G Protein-coupled Receptor Kinase Research Articles

Potential cardioprotective effect of paroxetine against ventricular remodeling in an animal model of myocardial infarction: a comparative study

BackgroundPost-myocardial infarction (MI) remodeling involves various structural and functional changes, such as inflammation and fibrosis. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is linked to the progression of cardiovascular diseases, including myocardial infarction. The inhibitory effects of paroxetine on GRK2 are recognized, yet its protective effect on post-MI remodeling has not been elucidated. Here, we investigated the cardioprotective effect of paroxetine in an animal model of MI, focusing on post-MI cardiac remodeling and comparing its effect to a β-blocker and an angiotensin receptor antagonist.MethodsAlbino Wistar rats were divided into five groups (control; untreated MI; and MI pre-treated with either paroxetine, metoprolol, or irbesartan). MI was induced using isoproterenol (100 mg.kg−1) on days 16 and 17. Cardioprotective effects were determined by assessing markers of cardiac injury, histopathology, inflammation, oxidative stress, and fibrosis. Statistical analysis performed using a one-way analysis of variance, followed by an appropriate post hoc test, the differences between the groups were considered significant when the (P < 0.05).ResultsParoxetine significantly attenuated cardiac injury biomarkers including serum Tn-I and CK-MB levels. In terms of cardiac remodeling, paroxetine significantly reduced the relative HW/BW index and the plasms FGF23 level. Furthermore, it modulated markers of fibrosis, inflammation, and oxidative stress.ConclusionThe current findings suggest that pre-treatment with paroxetine may exert a beneficial effect that protects against post-MI remodeling, including modulating fibrotic, inflammatory, and angiogenesis-related factors. Therefore, the current findings show the promising role of paroxetine as a cardioprotective that attenuates post-MI remodeling processes.

Signal profiles and spatial regulation of β-arrestin recruitment through Gβ5 and GRK3 at the μ-opioid receptor

The μ-opioid receptor (MOR) is a G-protein-coupled receptor (GPCR) that mediates both analgesic effects and adverse effects of opioid drugs. Despite extensive efforts to develop a signal-biased drug, drugs with sufficiently reduced side effects have not been established, in part owing to lack of comprehensive signal transducer profiles of MOR. In this study, by profiling the activity of signal transducers including G proteins and GPCR kinases (GRKs), we revealed an unprecedented mechanism of selective GRK3 activation by Gβ5, leading to β-arrestin recruitment. By utilizing multiple genome-edited cell lines and functional assays, we found that oliceridine, an FDA-approved G-protein-biased agonist, selectively activates Gαz- and GRK3-mediated signaling. Notably, among the five Gβ subtypes, only Gβ5 distinguishes GRK3 from GRK2. Using single-molecule imaging, we found that GRK3 is recruited to the plasma membrane upon MOR agonist stimulation by Gβ1 and Gβ5, yet their interaction dynamics with GRK3 and mechanisms of action are different. Furthermore, particle diffusion analysis suggests that Gβ5 is enriched in confined membrane domains, through which GRK3 is recruited to the plasma membrane in a freely diffusible state, thereby allowing GRK3 to efficiently interact with MOR. These findings provide a mechanism by which MOR agonists rely on a specific Gα–Gβ–GRK axis to induce β-arrestin recruitment.

A cross-tissue transcriptome-wide association study reveals GRK4 as a novel susceptibility gene for COPD

Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory disorder with environmental factors being the primary risk determinants. However, genetic factors also substantially contribute to the susceptibility and progression of COPD. Although genome-wide association studies (GWAS) have identified several loci associated with COPD susceptibility, the specific pathogenic genes underlying these loci, along with their biological functions and roles within regulatory networks, remain unclear. This lack of clarity constrains our ability to achieve a deeper understanding of the genetic basis of COPD. This study leveraged the FinnGen R11 genetic dataset, comprising 21,617 cases and 372,627 controls, along with GTEx V8 eQTLs data to conduct a cross-tissue transcriptome-wide association study (TWAS). Initially, we performed a cross-tissue TWAS analysis using the Unified Test for Molecular Signatures (UTMOST), followed by validation of the UTMOST findings in single tissues using the Functional Summary-based Imputation (FUSION) method and conditional and joint (COJO) analyses of the identified genes. Subsequently, candidate susceptibility genes were screened using Multi-marker Analysis of Genomic Annotation (MAGMA). The causal relationship between these candidate genes and COPD was further evaluated through summary data-based Mendelian randomization (SMR), colocalization analysis, and Mendelian randomization (MR). Additionally, the identified results were validated against the COPD dataset in the GWAS Catalog (GCST90399694). GeneMANIA was employed to further explore the functional significance of these susceptibility genes. In the cross-tissue TWAS analysis (UTMOST), we identified 17 susceptibility genes associated with COPD. Among these, a novel susceptibility gene, G protein-coupled receptor kinase 4 (GRK4), was validated through single-tissue TWAS (FUSION) and MAGMA analyses, with further confirmation via SMR, MR, and colocalization analyses. Moreover, GRK4 was validated in an independent dataset. This study identifies GRK4 as a potential novel susceptibility gene for COPD, which may influence disease risk by exacerbating inflammatory responses. The findings address gaps in previous single-tissue GWAS studies, revealing consistent expression and potential function of GRK4 across different tissues. However, considering the study’s limitations, further investigation and validation of GRK4’s role in COPD are warranted.

GRK2 mediates cisplatin-induced acute liver injury via the modulation of NOX4

BackgroundThe present study investigated the function of G protein-coupled receptor kinase 2 (GRK2) in acute liver injury (ALI) by cisplatin, and investigated the protective effect of pharmacological inhibition of GRK2.MethodsALI models were generated in global adult hemizygous (ALI-Grk2±) mice and wild-type (WT) mice. Liver biochemistry parameters and histopathology were used to evaluate the severity of ALI and the protective effect of pharmacological inhibition of GRK2. GRK2-siRNA was used to knock down the expression of GRK2 in AML12 cells in vitro.ResultsALI model mice exhibited increased blood levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and abnormal liver pathology accompanied by imbalanced L-glutathione (GSH) levels. Cisplatin administration upregulated GKR2, p-GRK2 and NADPH oxidase 4 (NOX4) expression in the liver tissues of ALI model mice. Compared to WT mice injected with cisplatin, Grk2± mice that received cisplatin showed significant improvements in liver function and pathological performance, decreased NOX4 levels, reduced endoplasmic reticulum (ER) stress, and diminished liver cell apoptosis. In vitro, the transfection of AML12 cells with siRNA significantly reduced NOX4 expression and inhibited cisplatin-induced reactive oxygen species production, ER stress (increased levels of GRP94, GRP78, p-elF2α and CHOP) and apoptotic death. Moreover, pharmacological treatment with drugs that inhibit GRK2 (CP-25 or paroxetine) significantly ameliorated cisplatin-induced ALI by improving liver pathological manifestations, inhibiting oxidative stress and ER stress, and reducing liver cell apoptosis. Similar results were observed in vitro.ConclusionsGRK2 mediates the development of cisplatin-induced ALI by modulating NOX4 and ER stress. Pharmacological inhibition of GRK2 with CP-25 or paroxetine effectively alleviated ALI. GRK2 can be used as a potential target for the prevention and treatment of liver injury.Graphical

Distinct role of GRK3 in platelet activation by desensitization of G protein-coupled receptors.

Many platelet agonists mediate their cellular effects through G protein-coupled receptors (GPCRs) to induce platelet activation, and GPCR kinases (GRKs) have been demonstrated to have crucial roles in most GPCR functions in other cell types. Here, we investigated the functional role of GRK3 and the molecular basis for the regulation of GPCR desensitization by GRK3 in platelets. We used mice lacking GRK3 as well as β-arrestin2 which has been shown to be important in GPCR function in platelets. Platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in both GRK3 -/- and β-arrestin2 -/- platelets compared to wild-type (WT) platelets, while non-GPCR agonist collagen-induced platelet aggregation and secretion were not affected. We have previously shown that GRK6 is not involved in the regulation of Gq-coupled 5HT2A and Gz-coupled a2A adrenergic receptors. Interestingly, in contrast to GRK6, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate 5-HT2A and a2A adrenergic receptors, respectively, was significantly potentiated in GRK3 -/- platelets, suggesting that GRK3 was involved in general GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP was restored in GRK3 -/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, confirming that GRK3 contributes to general GPCR desensitization. Finally, 2-MeSADP- and AYPGKF-induced AKT and ERK phosphorylation were significantly potentiated in GRK3 -/- platelets. GRK3 plays a crucial role in the regulation of platelet activation through general GPCR desensitization in platelets.

Role of the GRK2/3 N-terminus in discriminating the endocytic effects of opioid agonist drugs.

Endocytosis of the μ-type opioid receptor (MOR) is a fundamentally important cellular regulatory process that is characteristically driven less effectively by partial relative to full agonist ligands. Such agonist-selective endocytic discrimination depends on how strongly drugs promote MOR binding to β-arrestins and this, in turn, depends on how strongly they stimulate phosphorylation of the MOR cytoplasmic tail by GPCR kinases (GRKs) from the GRK2/3 subfamily. While these relatively 'downstream' steps in the agonist-selective endocytic pathway are now well defined, it remains unclear how agonist-bound receptors are distinguished 'upstream' by GRKs. Focusing on GRK2 as a prototype, we show that this single GRK subtype can distinguish the endocytic activities of different MOR agonists in cells lacking other GRKs, and that agonist-selectivity is introduced at the most upstream step of GRK2 binding to MOR. This interaction requires prior membrane recruitment of GRK2 by its conserved PH domain and is enhanced by phosphorylation of the MOR tail, but neither reaction can explain the high degree of agonist-selectivity in the observed interaction of GRK2 with MOR. We identify the N-terminal domain (NTD) of GRK2, which is identical in GRK3, as a discrete element required for the full agonist-selectivity of MOR-GRK2 interaction and show that the NTD is also required for GRK2 to promote MOR endocytosis after it is bound. We propose a simple cellular mechanism of upstream agonist discrimination that is organized as a series of biochemical checkpoints and utilizes the NTD as an agonist-selective sensor. Significance Statement This study investigates how GPCR kinases (GRKs) distinguish the effects of opioid agonist drugs on regulated endocytosis of the μ-opioid receptor (MOR). It shows that a single GRK subtype is sufficient to determine the agonist-selectivity of MOR internalization, agonists are distinguished by how strongly they promote GRK2 recruitment by MOR, and the GRK2/3 N-terminus is a key determinant of agonist discrimination.

CGMP-dependent pathway and a GPCR kinase are required for photoresponse in the nematode Pristionchus pacificus.

Light sensing is a critical function in most organisms and is mediated by photoreceptor proteins and phototransduction. Although most nematodes lack eyes, some species exhibit phototaxis. In the nematode Caenorhabditis elegans, the unique photoreceptor protein Cel-LITE-1, its downstream G proteins, and cyclic GMP (cGMP)-dependent pathways are required for phototransduction. However, the mechanism of light-sensing in other nematodes remains unknown. To address this question, we used the nematode Pristionchus pacificus, which was established as a satellite model organism for comparison with C. elegans. Similar to C. elegans, illumination with short-wavelength light induces avoidance behavior in P. pacificus. Opsin, cryptochrome/photolyase, and lite-1 were not detected in the P. pacificus genome using orthology and domain prediction-based analyses. To identify the genes related to phototransduction in P. pacificus, we conducted forward genetic screening for light-avoidance behavior and isolated five light-unresponsive mutants. Whole-genome sequencing and genetic mapping revealed that the cGMP-dependent pathway and Ppa-grk-2, which encodes a G protein-coupled receptor kinase (GRK) are required for light avoidance. Although the cGMP-dependent pathway is conserved in C. elegans phototransduction, GRK is not necessary for light avoidance in C. elegans. This suggests similarities and differences in light-sensing mechanisms between the two species. Using a reverse genetic approach, we showed that gamma-aminobutyric acid (GABA) and glutamate were involved in light avoidance. Through reporter analysis and suppression of synapse transmission, we identified candidate photosensory neurons. These findings advance our understanding of the diversity of phototransduction in nematodes even in the absence of eyes.

Slow dissociation kinetics of fentanyls and nitazenes correlates with reduced sensitivity to naloxone reversal at the μ-opioid receptor.

Fentanyls and nitazenes are μ-opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we determined the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues, compared to morphine and DAMGO. In vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ receptors to assess Gi protein activation and β-arrestin 2 recruitment. The apparent rate of agonist dissociation from the μ receptor varied: morphine, DAMGO, alfentanil and fentanyl dissociated rapidly, whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow apparent rate of dissociation was not because of G protein receptor kinase-mediated arrestin recruitment as its apparent rate of dissociation was not increased by inhibition of G protein-coupled receptor kinases (GRKs) with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. With fentanyls and nitazenes that slowly dissociate from the μ receptor, antagonism by naloxone is pseudo-competitive. In overdoses involving fentanyls and nitazenes, higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose.

GRK2 Protein Mediates the ANRIL, a lncRNA, to Affect the Proliferation and Apoptosis of Kasumi-1 Cells.

A long non-coding RNAs (LncRNAs) called antisense noncoding RNA in the INK4 locus (ANRIL), has emerged as substantial regulators of cell survival in acute myeloid leukemia (AML). However, its speciffc and potential mechanism is uncertain in AML. In this research, we investigated the role of ANRIL in cell proliferation, apoptosis, and the underlying mechanism in AML cells. ANRIL expression was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Kasumi-1 cells were transfected with LV-ANRIL plasmid to upregulate ANRIL expression, with or without co-transfection with a G Protein-Coupled Receptor Kinase 2 (GRK2) siRNA. Additionally, these cells were transfected with sh-ANRIL plasmid to inhibit ANRIL expression, with or without co-transfection with a GRK2 overexpression plasmid. Cell proliferation and apoptosis were determined using the cell counting kit-8 (CCK8) and flow cytometry. Protein expression levels of phosphatidylinositide 3-kinases (PI3K), protein kinase B (AKT), phosphorylated-Akt (p-AKT), Bcl-2-associated protein x (BAX), B-cell leukemia/lymphoma 2 protein (BCL-2), proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and GRK2 were detected by western blot. The RNA-binding protein immunoprecipitation (RIP) assay was conducted to investigate the interaction between ANRIL and GRK2. ANRIL expression was increased in Kasumi-1 cells. ANRIL upregulation expression promoted cell proliferation and inhibited apoptosis. Furthermore, its upregulation led to increased expressions of PI3K, AKT, p-AKT, PCNA, and BCL-2, and decreased expression of BAX in Kasumi-1 cells. Additionally, transfection with GRK2 siRNA attenuated the promoting effect of LV-ANRIL on Kasumi-1 cells proliferation and the PI3K/AKT pathway, increased BAX and cleaved caspase-3 expressions, and decreased BCL-2 and PCNA expressions. GRK2 overexpression reversed the inhibitory effect of sh-ANRIL on cell proliferation and the PI3K/AKT pathway. Furthermore, it promoted BCL-2 and PCNA expressions, and inhibited BAX and cleaved caspase-3 expressions. RIP assay confirmed the physical interaction between ANRIL and GRK2. The GRK2 protein-mediated ANRIL, increasing Kasumi-1 cell proliferation and inhibiting apoptosis by activating the PI3K/AKT/BCL-2 pathway.

Female Specific Restrictive Cardiomyopathy and Metabolic Dysregulation in transgenic mice expressing a Peptide of the Amino-Terminus of GRK2.

Cardiovascular disease and heart failure are a major health challenge, with sex differences in pathophysiology and treatment responses critically influencing patient outcomes. G protein-coupled receptor (GPCR) kinase 2 (GRK2) is a pivotal regulator of cellular signaling whose elevation is a hallmark of heart failure progression. Its complex network of protein interactions impact a wide range of physiological and pathophysiological processes including cardiac function. In this study, we examined the effects of cardiac-restricted expression of an amino-terminal peptide of GRK2 (βARKnt) in female mice subjected to acute and chronic pressure overload. Our findings reveal that that βARKnt affects hypertrophy development and cardiac function differently in female mice than in males, leading to a transition to heart failure not observed in control females or βARKnt males. Notably, the βARKnt female mice exhibited baseline hypertrophy with distinct left atrial morphology, increased fibrosis, and immune cell infiltration compared to the controls, which progressed under chronic stress, indicating adverse cardiac remodeling. Furthermore, βARKnt female mice, unlike males, exhibit impaired tissue respiration following acute pressure overload and altered glucose sensitivity and insulin tolerance, highlighting significant remodeling of cardiac and systemic metabolism.

Regulation of β-Adrenergic Receptors in the Heart: A Review on Emerging Therapeutic Strategies for Heart Failure.

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF.

The roles of G protein-coupled receptor kinase 2 in renal diseases.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) is an integrative node in many signalling network cascades. An emerging study indicates that GRK2 can interact with GPCRs and non-GPCR substrates in both kinase-dependent and -independent modes. Alterations in the functional levels of GRK2 have been found in a variety of renal diseases, such as hypertension-related kidney injury, sepsis-associated acute kidney injury (S-AKI), cardiorenal syndrome (CRS), acute kidney injury (AKI), age-related kidney injury or hyperglycemia-related kidney injury. Abnormal GRK2 expression contribute to the development of renal diseases, making them promising molecular targets for treating renal diseases. Blocking the prostaglandin E2 (PGE2)-EP1-Gaq-Ca2+ signal pathway in glomerular mesangial cells (GMCs) by internalizing prostaglandin E2 receptor 1 (EP1) with GRK2 may be a potential treatment for diabetic nephropathy (DN). In addition, GRK2 inhibition may have therapeutic effects in a variety of renal diseases, such as SLE-related kidney injury, DN, age-related kidney injury, hypertension-related kidney injury, and CRS. However, there is still a long way to go for the large-scale application of GRK2 inhibition in the field of renal diseases. In this review, we discuss recent updates in understanding the role of GRK2 in kidney dysfunction. Furthermore, we explore the potential of GRK2 as a possible therapeutic target for renal pathologies. We believe it will shed light on the future development of small-molecule inhibitors of GRK, as well as the clinical applications in renal diseases.

Uncovering conserved networks and global conformational changes in G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) are essential regulators of signaling pathways mediated by G protein-coupled receptors. Recent research suggests that GRK-mediated phosphorylation patterns dictate functional selectivity, leading to biased cellular responses. However, a comprehensive understanding of the structural mechanisms at the single-residue level remains elusive. This study aims to define the general conformational dynamics of GRKs with a particular focus on quantifying the transitions between the closed and open states. Specifically, we examined these transitions, classified based on the ionic lock between the regulatory G protein signaling homology domain and kinase domain. To facilitate a precise structural comparison, we assigned common labels to topologically identical positions across the 47 GRK structures retrieved from the Protein Data Bank. Our analysis identified both general and subfamily-specific dynamic movements within the networks and measured the conformational change scores between the two states. Elucidating these structural dynamics could provide significant insights into the regulatory mechanisms of GRK.

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 Gβγ 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 (βi) 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%; βi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; βi: 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 βi 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.

Abstract MP16: Expression of an AT 1 R Receptor Mutant which Genetically Biases Gαq Specifically in Vascular Smooth Muscle Cell Causes Hypertension and Enhanced Vascular Contraction

Angiotensin II (ANG) type 1 receptor (AT 1 R) belongs to the 7-membrane superfamily of G protein-coupled receptors (GPCR) which have the largest class of drug targets. AT 1 Rs can adopt different conformations that can bias G-protein coupling (particularly Gaq) or b-arrestin signaling. The b-arrestin pathway is reported to be cardioprotective while the canonical Gaq-dependent pathway is typically associated with the detrimental effects of ANG. We hypothesize that selective and inducible activation of the AT 1 R Gaq pathway in the absence of b-arrestin signaling in vascular smooth muscle (SMC) results in hypertension and vascular dysfunction via an AT 1 R-dependent mechanism. We generated an AT 1 R mutant (AT 1 R TSTS-AAAA ) which ablates the phosphorylation sites for GPCR kinases (GRK) in the C-terminal to impair b-arrestin recruitment and to induce a Gaq bias. Transgenic mice were made with a construct that induces both AT 1 R TSTS-AAAA and tdTomato expression in response to Cre-recombinase. Primary fibroblasts cultured from AT 1 R TSTS-AAAA mice exhibited a Cre-induced expression of tdTomato and marked increase in phosphorylated-extracellular signal-regulated kinase (P-ERK) in response to ANG when compared to mice cells expressing wildtype AT 1 R. Mice carrying inducible AT 1 R TSTS-AAAA were bred with tamoxifen-inducible SMC-specific CRE (SMC CreERT2 , S-TSTS) mice. Transgene expression in S-TSTS mice was successfully activated by tamoxifen as measured by the expression of the embedded tdTomato reporter in the aorta SMC. S-TSTS mice exhibited a trend of increased systolic blood pressure (SBP) 1 week after tamoxifen injection (147.76 ± 13.21 vs. 108.85 ± 4.58 mmHg). Candesartan (10 mg/kg/day in drinking water) was then administered to S-TSTS mice once hypertension was established. Candesartan treatment for 1 week reversed the hypertension in S-TSTS mice (100.00 ± 2.88 mmHg). Interestingly, 2 weeks after ending candesartan treatment, SBP was once again elevated in S-TSTS mice (142.01 ± 7.65 vs. 90.35 ± 1.14 mmHg, p&lt;0.05). In addition, mesenteric arteries from transgenic mice exhibited a robust increase in ANG-induced constriction when compared to control mice (ANG, max constriction 60.25 ± 4.78% vs. 14.12 ± 4.72%, p&lt;0.05). Our findings support the hypothesis that signaling through the Gaq-dependent pathway in SMC leads to increased vasoconstriction and hypertension through an AT 1 R-dependent mechanism.

G protein–coupled receptor kinase 5 regulates thrombin signaling in platelets

BackgroundOur prior genome-wide association study of thrombin-induced platelet aggregation identified a G protein–coupled receptor kinase 5 (GRK5) noncoding variant (rs10886430-G) that is strongly associated with increased platelet reactivity to thrombin. This variant predisposes to increased risk of stroke, pulmonary embolism, and venous thromboembolism. ObjectivesTo determine role of platelet specific GRK5 in platelet responses to agonists and injury. MethodsPlatelets from GRK5 mutant mice have been shown to have increased thrombin sensitivity, indicating that GRK5 may be a negative regulator of platelet activation. However, this has not been studied in a platelet-specific manner. We therefore used platelet-specific GRK5 mutant mice and models of thrombosis and pulmonary embolism. ResultsWe now demonstrate that mice lacking GRK5 specifically in platelets had a mild increase in thrombin responses in vitro and a shortened time to arterial thrombosis in vivo. In addition, platelet GRK5 mutant mice had increased thrombin but not collagen-induced thrombus burden in a mouse model of pulmonary embolism. ConclusionThese data indicate that platelet GRK5 has a significant role in limiting platelet responses to thrombin.

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.

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

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. 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.

G protein-coupled receptor kinase 2 as a novel therapeutic target for gland fibrosis of Sjögren's syndrome.

Sjogren's syndrome (SS) is a chronic, progressive autoimmune disorder characterized by gland fibrosis. We previously found a close correlation between gland fibrosis and the expression of G protein-coupled receptor kinase 2 (GRK2). In this study we explored the pathological and therapeutic significance of GRK2 in SS. Submandibular gland (SMG) antigen-induced SS mouse model was established in WT and GRK2+/- mice. We showed that the expression levels of GRK2 were significantly up-regulated in glandular tissue and positively correlated with fibrotic morphology in SS patients and mice. Hemizygous knockout of GRK2 significantly inhibited the gland fibrosis. In mouse salivary gland epithelial cells (SGECs), we demonstrated that GRK2 interacted with Smad2/3 to positively regulate the activation of TGF-β-Smad signaling with a TGF-β-GRK2 positive feedback loop contributing to gland fibrosis. Hemizygous knockout of GRK2 attenuated TGF-β-induced collagen I production in SGECs in vitro and hindered gland fibrosis in murine SS though preventing Smad2/3 nuclear translocation. Around 28 days post immunization with SMG antigen, WT SS mice were treated with a specific GRK2 inhibitor paroxetine (Par, 5 mg·kg-1·d-1, i.g. for 19 days). We found that Par administration significantly attenuated gland fibrosis and alleviated the progression of SS in mice. We conclude that genetic knockdown or pharmacological inhibition of GRK2 significantly attenuates gland fibrosis and alleviates the progression of SS. GRK2 binds to Smad2/3 and positively regulates the activation of TGF-β-Smad signaling. A TGF-β-GRK2 positive feedback loop contributes to gland fibrosis. Our research points out that GRK2 could be a promising therapeutic target for treating SS.

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

BackgroundAccording 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. PurposeThis study examines the effects of CIN on GRK2 and myocardial energy metabolism to elucidate its underlying mechanism to treat HF. MethodsThe 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). ResultsIn 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. ConclusionThis 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.