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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.

Efficient induction of humoral immune responses depends on the orchestrated migration of B cells within lymphoid organs, which is governed by G protein-coupled receptors (GPCRs) responding to chemoattractants, represented by chemokines. After ligand binding, GPCRs are phosphorylated by different GPCR kinases (GRKs) at distinct sites on the receptor C termini, which dictates functional outcomes of β-arrestin-mediated signaling, ranging from receptor inactivation to effector molecule activation. However, the molecular mechanisms by which individual GRKs are selectively targeted to GPCRs have been poorly understood. Our recent study revealed that a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and 8 (the COMMD3/8 complex) functions as an adaptor that recruits a specific GRK to chemoattractant receptors and plays an important role in the control of B-cell migration during humoral immune responses. In this review, we summarize the current understanding of chemoattractant receptor signaling in the context of humoral immunity and discuss the potential of the COMMD3/8 complex as a therapeutic target for autoimmune diseases. [ABSTRACT FROM AUTHOR]

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

G protein‐coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310. Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308, leading to a shift in ligand affinity from a high‐to‐low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1‐engaged conformations of β‐arrestin and subsequent alterations in signal transduction. Stimulation of the high‐affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2‐GRK pathway, resulting in a β‐arrestin‐bound low‐affinity state. This configuration, referred to as the "low‐LTB4‐induced complex," necessitates the finger loop region and the phosphoinositide‐binding motif of β‐arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low‐affinity BLT1 again binds to the ligand and triggers the generation of the low‐LTB4‐induced complex into a different form termed "high‐LTB4‐induced complex." This change is propelled by The308‐phosphorylation‐dependent basal phosphorylation by PKCs. Within the high‐LTB4‐induced complex, β‐arrestin adapts a unique configuration that involves additional N domain interaction to the low‐affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein β‐arrestins perform distinct functions. [ABSTRACT FROM AUTHOR]

The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R–β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R–β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation. [ABSTRACT FROM AUTHOR]

Introduction: Dysfunction of the endothelium impairs its’ protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses.Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells.Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells.Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.

The solubility of hydrogen sulfide (H2S) in ionic liquids was modeled at various temperatures and partial gas pressures using two approaches. The first approach as a thermodynamic model, is a generic Redlich–Kwong (GRK) cubic EoS and the other is a Group method of data handling (GMDH) as an artificial intelligence approach. Results of modeling using both approaches were obtained by estimation of the percent relative deviation as well as the regression coefficients (R2) and root mean square error. The GMDH model in this paper showed it is a reliable development to be an alternative method for the thermodynamic approach in modeling of H2S solubility data in ionic liquids in the extended ranges of pressure and temperatures. [ABSTRACT FROM AUTHOR]

Indonesia berkomitmen menurunkan emisi gas rumah kaca (GRK) melalui skema nationally determined contribution (NDC). Target yang hendak dicapai untuk menurunkan emisi GRK yaitu 29% melalui skema business as usual (BAU) atau 41% dengan bantuan internasional. Target ambisius ini membutuhkan transformasi, baik dalam sistem energi maupun sistem pangan dan tata guna lahan, yang perlu mengatasi potensi pertukaran di antara sejumlah besar target, seperti ketahanan pangan, ketahanan energi, menghindari deforestasi, konservasi keanekaragaman hayati, persaingan penggunaan lahan, serta penggunaan air tawar. Mitigasi dan adaptasi memiliki peran yang saling melengkapi dalam merespons perubahan iklim yang dilakukan pada skala temporal dan spasial. Makalah ini bertujuan untuk melakukan simulasi dan prediksi pada emisi manure management penghasil CO2eq dengan menggunakan metode pembelajaran mesin long short-term memory (LSTM) dan gated recurrent unit (GRU). Arsitektur lapisan tersembunyi yang digunakan berjumlah enam kombinasi, sedangkan dataset diperoleh dari repositori fao.org. Optimizer yang digunakan dalam makalah ini yaitu RMSprop, dengan graphical user interface menggunakan dashboard Streamlit. Hasil dari penelitian ini yaitu; (a) cattle dengan lima belas epoch menggunakan lapisan tersembunyi empat kombinasi (LSTM, GRU, LSTM, GRU) menghasilkan RMSE 450,601; (b) non-dairy cattle dengan lima belas epoch dan satu lapisan tersembunyi (GRU, GRU, GRU, GRU) menghasilkan nilai RMSE 361,421; (c) poultry birds dengan nilai dua belas epoch dan tiga lapisan tersembunyi (GRU, GRU, LSTM, LSTM) menghasilkan nilai RMSE 341,429. Tantangan yang dihadapi adalah penentuan epoch, kombinasi lapisan tersembunyi, serta karakteristik dataset yang berjumlah relatif sedikit. Hasil dari penelitian ini diharapkan dapat memberikan nilai tambah untuk mengembangkan alat dan model pendukung keputusan yang lebih baik untuk menilai tren emisi di sektor peternakan serta untuk mengembangkan strategi mitigasi emisi CO2eq yang mengarah pada praktik pengelolaan pupuk yang berkelanjutan.

GPCRs are inherently flexible molecules existing in an equilibrium of multiple conformations. Binding of GPCR agonists shifts this equilibrium. Certain agonists can increase the fraction of active-like conformations that predispose the receptor to coupling to a particular signal transducer or a select group of transducers. Such agonists are called biased, in contrast to balanced agonists that facilitate signaling via all transducers the receptor couples to. These biased agonists preferentially channel the signaling of a GPCR to particular G proteins, GRKs, or arrestins. Preferential activation of particular G protein or arrestin subtypes can be beneficial, as it would reduce unwanted on-target side effects, widening the therapeutic window. However, biasing GPCRs has two important limitations: a) complete bias is impossible due to inherent flexibility of GPCRs; b) receptor-independent functions of signal transducer proteins cannot be directly affected by GPCR ligands or differential receptor barcoding by GRK phosphorylation. This article is part of the Special Issue on "Ligand Bias". • Complete bias of GPCR signaling is impossible. • Bound signal transducers significantly affect GPCR conformation. • Preferential coupling to different G proteins constitutes bias. • Coupling to different arrestin subtypes constitutes bia. [ABSTRACT FROM AUTHOR]

Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, rods operate over many log units of light intensity. Several processes inside photoreceptor cells make this incredible light adaptation possible. Here, we added to our previously developed, fully space resolved biophysical model of rod phototransduction, some of the mechanisms that play significant roles in shaping the rod response under high illumination levels: the function of RGS9 in shutting off G protein transducin, and calcium dependences of the phosphorylation rates of activated rhodopsin, of the binding of cGMP to the lightregulated ion channel, and of two membrane guanylate cyclase activities. A well stirred version of this model captured the responses to bright, saturating flashes in WT and mutant mouse rods and was used to explain "Pepperberg plots," that graph the time during which the response is saturated against the natural logarithm of flash strength for bright flashes. At the lower end of the range, saturation time increases linearly with the natural logarithm of flash strength. The slope of the relation (τD) is dictated by the time constant of the rate-limiting (slowest) step in the shutoff of the phototransduction cascade, which is the hydrolysis of GTP by transducin. We characterized mathematically the X-intercept (Φo) which is the number of photoisomerizations that just saturates the rod response. It has been observed that for flash strengths exceeding a few thousand photoisomerizations, the curves depart from linearity. Modeling showed that the "upward bend" for very bright flash intensities could be explained by the dynamics of RGS9 complex and further predicted that there would be a plateau at flash strengths giving rise to more than ~107 photoisomerizations due to activation of all available PDE. The model accurately described alterations in saturation behavior of mutant murine rods resulting from transgenic perturbations of the cascade targeting membrane guanylate cyclase activity, and expression levels of GRK, RGS9, and PDE. Experimental results from rods expressing a mutant light-regulated channel purported to lack calmodulin regulation deviated from model predictions, suggesting that there were other factors at play. [ABSTRACT FROM AUTHOR]

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.

The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R–β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R–β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation.

This study investigated the release and proteomic profile of tissue factor microparticles (TFMPs) prospectively (up to 6 months) following a myocardial infarction (MI) in a chronic porcine model to establish their utility in tracking cellular level activities that predict physiologic outcomes. Our animal groups (n = 6 to 8 each) consisted of control, noninfarcted (negative control); infarcted only (positive control); and infarcted animals treated with cardiac resynchronization therapy (CRT) and a β-blocker (BB) (metoprolol succinate). The authors found different protein profiles in TFMPs between the control, infarcted only group, and the CRT + BB treated group with predictive impact on the outward phenotype of pathological remodeling after an MI within and between groups. This novel approach of monitoring cellular level activities by profiling the content of TFMPs has the potential of addressing a shortfall of the current crop of cardiac biomarkers, which is the inability to capture composite molecular changes associated with chronic maladaptive signaling in a spatial and temporal manner.

[Display omitted] Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB 1 receptor (CB 1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB 1 , and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB 1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and their signal transduction is tightly regulated by GPCR kinases (GRKs) and β-arrestins. In this review, we discuss novel aspects of the regulatory GRK/β-arrestin system. Therefore, we briefly revise the origin of the “barcode” hypothesis for GPCR/β-arrestin interactions, which states that β-arrestins recognize different receptor phosphorylation states to induce specific functions. We emphasize two important parameters which may influence resulting GPCR phosphorylation patterns: (A) direct GPCR–GRK interactions and (B) tissue-specific expression and availability of GRKs and β-arrestins. In most studies that focus on the molecular mechanisms of GPCR regulation, these expression profiles are underappreciated. Hence we analyzed expression data for GRKs and β-arrestins in 61 tissues annotated in the Human Protein Atlas. We present our analysis in the context of pathophysiological dysregulation of the GPCR/GRK/β-arrestin system. This tissue-specific point of view might be the key to unraveling the individual impact of different GRK isoforms on GPCR regulation.

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.

JC polyomavirus (JCPyV) infects the majority of the population, establishing a lifelong, asymptomatic infection in the kidney of healthy individuals. People who become severely immunocompromised may experience JCPyV reactivation, which can cause progressive multifocal leukoencephalopathy (PML), a neurodegenerative disease. Due to a lack of therapeutic options, PML results in fatality or signifi- cant debilitation among affected individuals. Cellular internalization of JCPyV is mediated by serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs) via clathrin-mediated endocytosis. The JCPyV entry process requires the clathrin-scaffolding proteins b-arrestin, adaptor protein 2 (AP2), and dynamin. Furthermore, a β-arrestininteracting domain, the Ala-Ser-Lys (ASK) motif, within the C terminus of 5-HT2AR is important for JCPyV internalization and infection. Interestingly, 5-HT2R subtypes A, B, and C equally support JCPyV entry and infection, and all subtypes contain an ASK motif, suggesting a conserved mechanism for viral entry. However, the role of the 5- HT2R ASK motifs and the activation of b-arrestin-associated proteins during internalization have not been fully elucidated. Through mutagenesis, the ASK motifs within 5-HT2BR and 5-HT2CR were identified as being critical for JCPyV internalization and infectivity. Furthermore, by using biochemical pulldown techniques, mutagenesis of the ASK motifs in 5-HT2BR and 5-HT2CR resulted in reduced b-arrestin binding. When small-molecule chemical inhibitors and RNA interference were used, G protein receptor kinase 2 (GRK2) was determined to be required for JCPyV internalization and infection by mediating interactions between b-arrestin and the ASK motif of 5-HT2Rs. These findings demonstrate that GRK2 and b-arrestin interactions with 5-HT2Rs are critical for JCPyV entry by clathrin-mediated endocytosis and the resultant infection. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is a leading cause of morbidity and mortality across the world. Cardiac fibrosis is associated with HF progression. Fibrosis is characterized by the excessive accumulation of extracellular matrix components. This is a physiological response to tissue injury. However, uncontrolled fibrosis leads to adverse cardiac remodeling and contributes significantly to cardiac dysfunction. Fibroblasts (FBs) are the primary drivers of myocardial fibrosis. However, until recently, FBs were thought to play a secondary role in cardiac pathophysiology. This review article will present the evolving story of fibroblast biology and fibrosis in cardiac diseases, emphasizing their recent shift from a supporting to a leading role in our understanding of the pathogenesis of cardiac diseases. Indeed, this story only became possible because of the emergence of FB-specific mouse models. This study includes an update on the advancements in the generation of FB-specific mouse models. Regarding the underlying mechanisms of myocardial fibrosis, we will focus on the pathways that have been validated using FB-specific, in vivo mouse models. These pathways include the TGF-β/SMAD3, p38 MAPK, Wnt/β-Catenin, G-protein-coupled receptor kinase (GRK), and Hippo signaling. A better understanding of the mechanisms underlying fibroblast activation and fibrosis may provide a novel therapeutic target for the management of adverse fibrotic remodeling in the diseased heart.

Every animal species expresses hundreds of different G protein-coupled receptors (GPCRs) that respond to a wide variety of external stimuli. GPCRs-driven signaling pathways are involved in pretty much every physiological function and in many pathologies. Therefore, GPCRs are targeted by about a third of clinically used drugs. The signaling of most GPCRs via G proteins is terminated by the phosphorylation of active receptor by specific kinases (GPCR kinases, or GRKs) and subsequent binding of arrestin proteins, that selectively recognize active phosphorylated receptors. In addition, GRKs and arrestins play a role in multiple signaling pathways in the cell, both GPCR-initiated and receptor-independent. Here we focus on the mechanisms of GRK- and arrestin-mediated regulation of GPCR signaling, which includes homologous desensitization and redirection of signaling to additional pathways by bound arrestins.

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3- methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4- chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with β-arrestin1 and that β-arrestin1 interacted with the β2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either β-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the β-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G proteincoupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells. [ABSTRACT FROM AUTHOR]

The Raf kinase inhibitor protein (RKIP) activates β-adrenoceptors (β-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated β-AR activation, chronic activation of β-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated β-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on β-AR to explore new strategies for β-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 – not GRK5 – as well as several RKIP binding sites within their N-termini (GRK21−185 and GRK31−185). Overexpression of these N-termini prevented β 2 -AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of β 2 -AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on β-AR signaling in HEK293 cells and in cardiomyocytes by their binding to β 2 -AR and, thus, provide important insights for the development of new strategies to modulate β 2 -AR signaling. • RKIP has a well-tolerated positive inotropic effect via β-adrenoceptor activation. • RKIP binds N-termini of cardiac GRK2 and GRK3 – but not GRK5. • GRK2/3 N-termini simulate RKIP effects on β-AR signaling by direct receptor interaction. • Interference strategy using GRK peptides seems to be a promising tool for receptor regulation. [ABSTRACT FROM AUTHOR]

Neurotensin (NT) is a 13-amino acid peptide acting as a neuromodulator in the CNS. NT immunoreactive cell bodies, synaptic terminals and receptors (NTS) are intimately associated with the dopaminergic system. In fact, NT exerts a stimulatory action on the dopaminergic (DAergic) neurons of substantia nigra pars compacta (SNpc) and ventral tegmental area by activating a mixed cation conductance, reducing D 2 -autoinhibition and modulating NMDA and AMPA transmission. In the present work, we describe an inhibitory effect of NT on metabotropic glutamate receptor I (mGluR I) actions in rat SNpc DAergic neurons. NTS and mGluR I share the same G αq/11 -PLC-IP 3 -Ca2+ intracellular pathway which causes either activation of unspecific cationic conductance or intracellular Ca2+ accumulation. We find that NT inhibits both inward current and the associated intracellular calcium elevation, elicited by the selective mGluR I agonist S-DHPG, in a concentration-dependent manner. This effect is mediated by type 1/2 NT receptors (NTS 1/2), as revealed by pharmacological analysis. Activation of other metabotropic receptors, such as muscarinic and GABA B , does not inhibit mGluR I inward currents. PKC, MEK 1–2, calcineurin, clathrin-dependent endocytosis and intracellular Ca2+ elevation are not involved in the NT-mediated modulation of mGluR I responses. Interestingly, inhibition of G-protein coupled receptor kinases (GRKs) 2/3 exacerbates the NT-induced mGluR I inhibition while sustaining the NT-induced inward current during repeated agonist stimulation. These data suggest that GRKs are key molecules regulating either the NT excitation or the cross-talk between NTS 1/2 and mGluR I in DAergic neurons of rat midbrain by tuning the degree of NTS 1/2 desensitization. Image 1 • Neurotensin (NT) inhibits mGluR I responses in dopaminergic neurons of rat substantia nigra pars compacta. • The NT-mediated inhibition of mGluR I involves NTS 1/2 receptors. • The NT-mediated inhibition of mGluR I is Ca2+-independent and does not involve common Gq-activated intracellular pathways. • NTS 1/2 undergo functional desensitization by G-protein coupled receptor kinases (GRKs) 2/3. • Inhibition of GRKs 2/3 strongly potentiates the NT-mediated inhibition of mGluR I responses. [ABSTRACT FROM AUTHOR]

Every animal species expresses hundreds of different G protein-coupled receptors (GPCRs) that respond to a wide variety of external stimuli. GPCRs-driven signaling pathways are involved in pretty much every physiological function and in many pathologies. Therefore, GPCRs are targeted by about a third of clinically used drugs. The signaling of most GPCRs via G proteins is terminated by the phosphorylation of active receptor by specific kinases (GPCR kinases, or GRKs) and subsequent binding of arrestin proteins, that selectively recognize active phosphorylated receptors. In addition, GRKs and arrestins play a role in multiple signaling pathways in the cell, both GPCR-initiated and receptor-independent. Here we focus on the mechanisms of GRK- and arrestin-mediated regulation of GPCR signaling, which includes homologous desensitization and redirection of signaling to additional pathways by bound arrestins. [ABSTRACT FROM AUTHOR]

Agonist activation of G protein-coupled receptors promotes sequential interaction of the receptor with heterotrimeric G proteins, G protein-coupled receptor kinases (GRKs), and arrestins. GRKs play a central role in mediating the switch from G protein to arrestin interaction and thereby control processes such as receptor desensitization and trafficking and arrestin-mediated signaling. In this review, I provide a historical perspective on some of the early studies that identified the family of GRKs with a primary focus on the non-visual GRKs. These studies included identification, purification, and cloning of the β-adrenergic receptor kinase in the mid- to late-1980s and subsequent cloning and characterization of additional members of the GRK family. This helped to lay the groundwork for ensuing work focused on understanding the structure and function of these important enzymes.

Many receptors for neurotransmitters, such as dopamine, norepinephrine, acetylcholine, and neuropeptides, belong to the superfamily of G protein-coupled receptors (GPCRs). A general model posits that GPCRs undergo two-step homologous desensitization: the active receptor is phosphorylated by kinases of the G protein-coupled receptor kinase (GRK) family, whereupon arrestin proteins specifically bind active phosphorylated receptors, shutting down G protein-mediated signaling, facilitating receptor internalization, and initiating distinct signaling pathways via arrestin-based scaffolding. Here, we review the mechanisms of GRK-dependent regulation of neurotransmitter receptors, focusing on the diverse modes of GRK-mediated phosphorylation of receptor subtypes. The immediate signaling consequences of GRK-mediated receptor phosphorylation, such as arrestin recruitment, desensitization, and internalization/resensitization, are equally diverse, depending not only on the receptor subtype but also on phosphorylation by GRKs of select receptor residues. We discuss the signaling outcome as well as the biological and behavioral consequences of the GRK-dependent phosphorylation of neurotransmitter receptors where known.

Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development of novel analgesic drugs and is particularly important for understanding the mechanisms responsible for opioid-induced tolerance and addiction. The family of G protein receptor kinases (GRKs) play a pivotal role in such processes, mediating phosphorylation of residues at the C-tail of opioid receptors. Numerous strategies, such as phosphosite specific antibodies and mass spectrometry have allowed the detection of phosphorylated residues and the use of mutant knock-in mice have shed light on the role of GRK regulation in opioid receptor physiology. Here we review our current understanding on the role of GRKs in the actions of opioid receptors, with a particular focus on the MOR, the target of most commonly used opioid analgesics such as morphine or fentanyl.

Abstract Generation of cAMP through G s -coupled G protein-coupled receptor (GPCR) [ e.g. β 2 -adrenoceptor (β 2 AR), adenosine A 2B receptor (A 2B R)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. G s -coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β 2 AR and A 2B R can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β 2 AR- and A 2B R-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β 2 AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A 2B R was regulated by GRK5, but not GRK2. β 2 AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A 2B R-AC signalling and attenuated A 2B R desensitization, while β 2 AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle. Graphical abstract Unlabelled Image Highlights • Depletion of GRK2 or GRK5 enhanced and prolonged β 2 AR adenylyl cyclase activity. • Combined GRK2/GRK5 knockdown further enhanced β 2 AR adenylyl cyclase activity. • Combined knockdown of GRK2 and GRK5 attenuated β 2 AR receptor desensitization. • Depletion of GRK5 enhanced acute adenosine A 2B -stimulated adenylyl cyclase activity. • Adenosine A 2B receptor desensitization was GRK5 and arrestin3 dependent. [ABSTRACT FROM AUTHOR]

We have recently demonstrated that the amino-terminal domain of G protein coupled receptor kinase (GRK) type 5, (GRK5-NT) inhibits NFκB activity in cardiac cells leading to a significant amelioration of LVH. Since GRK5-NT is known to bind calmodulin, this study aimed to evaluate the functional role of GRK5-NT in the regulation of calcium-calmodulin-dependent transcription factors. We found that the overexpression of GRK5-NT in cardiomyoblasts significantly reduced the activation and the nuclear translocation of NFAT and its cofactor GATA-4 in response to phenylephrine (PE). These results were confirmed in vivo in spontaneously hypertensive rats (SHR), in which intramyocardial adenovirus-mediated gene transfer of GRK5-NT reduced both wall thickness and ventricular mass by modulating NFAT and GATA-4 activity. To further verify in vitro the contribution of calmodulin in linking GRK5-NT to the NFAT/GATA-4 pathway, we examined the effects of a mutant of GRK5 (GRK5-NTPB), which is not able to bind calmodulin. When compared to GRK5-NT, GRK5-NTPB did not modify PE-induced NFAT and GATA-4 activation. In conclusion, this study identifies a double effect of GRK5-NT in the inhibition of LVH that is based on the regulation of multiple transcription factors through means of different mechanisms and proposes the amino-terminal sequence of GRK5 as a useful prototype for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Histamine H 3 receptors (H 3 Rs) signal through Gα i/o proteins and are found in neuronal cells as auto- and hetero-receptors. Alternative splicing of the human H 3 R (hH 3 R) originates 20 isoforms, and the mRNAs of two receptors of 445 and 365 amino acids (hH 3 R 445 and hH 3 R 365 ) are widely expressed in the human brain. We previously showed that the hH 3 R 445 stably expressed in CHO-K1 cells experiences homologous desensitization. The hH 3 R 365 lacks 80 residues in the third intracellular loop, and in this work we therefore studied whether this isoform also experiences homologous desensitization and the possible differences with the hH 3 R 445 . In clones of CHO-K1 cells stably expressing similar receptor levels (211 ± 12 and 199 ± 16 fmol/mg protein for hH 3 R 445 and hH 3 R 365 , respectively), there were no differences in receptor affinity for selective H 3 R ligands or for agonist-induced [ 35 S]-GTPγS binding to membranes and inhibition of forskolin-stimulated cAMP accumulation in intact cells. For both cell clones, pre-incubation with the H 3 R agonist RAMH (1 μM) resulted in functional receptor desensitization, as indicated by cAMP accumulation assays, and loss of receptors from the cell surface and reduced affinity for the agonist immepip in cell membranes, evaluated by radioligand binding. However, functional desensitization differed in the maximal extent (96 ± 15% and 58 ± 8% for hH 3 R 445 and hH 3 R 365 , respectively) and the length of pre-exposure required to reach the maximum desensitization (60 and 30 min, respectively). Furthermore, the isoforms differed in their recovery from desensitization. These results indicate that the hH 3 R 365 experiences homologous desensitization, but that the process differs between the isoforms in time-course, magnitude and re-sensitization. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are cell surface receptors that respond to a wide variety of stimuli, from light, odorants, hormones, and neurotransmitters to proteins and extracellular calcium. GPCRs represent the largest family of signaling proteins targeted by many clinically used drugs. Recent studies shed light on the conformational changes that accompany GPCR activation and the structural state of the receptor necessary for the interactions with the three classes of proteins that preferentially bind active GPCRs, G proteins, G protein-coupled receptor kinases (GRKs), and arrestins. Importantly, structural and biophysical studies also revealed activation-related conformational changes in these three types of signal transducers. Here, we summarize what is already known and point out questions that still need to be answered. Clear understanding of the structural basis of signaling by GPCRs and their interaction partners would pave the way to designing signaling-biased proteins with scientific and therapeutic potential. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases that regulate a large and diverse class of G protein-coupled receptors (GPCRs). Through GRK phosphorylation and ß-arrestin recruitment, GPCRs are desensitized and their signal terminated. Recent work on these kinases has expanded their role from canonical GPCR regulation to include noncanonical regulation of non-GPCR and nonreceptor substrates through phosphorylation as well as via scaffolding functions. Owing to these and other regulatory roles, GRKs have been shown to play a critical role in the outcome of a variety of physiological and pathophysiological processes including chemotaxis, signaling, migration, inflammatory gene expression, etc. This diverse set of functions for these proteins makes them popular targets for therapeutics. Role for these kinases in inflammation and inflammatory disease is an evolving area of research currently pursued in many laboratories. In this review, we describe the current state of knowledge on various GRKs pertaining to their role in inflammation and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background:S-equol, which is enantioselectively produced from daidzein by gut microbiota, has been suggested as a chemopreventive agent against type 2 diabetes mellitus (T2DM), but the underlying mechanisms remain unclear.Objective: We investigated the effects of S-equol on pancreatic β-cell function.Methods: β-Cell growth and insulin secretion were evaluated with male Institute of Cancer Research mice and isolated pancreatic islets from the mice, respectively. The mechanisms by which S-equol stimulated β-cell response were examined in INS-1 β-cells. The effect of S-equol treatment on β-cell function was assessed in low-dose streptozotocin-treated mice. S-equol was used at 10 μmol/L for in vitro and ex vivo studies and was administered by oral gavage (20 mg/kg, 2 times/d throughout the experimental period) for in vivo studies.Results:S-equol administration for 7 d increased Ki67-positive β-cells by 27% (P < 0.01) in mice. S-equol enantioselectively enhanced glucose-stimulated insulin secretion in mouse pancreatic islets by 41% (P < 0.001). In INS-1 cells, S-equol exerted stronger effects than daidzein on cell growth, insulin secretion, and cAMP-response element (CRE)-mediated transcription. These S-equol effects were diminished by inhibiting protein kinase A. The effective concentration of S-equol for stimulating cAMP production at the plasma membrane was lower than that for phosphodiesterase inhibition. S-equol-stimulated CRE activation was negatively controlled by the knockdown of G-protein α subunit group S (stimulatory) and positively controlled by that of G-protein-coupled receptor kinase-3 and -6. Compared with vehicle-treated controls, S-equol gavage treatment resulted in an increase in β-cell mass of 104% (P < 0.05), a trend toward high plasma insulin concentrations (by 118%; P = 0.06), and resistance to hyperglycemia after streptozotocin treatment (78% of AUC after glucose challenge; P < 0.01). S-equol administration significantly increased the number of Ki67-positive proliferating β-cells by 62% (P < 0.01) and decreased that of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic β-cells by 75% (P < 0.05).Conclusions: Our results show that S-equol boosts β-cell function and prevents hypoglycemia in mice, suggesting its potential for T2DM prevention. [ABSTRACT FROM AUTHOR]

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Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted “barcodes” that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or “ barcodes ” within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a “ sequence - dependent pathway ” anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second “ barcode ” imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the “ sequence - dependent pathway ”. Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second “ barcode ”. Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction pathway in membranes. [ABSTRACT FROM AUTHOR]

Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Birch, Wedegaertner, Orduña-Castillo, Gonzalez Ramirez, Qin and Trejo.)