Regulation of the pro-inflammatory G protein-coupled receptor GPR84.

GPR84 is an understudied rhodopsin-like class A G protein-coupled receptor, which is arousing particular interest from a therapeutic perspective. Not least this reflects that gpr84 expression is significantly up-regulated following acute inflammatory stimuli and in inflammatory diseases, and that receptor activation plays a role in regulating pro-inflammatory responses and migration of cells of the innate immune system such as neutrophils, monocytes, macrophages and microglia. Although most physiological responses of GPR84 reflect receptor coupling to Gαi/o-proteins, several studies indicate that agonist-activated GPR84 can recruit arrestin adaptor proteins and this regulates receptor internalisation and desensitisation. To date, little is known on the patterns of either basal or ligand regulated GPR84 phosphorylation and how these might control these processes. Here, we consider what is known about the regulation of GPR84 signalling with a focus on how G protein receptor kinase-mediated phosphorylation regulates arrestin protein recruitment and receptor function. LINKED ARTICLES: This article is part of a themed issue GPR84 Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.10/issuetoc.

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

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

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into ß-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors.

FFA1 and FFA4, two G protein-coupled receptors that are activated by long chain fatty acids, play crucial roles in mediating many biological functions in the body. As a result, these fatty acid receptors have gained considerable attention due to their potential to be targeted for the treatment of type-2 diabetes. However, the relative contribution of canonical G protein-mediated signalling versus the effects of agonist-induced phosphorylation and interactions with ß-arrestins have yet to be fully defined. Recently, several reports have highlighted the ability of ß-arrestins and GRKs to interact with and modulate different functions of both FFA1 and FFA4, suggesting that it is indeed important to consider these interactions when studying the roles of FFA1 and FFA4 in both normal physiology and in different disease settings. Here, we discuss what is currently known and show the importance of understanding fully how ß-arrestins and GRKs regulate the function of long chain fatty acid receptors.

GPCRs: The most promiscuous druggable receptor of the mankind.

All physiological events in living organisms originated as specific chemical/biochemical signals on the cell surface and transmitted into the cytoplasm. This signal is translated within milliseconds-hours to a specific and unique order required to maintain optimum performance and homeostasis of living organisms. Examples of daily biological functions include neuronal communication and neurotransmission in the process of learning and memory, secretion (hormones, sweat, and saliva), muscle contraction, cellular growth, differentiation and migration during wound healing, and immunity to fight infections. Among the different transducers for such life-dependent signals is the large family of G protein-coupled receptors (GPCRs). GPCRs constitute roughly 800 genes, corresponding to 2% of the human genome. While GPCRs control a plethora of pathophysiological disorders, only approximately one-third of GPCR families have been deorphanized and characterized. Recent drug data show that around 40% of the recommended drugs available in the market target mainly GPCRs. In this review, we presented how such system signals, either through G protein or via other players, independent of G protein, function within the biological system. We also discussed drugs in the market or clinical trials targeting mainly GPCRs in various diseases, including cancer.

The GRKs Reactome: Role in Cell Biology and Pathology.

G protein-coupled receptor kinases (GRKs) are protein kinases that function in concert with arrestins in the regulation of a diverse class of G protein-coupled receptors (GPCRs) signaling. Although GRKs and arrestins are key participants in the regulation of GPCR cascades, the complex regulatory mechanisms of GRK expression, its alternation, and their function are not thoroughly understood. Several studies together with the work from our lab in recent years have revealed the critical role of these kinases in various physiological and pathophysiological processes, including cardiovascular biology, inflammation and immunity, neurodegeneration, thrombosis, and hemostasis. A comprehensive understanding of the mechanisms underlying functional interactions with multiple receptor proteins and how these interactions take part in the development of various pathobiological processes may give rise to novel diagnostic and therapeutic strategies. In this review, we summarize the current research linking the role of GRKs to various aspects of cell biology, pathology, and therapeutics, with a particular focus on thrombosis and hemostasis.

Differential Involvement of ACKR3 C-Tail in ß-Arrestin Recruitment, Trafficking and Internalization.

Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits ß-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of ß-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of ß-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both ß-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for ß-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that ß-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for ß-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential ß-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when ß-arrestin recruitment is impaired or in the absence of ß-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced ß-arrestin recruitment, ACKR3 trafficking and internalization.

Transactivation of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs): Recent insights using luminescence and fluorescence technologies.

Alterations in signalling due to bidirectional transactivation of G protein-coupled receptor (GPCRs) and receptor tyrosine kinases (RTKs) are well established. Transactivation significantly diversifies signalling networks within a cell and has been implicated in promoting both advantageous and disadvantageous physiological and pathophysiological outcomes, making the GPCR/RTK interactions attractive new targets for drug discovery programmes. Transactivation has been observed for a plethora of receptor pairings in multiple cell types; however, the precise molecular mechanisms and signalling effectors involved can vary with receptor pairings and cell type. This short review will discuss the recent applications of proximity-based assays, such as resonance energy transfer and fluorescence-based imaging in investigating the dynamics of GPCR/RTK complex formation, subsequent effector protein recruitment and the cellular locations of complexes in living cells.

Sequence and Structure-Based Analysis of Specificity Determinants in Eukaryotic Protein Kinases.

Protein kinases lie at the heart of cell-signaling processes and are often mutated in disease. Kinase target recognition at the active site is in part determined by a few amino acids around the phosphoacceptor residue. However, relatively little is known about how most preferences are encoded in the kinase sequence or how these preferences evolved. Here, we used alignment-based approaches to predict 30 specificity-determining residues (SDRs) for 16 preferences. These were studied with structural models and were validated by activity assays of mutant kinases. Cancer mutation data revealed that kinase SDRs are mutated more frequently than catalytic residues. We have observed that, throughout evolution, kinase specificity has been strongly conserved across orthologs but can diverge after gene duplication, as illustrated by the G protein-coupled receptor kinase family. The identified SDRs can be used to predict kinase specificity from sequence and aid in the interpretation of evolutionary or disease-related genomic variants.

The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology.

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

The Roles of GRKs in Hemostasis and Thrombosis.

Along with cancer, cardiovascular and cerebrovascular diseases remain by far the most common causes of death. Heart attacks and strokes are diseases in which platelets play a role, through activation on ruptured plaques and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which make these receptors ideal targets for many antiplatelet drugs. However, little is known about the mechanisms that provide feedback regulation on GPCRs to limit platelet activation. Emerging evidence from our group and others strongly suggests that GPCR kinases (GRKs) are critical negative regulators during platelet activation and thrombus formation. In this review, we will summarize recent findings on the role of GRKs in platelet biology and how one specific GRK, GRK6, regulates the hemostatic response to vascular injury. Furthermore, we will discuss the potential role of GRKs in thrombotic disorders, such as thrombotic events in COVID-19 patients. Studies on the function of GRKs during platelet activation and thrombus formation have just recently begun, and a better understanding of the role of GRKs in hemostasis and thrombosis will provide a fruitful avenue for understanding the hemostatic response to injury. It may also lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.

Extended Human G-Protein Coupled Receptor Network: Cell-Type-Specific Analysis of G-Protein Coupled Receptor Signaling Pathways.

G-protein coupled receptors (GPCRs) mediate crucial physiological functions in humans, have been implicated in an array of diseases, and are therefore prime drug targets. GPCRs signal via a multitude of pathways, mainly through G-proteins and ß-arrestins, to regulate effectors responsible for cellular responses. The limited number of transducers results in different GPCRs exerting control on the same pathway, while the availability of signaling proteins in a cell defines the result of GPCR activation. The aim of this study was to construct the extended human GPCR network (hGPCRnet) and examine the effect that cell-type specificity has on GPCR signaling pathways. To achieve this, protein-protein interaction data between GPCRs, G-protein coupled receptor kinases (GRKs), Gα subunits, ß-arrestins, and effectors were combined with protein expression data in cell types. This resulted in the hGPCRnet, a very large interconnected network, and similar cell-type-specific networks in which, distinct GPCR signaling pathways were formed. Finally, a user friendly web application, hGPCRnet ( http://bioinformatics.biol.uoa.gr/hGPCRnet ), was created to allow for the visualization and exploration of these networks and of GPCR signaling pathways. This work, and the resulting application, can be useful in further studies of GPCR function and pharmacology.

Pharmacological antagonism of histamine H2R ameliorated L-DOPA-induced dyskinesia via normalization of GRK3 and by suppressing FosB and ERK in PD.

Parkinson's disease (PD) is often managed with L-3,4-dihydroxyphenylalanine (L-DOPA), which is still the gold standard to relieve the clinical motor symptoms of PD. However, chronic use of L-DOPA leads to significant motor complications, especially L-DOPA-induced dyskinesia (LID), which limit the therapeutic benefit. Few options are available for the pharmacological management of LID partly due to the inadequacy of our mechanistic understanding of the syndrome. We focused on the role of the histamine (HA) H2 receptor (H2R) in the striatum, which others have shown to be involved in the development of LID. We generated LID in a hemiparkinsonian mouse model and tested the signaling effects of ranitidine, an H2R antagonist. We used histidine decarboxylase deficient mice (Hdc-Ko) which lacks HA to study the role of G-protein-coupled receptor kinases (GRKs) in HA deficiency. Loss of HA in Hdc-Ko mice did not result in the downregulation of GRKs, especially GRK3 and GRK6, which were previously found to be reduced in hemiparkinsonian animal models. Ranitidine, when given along with L-DOPA, normalized the expression of GRK3 in the dopamine-depleted striatum thereby inhibiting LID in mice. The extracellular signal regulated kinase and ΔFosB signaling pathways were attenuated in the lesioned striatum when ranitidine was combined with L-DOPA than L-DOPA alone. These results demonstrate that ranitidine inhibits LID by normalizing the levels of GRK3, extracellular signal regulated kinase activation, and FosB accumulation in the dopamine-depleted striatum via HA H2R antagonism.

GPCRs and Signal Transducers: Interaction Stoichiometry.

Until the late 1990s, class A G protein-coupled receptors (GPCRs) were believed to function as monomers. Indirect evidence that they might internalize or even signal as dimers has emerged, along with proof that class C GPCRs are obligatory dimers. Crystal structures of GPCRs and their much larger binding partners were consistent with the idea that two receptors might engage a single G protein, GRK, or arrestin. However, recent biophysical, biochemical, and structural evidence invariably suggests that a single GPCR binds G proteins, GRKs, and arrestins. Here we review existing evidence of the stoichiometry of GPCR interactions with signal transducers and discuss potential biological roles of class A GPCR oligomers, including proposed homo- and heterodimers.

Blurring Boundaries: Receptor Tyrosine Kinases as functional G Protein-Coupled Receptors.

Receptor tyrosine kinases (RTKs) such as the insulin-like growth factor type 1 receptor (IGF-1R) control important biological activities as well as being involved in pathological processes. Due to their supportive nature in many human cancers they have long been considered attractive therapeutic targets. However, lessons learnt from early targeting trials highlight that a simple "active versus inactive" state model with classical kinase-only signaling is overly simplistic and does not describe reality. A vast amount of evidence exists disproving this model and hence provides a rational explanation for failure of many targeting agents designed under such a paradigm. In addition, substantial evidence exists that the IGF-1R and other RTKs make direct use of the G protein-coupled receptor (GPCR) components G proteins, GRKs, and ß-arrestins, outside of their traditional receptor family frame. In this chapter we review the evidence that RTKs can undertake a wide range of active conformations, capable of distinct downstream signal cascades and propose an RTK/GPCR functional hybrid model, while discussing the implications of such an update on therapeutic drug development pipelines.

Utilizing a structure-based docking approach to develop potent G protein-coupled receptor kinase (GRK) 2 and 5 inhibitors.

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the desensitization and internalization of GPCRs. Two of these, GRK2 and GRK5, are upregulated in heart failure and are promising targets for heart failure treatment. Although there have been several reports of potent and selective inhibitors of GRK2 there are few for GRK5. Herein, we describe a ligand docking approach utilizing the crystal structures of the GRK2-Gßγ·GSK180736A and GRK5·CCG215022 complexes to search for amide substituents predicted to confer GRK2 and/or GRK5 potency and selectivity. From this campaign, we successfully generated two new potent GRK5 inhibitors, although neither exhibited selectivity over GRK2.

The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

AIM: The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. METHODS: The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and ß-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. RESULTS: GPCR signaling plays an important role in T cell activation, homeostasis and function. CONCLUSION: GPCR signaling is critical in regulating T cell immunity.

Immunomodulatory Effects of CP-25 on Splenic T Cells of Rats with Adjuvant Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease in which T cells play an important role. Paeoniflorin-6-oxy-benzenesulfonate (CP-25) shows a strong anti-inflammatory and immunomodulatory effect in the joint of adjuvant arthritis (AA) rats, but the role of the spleen function is still unclear. The aim of this study was to research how CP-25 regulated spleen function of AA rats. Male Sprague-Dawley rats were administered with CP-25 (50 mg/kg) orally from day 17 to 29 after immunization. The spleen histopathological changes were analyzed by hematoxylin-eosin staining. G protein-coupled receptor kinases (GRKs) and prostaglandin receptor subtypes (EPs) were screened by Western blot and immunohistochemistry. The co-expression of GRK2 and EP2 as well as GRK2 and EP4 was measured by immunofluorescence and co-immunoprecipitation. The expression of GRK2 and EP4 in splenic T cells was further detected by immunofluorescence. CP-25 was found to relieve the secondary paw swelling, attenuate histopathologic changes, and downregulate GRK2, EP2 and EP4 expression in AA rats. Additionally, CP-25 not only downregulated the co-expression of GRK2 and EP4 but also downregulated GRK2, EP4 expression in splenic T cells of AA rats. From these results, we can infer that CP-25 play an anti-inflammatory and immune function by affecting the function of the splenic T cells.

G protein-coupled receptor kinases (GRKs) in tumorigenesis and cancer progression: GPCR regulators and signaling hubs.

Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus putting forward these kinases as potentially relevant modulators of the carcinoma-fibroblast-endothelial-immune cell network fostering tumor development and dissemination. A better understanding of the alterations in different GRK isoforms taking place during cancer development and metastasis in specific tumors and cell types and of its impact in signaling pathways would help to design novel therapeutic strategies.

Role of G protein-coupled receptor kinase-6 in Escherichia coli lung infection model in mice.

G protein-coupled receptor kinase-6 (GRK6) is a serine/threonine kinase that is important in inflammatory processes. In this study, we examined the role of GRK6 in Escherichia coli-induced lung infection and inflammation using GRK6 knockout (KO) and wild-type (WT) mice. Intratracheal instillation of E. coli significantly enhanced bacterial load in the bronchoalveolar lavage (BAL) of KO compared with WT mice. Reduced bacterial clearance in the KO mice was not due to an intrinsic defect in neutrophil phagocytosis or killing but as a result of reduced neutrophil numbers in the KO BAL. Interestingly, neutrophil numbers in the lung were increased in the KO compared with WT mice, suggesting a potential dysfunction in transepithelial migration of neutrophils from the lungs to the bronchoalveolar space. This effect was selective for lung tissue because peritoneal neutrophil numbers were similar between the two genotypes following peritoneal infection. Although neutrophil expression of CXCR2/CXCR3 was similar between WT and KO, IL-17A expression was higher in the KO compared with WT mice. These results suggest that enhanced neutrophil count in the KO lungs but reduced numbers in BAL are likely due to transepithelial migration defect and/or altered chemokines/cytokines. Together, our studies suggest a previously unrecognized and novel role for GRK6 in neutrophil migration specific to pulmonary tissue during bacterial infection.