of GRK2 in cardiac function and maladaptive pathology, and summarizes the ongoing and future research for targeting this critical kinase across cellular, animal and human models of cardiac dysfunction and HF.

Early Gßγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects.

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gßγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gßγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gßγ-GRK2 inhibition at the time of DMM by administering the Gßγ inhibitor "gallein" and the GRK2 inhibitor "paroxetine" daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gßγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gßγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gßγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gßγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.