G Protein-coupled Receptor Kinase Research Articles

Cryo-electron microscopy for GPCR research and drug discovery in endocrinology and metabolism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structuresresolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5-7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, Gprotein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathwayor an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of β-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and β-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of β-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of β-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

GRK2-YAP signaling is implicated in pulmonary arterial hypertension development.

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation of small pulmonary arterial vascular smooth muscle cells (PASMCs), endothelial dysfunction, and extracellular matrix remodeling. G protein-coupled receptor kinase 2 (GRK2) plays an important role in the maintenance of vascular tone and blood flow. However, the role of GRK2 in the pathogenesis of PAH is unknown. GRK2 levels were detected in lung tissues from healthy people and PAH patients. C57BL/6 mice, vascular smooth muscle cell-specific Grk2 -knockout mice ( Grk2ΔSM22 ), and littermate controls ( Grk2flox/flox ) were grouped into control and hypoxia mice ( n =8). Pulmonary hypertension (PH) was induced by exposure to chronic hypoxia (10%) combined with injection of the SU5416 (cHx/SU). The expression levels of GRK2 and Yes-associated protein (YAP) in pulmonary arteries and PASMCs were detected by Western blotting and immunofluorescence staining. The mRNA expression levels of Grk2 and Yes-associated protein ( YAP ) in PASMCs were quantified with real-time polymerase chain reaction (RT-PCR). Wound-healing assay, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, and 5-Ethynyl-2'-deoxyuridine (EdU) staining were performed to evaluate the proliferation and migration of PASMCs. Meanwhile, the interaction among proteins was detected by immunoprecipitation assays. The expression levels of GRK2 were upregulated in the pulmonary arteries of patients with PAH and the lungs of PH mice. Moreover, cHx/SU-induced PH was attenuated in Grk2ΔSM22 mice compared with littermate controls. The amelioration of PH in Grk2ΔSM22 mice was accompanied by reduced pulmonary vascular remodeling. In vitro study further confirmed that GRK2 knock-down significantly altered hypoxia-induced PASMCs proliferation and migration, whereas this effect was severely intensified by overexpression of GRK2 . We also identified that GRK2 promoted YAP expression and nuclear translocation in PASMCs, resulting in excessive PASMCs proliferation and migration. Furthermore, GRK2 is stabilized by inhibiting phosphorylating GRK2 on Tyr86 and subsequently activating ubiquitylation under hypoxic conditions. Our findings suggest that GRK2 plays a critical role in the pathogenesis of PAH, via regulating YAP expression and nuclear translocation. Therefore, GRK2 serves as a novel therapeutic target for PAH treatment.

GRK5 promoted renal fibrosis via HDAC5/Smad3 signaling pathway.

Renal fibrosis is a common pathological feature of chronic kidney diseases (CKD), poses a significant burden in the aging population, and is a major cause of end-stage renal disease (ESRD). In this study, we investigated the role of G protein-coupled receptor kinases (GRKs) 5 in the pathogenesis of renal fibrosis. GRK5 is a serine/threonine kinase that regulates G protein-coupled receptor (GPCR) signaling. GRK5 has been shown to play a role in various diseases including cardiac disorders and cancer. However, the role of GRK5 in renal fibrosis remains largely unknown. Our finding revealed that GRK5 was significantly overexpressed in renal fibrosis. Specifically, GRK5 was transferred into the nucleus via its nuclear localization sequence to regulate histone deacetylases (HDAC) 5 expression under renal fibrosis. GRK5 acted as an upstream regulator of HDAC5/Smad3 signaling pathway. HDAC5 regulated and prevented the transcriptional activity of myocyte enhancer factor 2A (MEF2A) to repress the transcription of Smad7 which leading to the activation of Smad3. These findings first revealed that GRK5 may be a potential therapeutic target for the treatment of renal fibrosis. Inhibition of GRK5 activity may be a promising strategy to attenuate the progression of renal fibrosis.

Biased Signaling in Mutated Variants of β2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations.

The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of β2-adrenergic receptors (β2AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor toward the β-arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of β-arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigated the allosteric mechanism of receptor bias in β2AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R1313.50 and Y3267.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. Moreover, partial dissociation of G protein from the receptor core is observed in the simulations of the triple mutant in contrast to the single mutant and wild-type receptor. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of β2AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side-effects and superior efficacy.

G protein-coupled receptor-biased signaling: potential drug discovery to facilitate treatment of metabolic diseases

G protein-coupled receptors (GPCRs) are important, potential drug targets for the treatment of metabolic disorders, such as obesity. GPCRs crosstalk with several transducers, including heterotrimeric G proteins, GPCR kinases (GRKs), and β-arrestins. GPCR-biased agonism has raised the potential of novel drug development to preferentially activate therapeutic signaling pathways over pathways that lead to unwanted side effects. The obesity epidemic and its metabolic complications continue to be a major global public health threat but effective treatments are limited. The accelerated development of structural techniques, like X-ray crystallography and cryo-electron microscopy, has paved the way to understanding how biased agonism measured at GPCRs results in specific downstream physiologic responses. Herein some well-validated GPCR targets are briefly summarized and several new and promising receptors for obesity treatment are outlined. This review highlights the significance of deciphering the role of GPCRs in obesity pathology and biased signaling for drug development. We anticipate the review will facilitate the development of novel GPCR-targeted anti-obesity drugs that lead to heightened therapeutic efficacy with decreased side effect profiles.

Nicotine Diminishes Alpha2-Adrenergic Receptor-Dependent Protection Against Oxidative Stress in H9c2 Cardiomyocytes.

Nicotine is a major component of cigarette smoke with various detrimental cardiovascular effects, including increased oxidative stress in the heart. Agonism of α2-adrenergic receptors (ARs), such as with dexmedetomidine, has been documented to exert cardioprotective effects against oxidative stress and related apoptosis and necroptosis. α2-ARs are membrane-residing G protein-coupled receptors (GPCRs) that primarily activate Gi/o proteins. They are also subjected to GPCR-kinase (GRK)-2-dependent desensitization, which entails phosphorylation of the agonist-activated receptor by GRK2 to induce its decoupling from G proteins, thus terminating α2AR-mediated G protein signaling. In the present study, we sought to examine the effects of nicotine on α2AR signaling and effects in H9c2 cardiomyocytes exposed to H2O2 to induce oxidative cellular damage. As expected, treatment of H9c2 cardiomyocytes with H2O2 significantly decreased cell viability and increased oxidative stress, as assessed by reactive oxygen species (ROS)-associated fluorescence levels (DCF assay) and superoxide dismutase activity. Both H2O2 effects were partly rescued by α2AR activation with brimonidine in control cardiomyocytes but not in cells pretreated with nicotine for 24 hours, in which brimonidine was unable to reduce H2O2-induced cell death and oxidative stress. This was due to severe α2AR desensitization, manifested as very low Gi protein activation by brimonidine, and accompanied by GRK2 upregulation in nicotine-treated cardiomyocytes. Finally, pharmacological inhibition of adenylyl cyclase (AC) blocked H2O2-dependent oxidative damage in nicotine-pretreated H9c2 cardiomyocytes, indicating that α2AR activation protects against oxidative injury via its classic coupling to Gai-mediated AC inhibition. Nicotine can negate the cardioprotective effects of α2AR agonists against oxidative injury, which may have important implications for patients treated with this class of drugs that are chronic tobacco smokers.

Interactions between AT1R and GRKs: the determinants for activation of signaling pathways involved in blood pressure regulation.

The success of Angiotensin II receptor blockers, specifically Angiotensin II type 1 receptor (AT1R) antagonists as antihypertensive drug emphasizes the involvement of AT1R in Essential hypertension. The structural insights and mutational studies of Ang II-AT1R have brought about the vision to design Ang II analogs that selectively activate the pathways with beneficial and cardioprotective effects such as cell survival and hinder the deleterious effects such as hypertrophy and cell death. AT1R belongs to G-protein coupled receptors and is regulated by G-protein coupled receptor kinases (GRKs) that either uncouples Gq protein for receptor desensitization or phosphorylate C-terminus to recruit β-arrestin for internalization of the receptor. The interaction of GRKs with ligand activated AT1R induces conformational changes and signal either Gq dependent or Gq independent pathways. These interactions might explain the complex regulatory mechanisms and offer promising ideas for hypertension therapeutics. This article reviews the functional role of AT1R, organization of GRK genes and regulation of AT1R by GRKs that play significant role in desensitization and internalization of the receptors.

Hepatic GRK2 is dispensable for glucose homeostasis and other key metabolic parameters in mice

ObjectiveG-protein-coupled receptor (GPCR) kinases (GRKs) abrogate GPCR signaling by promoting receptor desensitization and internalization. Accumulating evidence suggests that GRK2 represents an important regulator of GPCR-mediated effects on systemic glucose metabolism, obesity, and insulin resistance. Despite the key role of the liver in maintaining euglycemia, the potential metabolic relevance of hepatic GRK2 has yet to be examined. Thus, the goal of this study was to explore the potential role of hepatic GRK2 in maintaining glucose homeostasis and other key metabolic functions. MethodsTo address this question, we generated mice that showed a ∼90% reduction in GRK2 protein expression selectively in hepatocytes (Hep-GRK2-KO mice) and subjected these mice, together with their control littermates, to systematic metabolic phenotyping studies. ResultsWe found that Hep-GRK2-KO mice maintained on regular chow did not differ significantly from their control littermates in glycemia, glucose tolerance, insulin sensitivity, in vivo gluconeogenesis, and glucagon-induced hyperglycemia. We obtained similar findings when we analyzed Hep-GRK2-KO mice and control littermates consuming an obesogenic high-fat diet. Likewise, plasma levels of insulin, glucagon, free fatty acids, and ketone bodies remained unaffected by the lack of hepatocyte GRK2. The same was true when we examined the expression levels of key genes regulating hepatic glucose and fatty acid metabolism. ConclusionIn summary, our data suggest that hepatocyte GRK2 is dispensable for systemic glucose homeostasis and other key metabolic functions in both lean and obese mice. This finding suggests that drug development efforts aimed at inhibiting GRK2 to improve impaired glucose homeostasis and insulin sensitivity need to focus on other metabolically important tissues.

Pulsed radiofrequency on DRG inhibits hippocampal neuroinflammation by regulating spinal GRK2/p38 expression and enhances spinal autophagy to reduce pain and depression in male rats with spared nerve injury

Evidence indicates that microglial G protein-coupled receptor kinase 2 (GRK2) is a key regulator of the transition from acute to chronic pain mediated by microglial products via the p38 mitogen-activated protein kinase (MAPK) pathway in the spinal cord dorsal horn (SCDH). Increasing studies have shown that autophagic dysfunction in the SCDH and neuroinflammation in the hippocampus underlie NeP. However, whether GRK2/p38MAPK and autophagic flux in the SCDH and hippocampal neuroinflammation are involved in NeP and depression comorbidity has not been determined. Here, we explored the effects of high-voltage pulsed radiofrequency (PRF) (85 V-PRF; HV-PRF) to the dorsal root ganglion (DRG) on pain phenotypes in Wistar male rats with spared nerve injury (SNI) and the underlying mechanisms. The exacerbation of pain phenotypes was markedly relieved by PRF-DRG. The SNI-induced reduction in GRK2 expression, elevation of p-p38 MAPK levels in the SCDH, and increase in IL-1β and TNF-α levels in the hippocampus were reversed by PRF, which was accompanied by an increase in autophagic flux in spinal microglia. The beneficial effect of 85 V-PRF was superior to that of 45 V-PRF. In addition, the improvements elicited by 85 V-PRF were reversed by intrathecal injection of GRK2 antisense oligonucleotide, and these changes were accompanied by GRK2 downregulation and p-p38 upregulation in the SCDH, increased pro-inflammatory factor levels in the hippocampus, and excessive autophagy in spinal microglia. In conclusion, our data indicate that the application of HV-PRF to the DRG could serve as an excellent therapeutic technique for regulating neuroimmunity and neuroinflammation to relieve pain phenotypes.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

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-β1 (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, an Inhibitor of MALT1-Dependent Oncogenic Signaling, Is Downregulated By microRNA in ABC-DLBCL

Background: The “CBM” complex, composed of the scaffolding protein CARMA1, the adaptor protein BCL10, and the effector protein MALT1, performs multiple pivotal functions as a mediator of antigen receptor-dependent induction of the NF-κB transcription factor and subsequent lymphocyte activation. The effector molecule of the CBM complex, MALT1, regulates downstream signaling via two important functions: first, as a scaffold to recruit and activate components of the canonical NF-κB signaling machinery, and second, as a protease to enzymatically cleave and inactivate multiple substrates including several negative regulators of NF-κB signaling. Deregulated/constitutive activation of MALT1, which can result from gain-of-function mutations in either B cell receptor (BCR) or CARMA1 or from chromosomal translocation involving the MALT1 gene, underlies the pathogenesis of a variety of lymphoid malignancies including activated B-cell type-diffuse large B-cell lymphoma (ABC-DLBCL) and mucosa associated lymphoid tissue (MALT) lymphoma. We recently discovered that G-protein-coupled receptor kinase 2 (GRK2) binds to MALT1 and inhibits MALT1 scaffold and proteolytic activities. Further, we found that knockdown of GRK2 in ABC-DLBCL enhances tumor growth in vivo, suggesting that GRK2 may act as a tumor suppressor in MALT1-dependent lymphomas. Interestingly, we found that GRK2 mRNA levels are markedly lower in a subset of DLBCL cases in comparison to normal B cell controls and that lower GRK2 level is associated with reduced survival in ABC-DLBCL patients. We thus sought to investigate how GRK2 expression is regulated in ABC-DLBCL. Methods and Results: We surveyed 6 published DLBCL sequencing datasets (334 cases total) and identified only one case with a mutation/deletion in the ADRBK1 (GRK2) gene. Thus, we concluded that mutation/deletion could not account for the lower levels of GRK2 in a subset of DLBCL cases. We next investigated other potential mechanisms that could play a role in downregulating GRK2 in ABC-DLBCL, including the impact of microRNA (miRNA). Using DICER-knock-down (KD) and knock-out (KO) HEK 293T cells, we demonstrated that GRK2 protein level significantly increases when miRNA processing is impaired. Next, we performed a bioinformatic analysis which identified two candidate microRNAs, miR-148b and miR-331, whose levels inversely correlate with GRK2 mRNA in ABC-DLBCL patient tumor specimens. These two microRNAs were also identified among the top four candidate hits (miR-125b, miR-148b, miR-125a and miR-331) using mirDIP, a microRNA Data Integration Portal which we used to screen for candidate miRNAs predicted to target the 3'UTR of GRK2. We confirmed direct targeting of GRK2 by three top candidate miRNAs, miR-148b, miR-125a and miR-125b, using a GRK2 3'UTR reporter assay. Notably, we found that expression of these three microRNAs is higher in ABC-DLBCL cells (OCI-Ly3 and TMD8), which demonstrate lower GRK2 mRNA expression in comparison to primary B cells. We showed that overexpression of either miR-125a, miR-125b or miR-148 in OCI-Ly3 and TMD8 cells leads to reduced GRK2 protein level, increased MALT1 scaffolding and proteolytic activities and enhanced ABC-DLBCL cell proliferation. Conversely, anti-miRNA Locked Nucleic Acids (LNA) inhibitors of miR-125a, miR-125b or miR-148 each inhibited ABC-DLBCL cell proliferation and MALT1 activity. Further evaluation of the effect of anti-miRNA LNA inhibitors on tumor progression is under way, using an ABC-DLBCL xenograft animal model. Conclusions: Together, our data show that specific miRNAs (miR-148b, miR-125a and miR-125b) down-regulate GRK2 expression in ABC-DLBCL, which in turn enhances MALT1 scaffolding and proteolytic activities, leading to increased tumor cell proliferation. Future studies investigating miRNA inhibitors which enhance GRK2 expression and thereby suppress MALT1 activity may inform novel strategies for inhibiting MALT1-dependent lymphomagenesis.

Development of a new class of potent and highly selective G protein-coupled receptor kinase 5 inhibitors and structural insight from crystal structures of inhibitor complexes

G protein-coupled receptor kinase 5 (GRK5) is an important drug development target for heart failure, cardiac hypertrophy, and cancer. We have designed and developed a new class of highly selective, potent, and non-covalent GRK5 inhibitors. One of the inhibitors displayed GRK5 IC50 value of 10 nM and exhibited >100,000-fold selectivity over GRK2. The X-ray structure of a ketoamide-derived inhibitor-bound GRK5 showed the formation of a hemithioketal intermediate with active site Cys474 in the GRK5 active site and provided new insights into the ligand-binding site interactions responsible for high selectivity. The current studies serve as an important guide to therapeutic GRK5 inhibitor drug development.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p&amp;lt;.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p&amp;lt;.05). These results (i.e., GRK2 &amp; TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Paroxetine protects against bleomycin-induced pulmonary fibrosis by blocking GRK2/Smad3 pathway.

G protein-coupled receptor kinase-2 (GRK2) is involved in TGF-β1-induced activation of lung fibroblasts, which could give rise to the pathogenesis of pulmonary fibrosis. Paroxetine (PRXT) serves as a selective GRK2 inhibitor which is widely used to treat anxiety and depression for several decades. However, whether PRXT could inhibit TGF-β1-induced activation of lung fibroblasts and combat bleomycin-induced pulmonary fibrosis remains unclear. Here, we investigated the effects of PRXT on pulmonary fibrosis in C57/BL6 caused by bleomycin as well as on the activation of murine primary lung fibroblasts stimulated with TGF-β1. The results demonstrated that PRXT markedly improved the pulmonary function and 21-day survival in bleomycin-induced mice. Meanwhile, PRXT significantly decreased collagen deposition, inflammation, and oxidative stress in lung tissues from bleomycin-induced mice. Furthermore, we found that PRXT could inhibit the protein and mRNA expression of GRK2 and Smad3 in lung tissues from bleomycin-induced mice. In vitro experiments also PRXT could inhibit cell activation and collagen synthesis in a concentration-dependent manner in TGF-β1-induced lung fibroblasts. In addition, we found that Smad3 overexpression by adenovirus transfection could offset anti-fibrotic and antioxidative effects from PRXT in TGF-β1-induced lung fibroblasts, which showed no effects on the protein expression of GRK2. In conclusion, PRXT mediates the inhibition of GRK2, which further blocks the transcription of Smad3 in TGF-β1-induced lung fibroblasts, providing an attractive therapeutic target for pulmonary fibrosis.

Gαs is dispensable for β-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by β2-adrenergic receptor

β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293cells wildtype or CRISPR-Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Therapeutic potential and protective role of GRK6 overexpression in pulmonary arterial hypertension

Abnormal proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key mechanism in the development of pulmonary arterial hypertension (PAH). Signal transducer and activator of transcription 3 (STAT3) signalling plays a critical role in modulating PASMC proliferation, and G-protein-coupled receptor kinase 6 (GRK6) regulates the STAT3 pathway. However, the mechanism underlying the relationship between GRK6 and PAH remains unclear. In this study, we aimed to investigate the role of GRK6 in PAH and determine its potential as a therapeutic target. We utilised hypoxia- and SU5416-induced PAH mouse models and a monocrotaline-induced PAH rat model to analyse the involvement of GRK6. We conducted gain- and loss-of-function experiments using mouse PASMCs. Modulation of GRK6 expression was achieved via a lentiviral vector in vitro and an adeno-associated virus serotype 1 encoding GRK6 in vivo. GRK6 was significantly downregulated in the lung tissues of PAH mice and rats, predominantly in PASMCs. Knockout of GRK6 exacerbated PAH, while both therapeutic and prophylactic overexpression of GRK6 alleviated PAH, as evidenced by a reduction in right ventricular systolic pressure, right ventricular wall to left ventricular wall plus ventricular septum ratio, pulmonary vascular media thickness, and pulmonary vascular muscularisation. Mechanistically, GRK6 overexpression attenuated hypoxia-induced PASMC proliferation and STAT3 phosphorylation. Conversely, knockdown of GRK6 promoted hypoxia-induced proliferation, which was mitigated by a STAT3 inhibitor. Our findings highlight the potential protective and beneficial roles of GRK6 in PAH; we propose a lung-targeted GRK6 gene therapy utilizing adeno-associated virus serotype 1 as a potential treatment approach for patients with PAH.

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

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