G Protein-coupled Receptor Kinase Research Articles

G protein-coupled receptor kinase-2 (GRK-2) controls exploration through neuropeptide signaling in Caenorhabditis elegans.

Animals alter their behavior in manners that depend on environmental conditions as well as their developmental and metabolic states. For example, C. elegans is quiescent during larval molts or during conditions of satiety. By contrast, worms enter an exploration state when removed from food. Sensory perception influences movement quiescence (defined as a lack of body movement), as well as the expression of additional locomotor states in C. elegans that are associated with increased or reduced locomotion activity, such as roaming (exploration behavior) and dwelling (local search). Here we find that movement quiescence is enhanced, and exploration behavior is reduced in G protein-coupled receptor kinase grk-2 mutant animals. grk-2 was previously shown to act in chemosensation, locomotion, and egg-laying behaviors. Using neuron-specific rescuing experiments, we show that GRK-2 acts in multiple ciliated chemosensory neurons to control exploration behavior. grk-2 acts in opposite ways from the cGMP-dependent protein kinase gene egl-4 to control movement quiescence and exploration behavior. Analysis of mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to control exploration behavior. We find that GRK-2 controls exploration behavior in an opposite manner from the neuropeptide receptor NPR-1 and the neuropeptides FLP-1 and FLP-18. Finally, we show that secretion of the FLP-1 neuropeptide is negatively regulated by GRK-2 and that overexpression of FLP-1 reduces exploration behavior. These results define neurons and molecular pathways that modulate movement quiescence and exploration behavior.

Gender differences in GRK2 in cardiovascular diseases and its interactions with estrogen.

G protein-coupled receptor kinase 2 (GRK2) is a multifunctional protein involved in regulating G protein-coupled receptor (GPCR) and non-GPCR signaling in the body. In the cardiovascular system, increased expression of GRK2 has been implicated in the occurrence and development of several cardiovascular diseases (CVDs). Recent studies have found gender differences in GRK2 in the cardiovascular system under physiological and pathological conditions, where GRK2's expression and activity are increased in males than in females. The incidence of CVDs in premenopausal women is lower than in men of the same age, which is related to estrogen levels. Given the shared location of GRK2 and estrogen receptors, estrogen may interact with GRK2 by modulating vital molecules such as calmodulin (CaM), caveolin, RhoA, nitrate oxide (NO), and mouse double minute 2 homolog (Mdm2), via signaling pathways mediated by estrogen's genomic (ERα and ERβ), and non-genomic (GPER) receptors, conferring cardiovascular protection in females. Highlighting the gender differences in GRK2 and understanding its interaction with estrogen in the cardiovascular system is pertinent in treating gender-related CVDs. As a result, this article explores the gender differences of GRK2 in the cardiovascular system and its relationship with estrogen during disease conditions. Estrogen's protective and therapeutic effects and its mechanism on GRK2-related cardiovascular diseases have also been discussed.

Bioinformatics analysis identifies key genes of prognostic value in lung cancer

Lung cancer is the most common human malignancy worldwide and can be divided into different types of carcinomas depending on their pathological features. Advances in medical science and technology have led to the identification of some lung cancer-related marker genes, including EGFR (epidermal growth factor receptor), BRAF (B-Raf proto-oncogene), RAS (RAS proto-oncogene, GTPase) and HER2 (human epidermal growth factor receptor 2). However, the underlying biomarker and key genes associated with different types of lung cancer are still poorly understood. In this study, we analyzed a GEO (Gene Expression Omnibus) dataset and identified 28 upregulated intersection DEGs (different expression genes) and 125 downregulated intersection DEGs among AC (adenocarcinoma), PTC (primary typical carcinoid), PLCC (primary large cell carcinoma), PLCNC (primary large cell lung carcinoma) and PSCLC (primary small cell lung carcinoma). Through PPI (protein-protein interaction) network analysis, we identified 14 genes among the DEGs, namely MFAP4 (microfibril-associated protein 4), PDZD2 (PDZ domain containing 2), FBLN1 (fibulin 1), FBLN5 (fibulin 5), EFEMP1 (EGF containing fibulin extracellular matrix protein 1), KDR (kinase insert domain receptor), S1PR1 (sphingosine-1-phosphate receptor 1), CAV1 (caveolin 1), GRK5 (G protein-coupled receptor kinase 5), EDNRA (endothelin receptor type A), EDNRB (endothelin receptor type B), CALCRL (calcitonin receptor-like receptor), PTGER4 (prostaglandin E receptor 4), and ADRB1 (adrenoceptor beta 1), which were found to be downregulated in different subtypes of lung cancer and associated with poor survival outcomes. In addition, most of the screened DEGs demonstrated good predictive ability in LUAD (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma). Among them, MFAP4 was found to promote cell proliferation while also suppressing cell migration and angiogenesis. In summary, we propose MFAP4, PDZD2, FBLN1, FBLN5, EFEMP1, KDR, S1PR1, CAV1, GRK5, EDNRA, EDNRB, CALCRL, PTGER4 and ADRB1 as potential prognostic markers in lung cancer patients.

G Protein-Coupled Receptor Kinases Take Central Stage

The relevance of the family of G protein-coupled receptor kinases (GRKs) is based on its key participation in the regulation and intracellular dynamics of the largest family of membrane receptors, namely G protein-coupled receptors (GPCRs) [...].

Cardiac β-Adrenoceptor Signaling: The New Insight on An Old Target in the Therapy of Cardiovascular Disease

Review Cardiac β-Adrenoceptor Signaling: The New Insight on An Old Target in the Therapy of Cardiovascular Disease Ying Song 1, Anthony Yiu-Ho Woo 2,*, Yan Zhang 3,4,*, and Ruiping Xiao 5,6,7,8 1 Center of Basic Medical Research, Institute of Medical Innovation and Research, Peking University Third Hospital, Beijing, 100191, China. 2 School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, China. 3 Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovascular Sciences, School of Basic Medical Sciences, Ministry of Education, Peking University Health Science Center, Beijing, 100191, China. 4 Beijing Key Laboratory of Cardiovascular Receptors Research, Peking University, Beijing, 100191, China. 5 State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, 100871, China. 6 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. 7 Beijing City Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing, 100871, China. 8 PKU-Nanjing Joint Institute of Translational Medicine, Nanjing, 210000, China. * Correspondence: yiuhowoo@syphu.edu.cn (Anthony Yiu-Ho Woo), Tel.: +86-24-23986375; zhangyan9876@pku.edu.cn (Yan Zhang), Tel.: +86-10-82805945. Received: 19 October 2022 Accepted: 28 October 2022 Published: 21 December 2022 Abstract: A variety of G protein-coupled receptors (GPCRs) are involved in the regulation of cardiovascular function. The β-adrenoceptors (β-ARs), with three subtypes, are the dominant receptor species in the heart, in which the β1-AR and the β2-AR are considered functional. Stimulation of the β-ARs produces myocardial inotropy via activation of the Gs-cAMP-PKA signaling cascade. Prolonged stimulation of the β1-AR is cardiac harmful because the stimulated β1-AR couples only to Gs proteins and it mediates a cardiotoxic signal. On the other hand, the β2-AR couples dually to both Gs and Gi proteins and the β2-AR-Gi pathway is antiapoptotic. The activated Gi signal also counteracts the β-AR-Gs-promoted positive inotropic effect. Other key players in cardiac β-AR signaling include Ca2+/calmodulin-dependent protein kinases (CaMKs), GPCR kinases (GRKs), β-arrestins and phosphodiesterases. During heart failure, excessive sympathetic stimulation results in the activation of the cardiotoxic β1-AR-CaMKIIδ pathway and the upregulation of GRK2 and Gi in the heart. GRK2 promotes the desensitization of β-ARs and enhances a β2-AR-mediated Gi signaling. These signal transduction processes accompanying the downregulation of the β1-AR are involved in cardiac dysfunction, maladaptive cardiac remodeling, and the progression of chronic heart failure. β-Blockers are widely used in the treatment of cardiovascular disease. They have established their position as one of the “four pillars of heart failure” thirty years ago. In the present review, we provide an overview of the recent progress in the basic research of GPCRs focusing on cardiac β-AR signal transduction.

596 TARGETING GRK2 TO PREVENT AORTIC VALVE CALCIFICATION

Abstract Introduction Calcific aortic valve stenosis (CAVS) is a clinically relevant issue due to the lack of drugs for prevention or treatment. CAVS is driven by endothelial dysfunction and inflammation. A novel therapeutic strategy should target specific molecules involved in the regulation of both endothelial function and immune responses. G protein-coupled receptor kinase 2 (GRK2) regulates desensitization and downregulation of G protein-coupled receptors, and is able to interact with an extensive repertoire of proteins. We previously demonstrated that the lack of this protein in the endothelium promotes vascular inflammation and atherosclerosis in mice due to increased mitochondrial reactive oxygen species (ROS). Aim This study aimed to evaluate the role of GRK2 in Aortic Valve Calcification (AVC) by in vivo and in vitro studies. Methods To reach our purpose we first evaluated the expression of GRK2 in aortic valve of patient displaying fibrotic or calcific lesions of valve leaflets. Then, we evaluated GRK2 expression in mitochondria fraction from EC isolated from CAVS patients (VEC) vs control ECs upon Angiotensin II (AngII) stimulation (1µM). We also performed histological analysis by using 12 months old mice with selective endothelial knock-out of GRK2 (Tie2CRE-GRK2fl/fl) compared to control (GRK2fl/fl) to evaluate the presence of microcalcification in the aortic valve. Finally, we cloned a small sequence of the PH domain of ßARKct into the pcDNA3 to induce GRK2 localization into mitochondria. Results Immunofluorescence staining of aortic valve leaflets revealed that GRK2expression is more abundant in calcific lesion instead of fibrotic valve. In vitro, we observed that AngII is able to upregulate GRK2 mitochondrial localization in a time-dependent manner in ECs. Otherwise in VEC the stimulation with Ang II is not able to further induce GRK2 mitochondrial localization. Histological analysis revealed that Tie2CRE-GRK2fl/fl mice of 12 months-old display presence of microcalcification, more pronounced than GRK2fl/fl thus demonstrating that the lack of GRK2 in the EC accelerates the calcific degeneration of the aortic valve in mice. Finally, we cloned several small sequences of the PH domain of ßARKct into the pcDNA3.1 plasmid, named as PH#1-4. We found that the transfection into HEK293 cells of the PH#3 potently increased GRK2 localization into the mitochondria as compared to ßARKct, PH4, and pcDNA3.1 as control. PH3 also determined increased biogenesis and reduced ROS production after AngII stimulation. These data support the concept that a smaller portion of the PH domain of ßARKct can reproduce its biological effect Conclusions In conclusion, our data suggest a direct involvement of GRK2 in the pathogenesis of CAVS. Intracellular re-localization of GRK2 could be a novel strategy to prevent AVC in a pathophysiological condition such as ageing

Genome-wide identification of SNPs associated with body weight in yak

BackgroundThe yak is the most important livestock in the Qinghai-Tibet Plateau, and body weight directly affects the economic values of yak. Up to date, the genome-wide profiling of single-nucleotide polymorphisms (SNPs) associating with body weight has not been reported in yak. In the present study, the SNPs in 480 yaks from three breeds were analyzed using the commercial high-density (600 K) yak SNP chips.ResultsThe results identified 12 and 4 SNPs potentially associated with body weight in male and female yaks, respectively. Among them, 9 and 2 SNPs showed significant difference in yak body weight between different genotypes at each locus in male and female yaks, respectively. Further exploration found 33 coding genes within the 100 kbp upstream or downstream to the SNP loci, which might be potentially affected by the variation of SNPs. Among them, G protein-coupled receptor kinase 4 (GRK4) might be potentially affected by the SNP AX-174555047, which has been reported to affect the functioning of two body-weight associated hormones (parathyroid hormone, PTH, and adrenomedullin, ADM). Determination of PTH and ADM levels in yak revealed positive relationship between PTH level and body weight, negative relationship between ADM level and body weight along with the variation of AX-174555047 mutation.ConclusionsThese results suggested that the SNP AX-174555047 might potentially affect body weight through mediating GRK4 expression and then PTH and ADM functioning. Thus, the SNP AX-174555047 might be used as a biomarker for molecular breeding of yak. More investigations are required to validate this point.

Control of Gαq signaling dynamics and GPCR cross-talk by GRKs.

Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.

Prostaglandin EP3 receptor activation is antinociceptive in sensory neurons via PI3Kγ, AMPK and GRK2.

Prostaglandin E2 is considered a major mediator of inflammatory pain, by acting on neuronal Gs protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is Gi protein-coupled and antagonizes the pronociceptive prostaglandin E2 effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons. Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)-/- , and PI3Kγkinase dead (KD)/KD mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry. Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding. Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E2 . These new insights hold the potential for a novel approach in pain therapy.

Vinculin phosphorylation impairs vascular endothelial junctions promoting atherosclerosis.

Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated. Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs). A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE-/-) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE-/- mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis. The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis.

A Common Variant in GRK5 Reveals a PAR1-Specific Mechanism for Its Regulation of Platelet Function

Two separate genome wide association studies (GWAS), recently conducted by our group and another group, have identified the variant rs10886430 in the G protein-coupled receptor kinase 5 (GRK5) gene with a minor allele frequency of ~14% in Caucasians. Both studies show that the variant resides in a megakaryocyte-specific enhancer that alters the binding of the transcription factors GATA1 and MEIS1. In contrast to the major allele (A), the minor allele (G) is associated with decreased GRK5 mRNA expression, increased platelet reactivity, and greater risk of cardiovascular disease. However, the effect of the variant on protein expression has not been investigated by either group. In addition, there is an inconsistency between the findings of each of the studies regarding the specific role of GRK5 in thrombin receptor PAR1- and PAR4-mediated platelet activation. Further studies are needed to clarify whether, and how, GRK5 regulates platelet activity via PAR1, PAR4, or both. Here we introduce the A>G mutation into a megakaryoblastic cell line, Meg-01, by CRISPR-Cas9 genome editing. GRK5 protein expression is decreased by 80% in A>G mutant Meg-01 cells compared to WT control cells. To determine whether GRK5 regulates PAR1, PAR4, or both, we have isolated human platelets and specifically blocked GRK5 activity with compound CCG273261 to compare platelet responses to PAR1- and PAR4-specific agonists. Our data show that pharmacological inhibition of GRK5 increases markers of platelet activation such as integrin activation and P-selectin surface exposure in response to PAR1 receptor agonist peptide. Platelet activation in response to PAR4 receptor agonist peptide is normal. Furthermore, this inhibition increases Ca2+ mobilization in response to PAR1 receptor agonist, but not to PAR4 receptor agonist. Consistent with the results of our functional assays, mass spectrometry analysis revealed an interaction between GRK5 and PAR1 but did not detect an interaction with PAR4. Collectively, our data show that 1) this GRK5 intronic variant regulates GRK5 expression at both transcriptional and translational levels, 2) GRK5 preferentially regulates PAR-1 over PAR4-mediated signaling, and 3) GRK5 has a binding interaction with PAR1 (Fig 1). Our findings will enable us to develop superior therapeutics for cardiovascular risk groups especially those individuals who carry the intronic variant in GRK5 and as a result are more likely to experience thrombotic events. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

Abstract 11977: Dapagliflozin Downregulates Adrenal G Protein-Coupled Receptor-Kinase-2 to Exert Sympatholysis in Heart Failure

Introduction: Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor exerting several cardiorenal protective effects independently of blood glucose regulation. Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity, especially in heart failure (HF). GRK2 severely dysregulates adrenal sympatho-inhibitory α 2 -adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Hypothesis: SGLT2 inhibition with dapagliflozin lowers SNS hyperactivity in part by opposing adrenal GRK2 actions. Methods: We used the rat pheochromocytoma PC12 cell line expressing human α 2A AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Results: 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<.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, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2 AR 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<.05). GRK2 and TH downregulation were also observed in PC12 cells treated with 5 μM dapagliflozin for 24 hours. Importantly, α 2 AR-dependent inhibition of CA secretion was markedly enhanced 24 hrs post-dapagliflozin application. This was due to reduced GRK2-dependent receptor desensitization induced by dapagliflozin, since this effect was abolished in PC12 cells overexpressing GRK2. Conclusions: Dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, thus reducing CA biosynthesis, and GRK2, thus reducing α 2 AR desensitization to lower CA secretion. This adrenal-mediated sympatholysis may be part of the increasingly apparent cardiovascular benefits of SGLT2 inhibitors in human HF irrespective of diabetic status.

Abstract 11967: Microrna-150 Overexpression Blunts Maladaptive Cardiac Remodeling in Mice With Cardiomyocyte-Specific Loss of β-Arrestin-Mediated β 1 -Adrenergic Receptor Signaling

Background and Aims: β-arrestins transduce alternative pathways independent of G protein signaling, a concept known as β-arrestin-biased signaling that is promoted by G protein-coupled receptor kinase (GRK) 5/6 phosphorylation on β-adrenergic receptors (βARs). The β 1 AR is found primarily in the heart (mainly in cardiomyocytes [CMs]) and β-arrestin-mediated β 1 AR signaling elicits cardioprotection by CM survival. We showed that a small noncoding RNA (ncRNA), microRNA-150 (miR-150) is upregulated by the β-blocker carvedilol acting through β-arrestin-mediated β 1 AR signaling, and that CM miR-150 confers cardioprotection (see Figure). Here, we investigate if miR-150 in part rescues maladaptive cardiac remodeling seen in mice with CM-specific abrogation of β-arrestin-mediated β 1 AR signaling. Methods and Results: Using CM-specific transgenic (TG) mice expressing a mutant β 1 AR (i.e. GRK - β 1 AR exhibiting impairment in β-arrestin-mediated β 1 AR signaling and miR-150 activation by carvedilol), we first develop a novel double TG (DTG) mouse line overexpressing miR-150 and CM-specific GRK - β 1 AR. We demonstrate that miR-150 improves maladaptive cardiac remodeling observed in CM-specific GRK - β 1 AR TG mice after chronic catecholamine stimulation. Genome-wide circular RNA, long ncRNA (lncRNA) and mRNA profiling analyses in DTG mouse hearts unveil a subset of cardiac ncRNAs and genes as heretofore unrecognized mechanisms for miR-150’s beneficial effects. We further show that lncRNA Gm41664 and Gdap1l1 have miR-150 binding sites, and that they are direct novel upstream and downstream regulators of miR-150. Lastly, protective roles of miR-150 in CMs are in part attributed to the functional repression of pro-apoptotic Gdap1l1 and are mitigated by pro-apoptotic Gm41664 (see Figure). Conclusions: Our findings support that miR-150 contributes significantly to β 1 AR/β-arrestin-mediated cardioprotection by regulating a unique ncRNA and gene signature in CMs.

Grk7 but not Grk1 undergoes cAMP-dependent phosphorylation in zebrafish cone photoreceptors and mediates cone photoresponse recovery to elevated cAMP

In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases (GRKs) is essential for sustained visual function. Previous invitro analysis demonstrated that GRK1 and GRK7 are phosphorylated by PKA, resulting in a reduced capacity to phosphorylate rhodopsin. Invivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones invivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation invivo. Lastly, immunoblot analyses of rod grk1a-/- and cone grk1b-/- zebrafish and Nrl-/- mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation invivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.

M2 macrophages-derived exosomal miR-3917 promotes the progression of lung cancer via targeting GRK6.

Macrophages in the tumor microenvironment (TME) can serve as potential targets for therapeutic intervention. The aim of this study was to investigate the molecular mechanism by which M2 macrophage-derived exosomes (M2-Ex) affect lung cancer progression through miRNA transport. The THP-1 cells were differentiated into M0 and M2 macrophages. M2-Ex were isolated and identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Cancer tissues and adjacent tissues of non-small-cell lung cancer (NSCLC) patients were collected. H1299 and A549 cells were co-cultured with M2-Ex. Subcutaneous xenograft mouse model was established. miR-3917 is highly expressed in lung cancer tissues and M2-Ex. Interference of miR-3917 in M2-Ex inhibits H1299 cell proliferation, migration and invasion, while overexpression of miR-3917 had the opposite effect in A549 cells. M2-Ex promote tumor growth by delivering miR-3917 in vivo. miR-3917 could target G protein-coupled receptor kinase 6 (GRK6), and interference of miR-3917 in M2-Ex inhibits H1299 cells proliferation, migration and invasion by up-regulating GRK6 level, while overexpression of miR-3917 had the opposite effect in A549 cells. M2-Ex can transfer miR-3917 into lung cancer cells and promote lung cancer progression, providing theoretical basis for the diagnosis and effective treatment of lung cancer.

GRK2 inhibitors, paroxetine and CCG258747, attenuate IgE-mediated anaphylaxis but activate mast cells via MRGPRX2 and MRGPRB2.

G protein-coupled receptor (GPCR) kinase 2 (GRK2), which phosphorylates agonist-occupied GPCRs to promote their desensitization, has been investigated as an attractive therapeutic target for cardiovascular and metabolic diseases. Several GRK2-targeted inhibition strategies have been reported including the use of direct pharmacological inhibitors such as paroxetine (a widely prescribed antidepressant) and its analogs such as compound CCG258747. Cross-linking of high affinity IgE receptor (FcϵRI) on mast cells (MCs) and the resulting degranulation causes anaphylaxis and allergic asthma. Using gene silencing strategy, we recently showed that GRK2 contributes to FcεRI signaling and MC degranulation. The purpose of this study was to determine if the GRK2 inhibitors paroxetine and CCG258747 modulate FcεRI-mediated MC responses in vitro and in vivo. Utilizing rat basophilic leukemia (RBL-2H3) cells and primary mouse lung MCs (LMCs), we found that paroxetine and CCG258747 inhibit FcϵRI-mediated calcium mobilization and degranulation. Furthermore, intravenous administration of paroxetine and CCG258747 in mice resulted in substantial reduction of IgE-mediated passive cutaneous anaphylaxis. Unlike LMCs, human cutaneous MCs abundantly express a novel GPCR known as MRGPRX2 (mouse; MRGPRB2). We found that in contrast to their inhibitory effects on FcεRI-mediated MC responses, both paroxetine and CCG258747 induce calcium mobilization and degranulation in RBL-2H3 cells stably expressing MRGPRX2 but not in untransfected cells. Furthermore, paroxetine and CCG258747 induced degranulation in peritoneal MCs from Wild-type (WT) mice in vitro and caused increased cutaneous vascular permeability in vivo, but these responses were substantially reduced in Mrgprb2−/− mice. Additionally, upon intradermal injection, paroxetine also induced neutrophil recruitment in WT but not Mrgprb2−/− mice. These findings suggest that in addition to their potential therapeutic utility against cardiovascular and metabolic disorders, paroxetine-based GRK2-inhibitors may serve to modulate IgE-mediated anaphylaxis and to enhance cutaneous host defense by harnessing MC’s immunomodulatory property through the activation of MRGPRX2/MRGPRB2.

Crosstalk between CXCR4/ACKR3 and EGFR Signaling in Breast Cancer Cells

A better understanding of the complex crosstalk among key receptors and signaling pathways involved in cancer progression is needed to improve current therapies. We have investigated in cell models representative of the major subtypes of breast cancer (BC) the interplay between the chemokine CXCL12/CXCR4/ACKR3 and EGF receptor (EGFR) family signaling cascades. These cell lines display a high heterogeneity in expression profiles of CXCR4/ACKR3 chemokine receptors, with a predominant intracellular localization and different proportions of cell surface CXCR4+, ACKR3+ or double-positive cell subpopulations, and display an overall modest activation of oncogenic pathways in response to exogenous CXCL12 alone. Interestingly, we find that in MDA-MB-361 (luminal B subtype, Her2-overexpressing), but not in MCF7 (luminal A) or MDA-MB-231 (triple negative) cells, CXCR4/ACKR3 and EGFR receptor families share signaling components and crosstalk mechanisms to concurrently promote ERK1/2 activation, with a key involvement of the G protein-coupled receptor kinase 2 (GRK2) signaling hub and the cytosolic tyrosine kinase Src. Our findings suggest that in certain BC subtypes, a relevant cooperation between CXCR4/ACKR3 and growth factor receptors takes place to integrate concurrent signals emanating from the tumor microenvironment and foster cancer progression.

Emerging Therapeutic Targets for Heart Failure.

Heart failure is a global epidemic that affects at least 26 million individuals globally and is becoming more prevalent. Despite advances in treatment strategies, survival and symptom management in individuals with heart failure remain exceptionally low. This review discusses emerging targets for the treatment of heart failure. Recently, a number of targets are being investigated as prospective treatment possibilities for heart failure. These include targets like Runx1 transcription factor (RUNX1), milk fact globule-EFG factor 8 (MFGE8) protein and enzymes such as neuraminidase 1 (NEU1), G protein-coupled receptor kinase 5 (GRK5), G protein-coupled oestrogen receptor 1 (GPER1), urotensin-II receptor (UTR), cluster of differentiation 47 (CD47) and relaxin receptor 1 (RXFP1). On a worldwide level, heart failure is a developing epidemic with substantial morbidity and death. The number of individuals diagnosed with chronic heart failure is rising, and it is anticipated to surge by 46% by 2030. Appropriate heart failure treatment can have the greatest influence on prolonging patients' lives in the coming year. Targets discussed in this review may provide new therapeutic approaches for the treatment of heart failure.