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.

Reversing T Cell Dysfunction to Boost Glioblastoma Immunotherapy by Paroxetine-Mediated GRK2 Inhibition and Blockade of Multiple Checkpoints through Biomimetic Nanoparticles.

T cell dysfunction-induced tumor immune escape is particularly severe in glioblastoma (GBM), and significantly affects the efficacy of immunotherapy. It is crucial to innovatively reverse the T cell dysfunction for improving GBM immunotherapy. Herein, T cell dysfunction is remarkably reversed and immunotherapy of GBM is boosted by repurposing the U. S. Food and Drug Administration-approved antidepressant paroxetine (PX) with biomimetic nanoparticles (CS-J@CM/6 NPs). The PX is successfully applied to abrogate T cell sequestration in the bone marrow of GBM-bearing mice and increase their infiltration in tumor. The biomimetic NPs are composed of ultrasmall Cu2- x Se NPs, JQ1, and tumor cell membrane modified with CD6, and are efficiently delivered into tumor through the specific interactions between CD6 and activated leukocyte cell adhesion molecule. They ameliorate the T cell dysfunction through the double roles of loaded JQ1, which simultaneously decreases the expression of PD-1 and TIM-3 on T cells, and the expression of PD-L1 on tumor cells. The NP also induces the immunogenic cell death of tumor cells to activate immune response. The synergistic roles of PX and biomimetic CS-J@CM/6 NPs notably enhance the survival of GBM-bearing mice. This work provides new insights into tumor immunotherapy by repurposing "old drugs" with advanced NPs.

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.

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.

G protein-coupled receptor kinase type 2 and ß-arrestin2: Key players in immune cell functions and inflammation.

G protein-coupled receptor kinase type 2 (GRK2) and ß-arrestin2 are representative proteins that regulate the transduction and trafficking of G protein-coupled receptor (GPCR) signaling. The kinase GRK2 and the multifunctional scaffolding protein ß-arrestin2 are key integrated signaling nodes in various biological processes, and both of them regulate cell proliferation and promote cell invasion and migration. GRK2/ß-arrestin2 play multiple roles in the pathological mechanisms of a wide range of diseases including heart failure, cancer, and inflammatory diseases. This review summarizes the roles of GRK2/ß-arrestin2 in immune cell function and focuses on the pathological implications of GRK2/ß-arrestin2 in various inflammatory diseases.

The RAF Kinase Inhibitor Protein (RKIP): Good as Tumour Suppressor, Bad for the Heart.

The RAF kinase inhibitor protein, RKIP, is a dual inhibitor of the RAF1 kinase and the G protein-coupled receptor kinase 2, GRK2. By inhibition of the RAF1-MAPK (mitogen-activated protein kinase) pathway, RKIP acts as a beneficial tumour suppressor. By inhibition of GRK2, RKIP counteracts GRK2-mediated desensitisation of G protein-coupled receptor (GPCR) signalling. GRK2 inhibition is considered to be cardioprotective under conditions of exaggerated GRK2 activity such as heart failure. However, cardioprotective GRK2 inhibition and pro-survival RAF1-MAPK pathway inhibition counteract each other, because inhibition of the pro-survival RAF1-MAPK cascade is detrimental for the heart. Therefore, the question arises, what is the net effect of these apparently divergent functions of RKIP in vivo? The available data show that, on one hand, GRK2 inhibition promotes cardioprotective signalling in isolated cardiomyocytes. On the other hand, inhibition of the pro-survival RAF1-MAPK pathway by RKIP deteriorates cardiomyocyte viability. In agreement with cardiotoxic effects, endogenous RKIP promotes cardiac fibrosis under conditions of cardiac stress, and transgenic RKIP induces heart dysfunction. Supported by next-generation sequencing (NGS) data of the RKIP-induced cardiac transcriptome, this review provides an overview of different RKIP functions and explains how beneficial GRK2 inhibition can go awry by RAF1-MAPK pathway inhibition. Based on RKIP studies, requirements for the development of a cardioprotective GRK2 inhibitor are deduced.

Novel inhibitors of phosphorylation independent activity of GRK2 modulate cAMP signaling.

G protein-coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2-mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates' ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation-independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice.

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and downregulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Lycium barbarum polysaccharide antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury, and salvage mitochondrial fission/fusion imbalance and AKT/eNOS signaling.

Ischemia-reperfusion (I/R) injury is the main reason why infarct size continues to progress during the process of restoring myocardial perfusion, and it significantly increases the risk of death. At present, the therapeutic effects of clinically used drugs are limited. Therefore, it is particularly necessary to explore myocardial-protective agents that effectively prevent I/R injury. Lycium barbarum polysaccharide (LBP) is a water-soluble polysaccharide extracted from wolfberry fruit. In this study, we found that LBP limited myocardial infarct size, improved adverse remodeling, and reduced cell death and oxidative stress. G protein-coupled receptor kinase-2 (GRK2) is a key molecule involved in myocardial I/R injury. In vivo and in vitro experiments showed that LBP inhibited the upregulation of GRK2 expression induced by I/R injury, which was related to the antiapoptotic effect of LBP. In addition, we found that LBP partially restored I/R-induced mitochondrial fission/fusion imbalance, as well as levels of phosphorylated protein kinase B (p-AKT) and phosphorylated endothelial cell nitric oxide synthase (p-eNOS), and this restorative effect could be attenuated by overexpression of GRK2. Overall, our findings suggest that LBP antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury and saves mitochondrial fission/fusion imbalance and AKT/eNOS signaling. This study may provide new ideas for the study of I/R injury and the rational application of the herbal medicine LBP.

TNF-α impairs EP4 signaling through the association of TRAF2-GRK2 in primary fibroblast-like synoviocytes.

Our previous study showed that chronic treatment with tumor necrosis factor-α (TNF-α) decreased cAMP concentration in fibroblast-like synoviocytes (FLSs) of collagen-induced arthritis (CIA) rats. In this study we investigated how TNF-α impairs cAMP homeostasis, particularly clarifying the potential downstream molecules of TNF-α and prostaglandin receptor 4 (EP4) signaling that would interact with each other. Using a cAMP FRET biosensor PM-ICUE3, we demonstrated that TNF-α (20 ng/mL) blocked ONO-4819-triggered EP4 signaling, but not Butaprost-triggered EP2 signaling in normal rat FLSs. We showed that TNF-α (0.02-20 ng/mL) dose-dependently reduced EP4 membrane distribution in normal rat FLS. TNF-α significantly increased TNF receptor 2 (TNFR2) expression and stimulated proliferation in human FLS (hFLS) via ecruiting TNF receptor-associated factor 2 (TRAF2) to cell membrane. More interestingly, we revealed that TRAF2 interacted with G protein-coupled receptor kinase (GRK2) in the cytoplasm of primary hFLS and helped to bring GRK2 to cell membrane in response of TNF-α stimulation, the complex of TRAF2 and GRK2 then separated on the membrane, and translocated GRK2 induced the desensitization and internalization of EP4, leading to reduced production of intracellular cAMP. Silencing of TRAF2 by siRNA substantially diminished TRAF2-GRK2 interaction, blocked the translocation of GRK2, and resulted in upregulated expression of membrane EP4 and intracellular cAMP. In CIA rats, administration of paroxetine to inhibit GRK2 effectively improved the symptoms and clinic parameters with significantly reduced joint synovium inflammation and bone destruction. These results elucidate a novel form of cross-talk between TNFR (a cytokine receptor) and EP4 (a typical G protein-coupled receptor) signaling pathways. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of inflammatory diseases.

Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice.

BACKGROUND: The main symptoms of chemotherapy-induced peripheral neuropathy (CIPN) include pain and numbness. Neuronal G protein-coupled receptor kinase 2 (GRK2) plays an important role in various pain models. Cisplatin treatment can induce the activation of proinflammatory microglia in spinal cord. The purpose of this study was to investigate the role of spinal neuronal GRK2 in cisplatin-induced CIPN and in the prevention of CIPN by electroacupuncture (EA). METHODS: The pain and sensory deficit behaviors of mice were examined by von Frey test and adhesive removal test. The expression of neuronal GRK2 in the spinal cord is regulated by intraspinal injection of adeno-associated virus (AAV) containing neuron-specific promoters. The protein levels of GRK2, triggering receptor expressed on myeloid cells 2 (TREM2), and DNAX-activating protein of 12 kDa (DAP12) in spinal dorsal horn were detected by Western blot, the density of intraepidermal nerve fibers (IENFs) was detected by immunofluorescence, and microglia activation were evaluated by real-time polymerase chain reaction (PCR). RESULTS: In this study, cisplatin treatment led to the decrease of GRK2 expression in the dorsal horn of spinal cord. Overexpression of neuronal GRK2 in spinal cord by intraspinal injection of an AAV vector expressing GRK2 with human synapsin (hSyn) promotor significantly inhibited the loss of IENFs and alleviated the mechanical pain and sensory deficits induced by cisplatin. Real-time PCR analysis showed that the overexpression of neuronal GRK2 significantly inhibited the messenger RNA (mRNA) upregulation of proinflammatory cytokine interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase (iNOS), and M1 microglia marker cluster of differentiation (CD)16 induced by cisplatin. Furthermore, the TREM2 and DAP12, which has been demonstrated to play a role in microglia activation and in the development of CIPN, were also downregulated by overexpression of neuronal GRK2 in this study. Interestingly, preventive treatment with EA completely mimics the effect of overexpression of neuronal GRK2 in the spinal cord in this mouse model of cisplatin-induced CIPN. EA increased GRK2 level in spinal dorsal horn after cisplatin treatment. Intraspinal injection of AAV vector specifically downregulated neuronal GRK2, completely reversed the regulatory effect of EA on CIPN and microglia activation. All these indicated that the neuronal GRK2 mediated microglial activation contributed to the process of CIPN. CONCLUSIONS: Neuronal GRK2 in the spinal cord contributed to the preventive effect of EA on CIPN. The neuronal GRK2 may be a potential target for CIPN intervention.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and down-regulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

IGF1-driven induction of GPCR kinase 2 in the primary afferent neuron promotes resolution of acute hyperalgesia.

Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery.

G protein-coupled receptor kinase-2 confers isoform-specific calcium sensitivity to dopamine D2 receptor desensitization.

The dopamine D2 receptor (D2 R) functions as an autoreceptor on dopaminergic cell bodies and terminals and as a postsynaptic receptor on a variety of neurons in the central nervous system. As a result of alternative splicing, the D2 R is expressed as two isoforms: long (D2L R) and short (D2S R) differing by a stretch of 29 residues in the third intracellular loop, with D2S R being the predominant presynaptic isoform. Recent reports described a Ca2+ sensitivity of the desensitization time course of potassium currents elicited via D2S R, but not via D2L R, when either isoform was selectively expressed in dopaminergic neurons. Here, we aimed to study the mechanism behind this subtype-specific Ca2+ sensitivity. Thus, we measured the desensitization of potassium channel responses evoked by D2L R and D2S R using two-electrode voltage clamp in Xenopus oocytes in the absence and presence of different amounts of ß-arrestin2 and G protein-coupled receptor kinase-2 (GRK2), both of which are known to play important roles in D2 R desensitization in native cells. We found that co-expression of both GRK2 and ß-arrestin2 was necessary for reconstitution of the Ca2+ sensitivity of D2S R desensitization, while D2L R did not display Ca2+ sensitivity under these conditions. The effect of Ca2+ chelation by BAPTA-AM to slow the rate of D2S R desensitization was mimicked by the GRK2 inhibitor, Cmpd101, and by the kinase-inactivating GRK2 mutation, K220R, but not by the PKC inhibitor, Gö6976, nor by the calmodulin antagonist, KN-93. Thus, Ca2+ -sensitive desensitization of D2S R appears to be mediated via a GRK2 phosphorylation-dependent mechanism.

The protective effect of allicin on myocardial ischemia-reperfusion by inhibition of Ca2+ overload-induced cardiomyocyte apoptosis via the PI3K/GRK2/PLC-γ/IP3R signaling pathway.

PURPOSE: To investigate the protective effect and mechanism of allicin on myocardial ischemia-reperfusion (MI/R) injury. METHODS: We investigated the mechanisms by which allicin attenuated the MI/R injury by focusing on phosphoinositide 3-kinase, G protein coupled receptor kinases 2, phospholipase Cγ and cardiomyocyte apoptosis. Sixty male mice were randomly assigned into three groups: repeated MI/R (model), sham-operated (control), and MI/R+ allicin group (allicin). Ultrasound examination was used to examine the cardiac function. Masson staining was used to evaluate the myocardial infarct area. TUNEL assay was performed to examine the anti-apoptotic effect of allicin. Differentially expressed genes (DEGs) and pathways were analyzed by mRNA microarray analysis. Immunofluorescence staining and western blot were carried out to detect the effect of allicin on the PI3K. A pan-PLC activator, m-3M3FBS, was applied to investigate whether allicin induced cardiomyocyte apoptosis was via the GRK2/PLC/IP3R signaling pathway. RESULTS: Masson staining and the TUNEL assay revealed that allicin reduced infarct size and played an anti-apoptotic role in M/IR. Ultrasound examination revealed that allicin improved cardiac function after M/IR injury. Gene ontology analysis indicated that the calcium signaling pathway and PI3KCA(PI3K) were selected. Immunofluorescence staining and western blot exposed that PI3K was activated by allicin during MI/R injury. Fura-2AM staining revealed that the PI3K -mediated GRK2/PLC-γ/IP3R pathway may be involved in the protective effect of allicin on MI/R injury. CONCLUSIONS: Allicin has a protective effect on MI/R injury. This effect might be associated with the inhibition of Ca2+ overload-induced apoptosis and the inhibition of the PI3K -mediated GRK2/PLC-γ/IP3R signaling pathway.

Involvement of GRK2 in modulating nalfurafine-induced reduction of excessive alcohol drinking in mice.

Though it is well known that G protein-coupled receptor kinase 2 [GRK2] is involved in regulation of mu opioid receptor [MOR] desensitization and morphine-related behaviors, the potential role of GRK2 in regulation of kappa opioid receptor [KOR] functions in vivo has not been established yet. A couple of recent studies have found that GRK2 activity desensitizes KOR functions via decreasing G protein-coupled signaling with sensitizing arrestin-coupled signaling. Nalfurafine, a G protein-biased KOR full agonist, produces an inhibitory effect on alcohol intake in mice, with fewer side effects (sedation, aversion, or anxiety/depression-like behaviors). Using RNA sequencing (RNA-seq) analysis, we first identified that nuclear transcript level of grk2 [adrbk1] (but not other grks) was significantly up-regulated in mouse nucleus accumbens shell (NAcs) after chronic excessive alcohol drinking, suggesting alcohol specifically increased NAcs grk2 expression. We then tested whether selective GRK2/3 inhibitor CMPD101 could alter alcohol intake and found that CMPD101 alone had no effect on alcohol drinking. Therefore, we hypothesized that the grk2 increase in the NAcs could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Nalfurafine decreased alcohol drinking in a dose-related manner, and pretreatment with CMPD101 enhanced the reduction in alcohol intake induced by nalfurafine, indicating an involvement of GRK2/3 blockade in modulating G protein-biased KOR agonism of nalfurafine. Together, our study provides initial evidence relevant to the transcriptional change of grk2 gene in the NAc shell after excessive alcohol drinking. Pharmacological GRK2/3 blockade enhanced nalfurafine's efficacy, suggesting a GRK2/3-mediated mechanism, probably through the G protein-mediated KOR signaling.