G Protein-coupled Receptor Kinase Research Articles

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/β-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Ubiquitination of GRK2 Is Required for the β-Arrestin-Biased Signaling Pathway of Dopamine D2 Receptors to Activate ERK Kinases.

A class-A GPCR dopamine D2 receptor (D2R) plays a critical role in the proper functioning of neuronal circuits through the downstream activation of both G-protein- and β-arrestin-dependent signaling pathways. Understanding the signaling pathways downstream of D2R is critical for developing effective therapies with which to treat dopamine (DA)-related disorders such as Parkinson's disease and schizophrenia. Extensive studies have focused on the regulation of D2R-mediated extracellular-signal-regulated kinase (ERK) 1/2 signaling; however, the manner in which ERKs are activated upon the stimulation of a specific signaling pathway of D2R remains unclear. The present study conducted a variety of experimental techniques, including loss-of-function experiments, site-directed mutagenesis, and the determination of protein interactions, in order to investigate the mechanisms underlying β-arrestin-biased signaling-pathway-mediated ERK activation. We found that the stimulation of the D2R β-arrestin signaling pathway caused Mdm2, an E3 ubiquitin ligase, to move from the nucleus to the cytoplasm and interact with tyrosine phosphorylated G-protein-coupled receptor kinase 2 (GRK2), which was facilitated by Src, a non-receptor tyrosine kinase. This interaction led to the ubiquitination of GRK2, which then moved to the plasma membrane and interacted with activated D2R, followed by the phosphorylation of D2R as well as the mediation of ERK activation. In conclusion, Mdm2-mediated GRK2 ubiquitination, which is selectively triggered by the stimulation of the D2R β-arrestin signaling pathway, is necessary for GRK2 membrane translocation and its interaction with D2R, which in turn mediates downstream ERK signaling. This study is primarily novel and provides essential information with which to better understand the detailed mechanisms of D2R-dependent signaling.

Conformational flexibility of β-arrestins - How these scaffolding proteins guide and transform the functionality of GPCRs.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and β-arrestins. To influence specific GPCR signaling behaviors, β-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within β-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess β-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.

Overexpression of GRK2 in vascular smooth muscle leads to inappropriate hypertension and acute heart failure as in clinical scenario 1

Clinical scenario 1 (CS1) is acute heart failure (HF) characterized by transient systolic blood pressure (SBP) elevation and pulmonary congestion. Although it is managed by vasodilators, the molecular mechanism remains unclear. The sympathetic nervous system plays a key role in HF, and desensitization of cardiac β-adrenergic receptor (AR) signaling due to G protein-coupled receptor kinase 2 (GRK2) upregulation is known. However, vascular β-AR signaling that regulates cardiac afterload remains unelucidated in HF. We hypothesized that upregulation of vascular GRK2 leads to pathological conditions similar to CS1. GRK2 was overexpressed in vascular smooth muscle (VSM) of normal adult male mice by peritoneally injected adeno-associated viral vectors driven by the myosin heavy chain 11 promoter. Upregulation of GRK2 in VSM of GRK2 overexpressing mice augmented the absolute increase in SBP (+ 22.5 ± 4.3 mmHg vs. + 36.0 ± 4.0 mmHg, P < 0.01) and lung wet weight (4.28 ± 0.05 mg/g vs. 4.76 ± 0.15 mg/g, P < 0.01) by epinephrine as compared to those in control mice. Additionally, the expression of brain natriuretic peptide mRNA was doubled in GRK2 overexpressing mice as compared to that in control mice (P < 0.05). These findings were similar to CS1. GRK2 overexpression in VSM may cause inappropriate hypertension and HF, as in CS1.

DANGER Signals Activate G -Protein Receptor Kinases Suppressing Neutrophil Function and Predisposing to Infection After Tissue Trauma.

Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition. Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation. We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models. Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury. Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Endocytic proteins mediating GPR15 receptor internalisation 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 (PKCs), arrestins, clathrin, caveolin, and dynamin in GPR15 internalisation. The results demonstrate that GPR15 internalisation 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 internalisation mechanisms for the recently deorphanized GPR15.

GRK2 Mediates Macrophage Polarization by Regulating EP4-cAMP-pCREB Signaling in Ulcerative Colitis and the Therapeutic Effect of Paroxetine on Mice with DSS-Induced Colitis.

G protein-coupled receptor kinase 2 (GRK2) is one of the cytosolic enzymes, and GRK2 translocation induces prostaglandin E2 receptor 4 (EP4) over-desensitization and reduces the level of cyclic adenosine monophosphate (cAMP) to regulate macrophage polarization. However, the role of GRK2 in the pathophysiology of ulcerative colitis (UC) remains unclear. In this study, we investigated the role of GRK2 in macrophage polarization in UC, using biopsies from patients, a GRK2 heterozygous mouse model with dextran sulfate sodium (DSS)-induced colitis, and THP-1 cells. The results showed that a high level of prostaglandin E2 (PGE2) stimulated the receptor EP4 and enhanced the transmembrane activity of GRK2 in colonic lamina propria mononuclear cells (LPMCs), resulting in a down-regulation of membrane EP4 expression. Then, the suppression of cAMP-cyclic AMP responsive element-binding (CREB) signal inhibited M2 polarization in UC. Paroxetine is acknowledged as one of the selective serotonin reuptake inhibitors (SSRI), which is also considered as a potent GRK2 inhibitor with a high selectivity for GRK2. We found that paroxetine could alleviate symptoms of DSS-induced colitis in mice by regulating GPCR signaling to affect macrophage polarization. Taken together, the current results show that GRK2 may act as a novel therapeutic target in UC by regulating macrophage polarization, and paroxetine as a GRK2 inhibitor may have therapeutic effect on mice with DSS-induced colitis.

Function and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor

Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The β2 adrenergic receptor’s (β2AR) 71 amino acid CT is a substrate for GPCR kinases and binds β-arrestins to regulate signaling. Here we show that the β2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking β-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged β2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

GRK2 participation in cardiac hypertrophy induced by isoproterenol through the regulation of Nrf2 signaling and the promotion of NLRP3 inflammasome and oxidative stress.

In cases of heart failure, cardiac hypertrophy may be caused by the upregulation of G-protein-coupled receptor kinase 2 (GRK2). Both NLRP3 inflammasome and oxidative stress contribute to cardiovascular disease. In this study, we clarified the effect of GRK2 on cardiac hypertrophy in H9c2 cells induced by isoproterenol (ISO) and examined the underlying mechanisms. We randomly categorized H9c2 cells into five groups: an ISO group, a paroxetine plus ISO group, a GRK2 small-interfering RNA (siRNA) plus ISO group, a GRK2 siRNA combined with ML385 plus ISO group, and a control group. To determine the effect of GRK2 on cardiac hypertrophy induced by ISO, we carried out CCK8 assays, RT-PCR, TUNEL staining, ELISA assay, DCFH-DA staining, immunofluorescence staining, and western blotting. By using paroxetine or siRNA to inhibit GRK2, we significantly decreased cell viability; reduced the mRNA levels of ANP, BNP, and β-MHC; and limited the apoptosis rate and protein levels of cleaved caspase-3 and cytochrome c in H9c2 cells treated with ISO. We also found that oxidative stress induced by ISO could be mitigated with paroxetine or GRK2 siRNA. This result was validated by decreased activities of the antioxidant enzymes CAT, GPX, and SOD and increased MDA levels and ROS production. We observed that the protein expression of NLRP3, ASC, and caspase-1 and the intensity of NLRP3 could be inhibited by paroxetine or GRK2 siRNA. Both paroxetine and GRK2 siRNA were able to abolish the increase in GRK2 expression induced by ISO. They also could increase protein levels of HO-1, nuclear Nrf2, and Nrf2 immunofluorescence intensity; however, they could not change the protein level of cytoplasmic Nrf2. By combining treatment with ML385, we were able to reverse GRK2 inhibition on H9c2 cells treated with ISO. According to the results of this study, GRK2 participated in cardiac hypertrophy induced by ISO by mitigating NLRP3 inflammasome and oxidative stress through the signaling of Nrf2 in H9c2 cells.

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

GRK2 differentially regulates FcεRI and MRGPRB2-mediated responses in mast cells.

In addition to high-affinity IgE receptor (FcεRI), a subtype of mouse mast cells (MCs) expresses a G protein-coupled receptor known as Mas-related G protein-coupled receptor (GPCR)-B2 (MRGPRB2; human ortholog MRGPRX2). GPCR kinase 2 (GRK2) is a Serine/Threonine kinase that phosphorylates GPCRs to promote their desensitization and internalization. We previously showed that silencing GRK2 expression in mouse bone marrow-derived MCs (BMMCs) blocks IgE-mediated degranulation. Compound 48/80 (C48/80), substance P (SP) and LL-37 cause degranulation in human and mouse MCs via MRGPRX2 and MRGPRB2, respectively. We also reported that C48/80 and SP cause desensitization and internalization of MRGPRX2, but LL-37 does not. Here, we generated mice with MC-specific deletion of Grk2 (Cpa3Cre+/Grk2fl/fl ) to determine its role on IgE-mediated responses and to assess whether it differentially regulates degranulation in response to LL-37, C48/80 and SP. Absence of GRK2 substantially inhibited IgE-mediated tyrosine phosphorylation of STAT5, calcium mobilization, and degranulation in mouse primary lung-derived MCs (PLMCs). By contrast, peritoneal MCs (PMCs) from Cpa3Cre+/Grk2fl/fl mice demonstrated significant enhancement of degranulation in response to C48/80 and SP, but not LL-37. Deletion of Grk2 in MCs attenuated IgE-mediated passive cutaneous anaphylaxis (PCA) and itch but not passive systemic anaphylaxis (PSA). Surprisingly, PSA was significantly reduced in Mrgprb2-/- mice. These findings suggest that GRK2 contributes to PCA and itch but not PSA. By contrast, GRK2 desensitizes MRGPRX2/B2-mediated responses to C48/80 and SP but not LL-37. However, IgE-mediated PSA likely involves the activation of MRGPRB2 by LL-37 or a similar agonist, whose function is resistant to modulation by GRK2.

Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory.

G protein-coupled receptors (GPCRs) promote the expression of immediate early genes required for learning and memory. Here, we showed that β2-adrenergic receptor (β2AR) stimulation induced the nuclear export of phosphodiesterase 4D5 (PDE4D5), an enzyme that degrades the second messenger cAMP, to enable memory consolidation. We demonstrated that the endocytosis of β2AR phosphorylated by GPCR kinases (GRKs) mediated arrestin3-dependent nuclear export of PDE4D5, which was critical for promoting nuclear cAMP signaling and gene expression in hippocampal neurons for memory consolidation. Inhibition of the arrestin3-PDE4D5 association prevented β2AR-induced nuclear cAMP signaling without affecting receptor endocytosis. Direct PDE4 inhibition rescued β2AR-induced nuclear cAMP signaling and ameliorated memory deficits in mice expressing a form of the β2AR that could not be phosphorylated by GRKs. These data reveal how β2AR phosphorylated by endosomal GRK promotes the nuclear export of PDE4D5, leading to nuclear cAMP signaling, changes in gene expression, and memory consolidation. This study also highlights the translocation of PDEs as a mechanism to promote cAMP signaling in specific subcellular locations downstream of GPCR activation.

Aged G Protein-Coupled Receptor Kinase 3 (Grk3)-Deficient Mice Exhibit Enhanced Osteoclastogenesis and Develop Bone Lesions Analogous to Human Paget's Disease of Bone.

Paget's Disease of Bone (PDB) is a metabolic bone disease that is characterized by dysregulated osteoclast function leading to focal abnormalities of bone remodeling. It can lead to pain, fracture, and bone deformity. G protein-coupled receptor kinase 3 (GRK3) is an important negative regulator of G protein-coupled receptor (GPCR) signaling. GRK3 is known to regulate GPCR function in osteoblasts and preosteoblasts, but its regulatory function in osteoclasts is not well defined. Here, we report that Grk3 expression increases during osteoclast differentiation in both human and mouse primary cells and established cell lines. We also show that aged mice deficient in Grk3 develop bone lesions similar to those seen in human PDB and other Paget's Disease mouse models. We show that a deficiency in Grk3 expression enhances osteoclastogenesis in vitro and proliferation of hematopoietic osteoclast precursors in vivo but does not affect the osteoclast-mediated bone resorption function or cellular senescence pathway. Notably, we also observe decreased Grk3 expression in peripheral blood mononuclear cells of patients with PDB compared with age- and gender-matched healthy controls. Our data suggest that GRK3 has relevance to the regulation of osteoclast differentiation and that it may have relevance to the pathogenesis of PDB and other metabolic bone diseases associated with osteoclast activation.

CXCL9, 10, 11/CXCR3 Axis Contributes to the Progress of Primary Sjogren's Syndrome by Activating GRK2 to Promote T Lymphocyte Migration.

Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that causes dysfunction of secretory glands and the specific pathogenesis is still unknown. The CXCL9, 10, 11/CXCR3 axis and G protein-coupled receptor kinase 2 (GRK2) involved in many inflammation and immunity processes. We used NOD/Ltj mice, a spontaneous SS animal model, to elucidate the pathological mechanism of CXCL9, 10, 11/CXCR3 axis promoting T lymphocyte migration by activating GRK2 in pSS. We found that CD4 + GRK2, Th17 + CXCR3 was apparently increased and Treg + CXCR3 was significantly decreased in the spleen of 4W NOD mice without sicca symptom compared to ICR mice (control group). The protein levels of IFN-γ, CXCL9, 10, 11 increased in submandibular gland (SG) tissue accompanied by obvious lymphocytic infiltration and Th17 cells overwhelmingly infiltrated relative to Treg cells at the sicca symptom occurs, and we found that the proportion of Th17 cells was increased, whereas that of Treg cells was decreased in spleen. In vitro, we used IFN-γ to stimulate human salivary gland epithelial cells (HSGECs) co-cultured with Jurkat cells, and the results showed that CXCL9, 10, 11 was increased by IFN-γ activating JAK2/STAT1 signal pathway and Jurkat cell migration increased with the raised of cell membrane GRK2 expression. HSGECs with tofacitinib or Jurkat cells with GRK2 siRNA can reduce the migration of Jurkat cells. The results indicate that CXCL9, 10, 11 significantly increased in SG tissue through IFN-γ stimulating HSGECs, and the CXCL9, 10, 11/CXCR3 axis contributes to the progress of pSS by activating GRK2 to promote T lymphocyte migration.

Genetic Arg-304-His substitution in GRK5 protects against sepsis progression by alleviating NF-κB-mediated inflammation

Previous studies have demonstrated that G protein-coupled receptor kinase 5 (GRK5) exerts a pivotal regulatory effect on the inflammation associated with sepsis. The present study aimed to investigate the clinical association of GRK5 genetic variants with sepsis and to further explore the underlying genetic mechanisms involved in regulating sepsis-induced inflammatory responses and the pathogenesis of sepsis. This case-control study enrolled 1081 septic patients and 1147 matched controls for genotyping of GRK5 rs2230349 and rs2230345 polymorphisms. The effect of these genetic variants on GRK5-mediated inflammatory responses was analyzed in peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages. A clinically relevant polymicrobial sepsis model was established by subjecting wild-type (WT) and GRK5-knockout mice to cecal ligation and puncture (CLP) to evaluate the role of GRK5 in sepsis. We identified significant differences in the genotype/allele distribution of rs2230349 G>A, but not rs2230345, between the sepsis subtype and septic shock subgroups (GA+AA vs. GG genotype, OR=0.698, 95% CI=0.547-0.893, P=0.004; A vs. G allele, OR=0.753, 95% CI=0.620-0.919, P=0.005) and between the survivor and nonsurvivor subgroups (GA+AA vs. GG genotype, OR=0.702, 95% CI=0.531-0.929, P=0.015; A vs. G allele, OR=0.753, 95% CI=0.298-0.949, P=0.017). PBMCs carrying the sepsis-associated protective A allele produced significantly lower levels of TNF-α and IL-1β upon LPS stimulation. The results from the in vitro experiment showed that the Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 significantly decreased the LPS-induced production of several proinflammatory cytokines, such as TNF-α, IL-6, IL-1β and MCP-1, via the IκB-α/NF-κB signaling pathway in THP-1 macrophages. Furthermore, GRK5-knockout mice exhibited a significant decrease in IκB-α phosphorylation/degradation, the p-p65/p65 ratio, the p-p50/p50 ratio, p65 nuclear translocation and downstream cytokine (TNF-α, IL-6, IL-1β and VCAM-1) production compared to WT mice after CLP surgery. A significant improvement in 7-day survival rate in GRK5-KO septic mice was observed in the presence of antibiotics. The Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 might disrupt GRK5 function and alleviate IKB-α/NF-κB-mediated inflammatory responses, which ultimately conferred a genetic protective effect against susceptibility to sepsis progression and mortality. These results may, to some extent, explain the heterogeneity of the clinical prognoses of septic patients and provide novel opportunities for individualized approaches for sepsis treatment.

Atypical regulation of an atypical chemokine receptor: ACKR3 'senses' CXCR4 activation through phosphorylation.

ACKR3 is an arrestin-biased 7-transmembrane receptor that is unable to activate G proteins. The receptor shares the chemokine agonist, CXCL12, with the canonical G protein-coupled receptor (GPCR), CXCR4. The receptor pair plays a critical role in mediating cell migration during cardiac and CNS development as well as contributing to cancer growth and metastasis. Whereas CXCR4 drives cellular migration through G protein signaling, ACKR3 primarily acts to scavenge chemokines, which indirectly regulates migration through shaping the extracellular chemokine gradient. Scavenging is driven by constitutive internalization and recycling of the receptor and concomitant shuttling of CXCL12 to lysosomes for degradation. Since internalization of GPCRs is often dependent on phosphorylation and β-arrestins, we set out to resolve how GPCR kinases (GRKs) and β-arrestins influence ACKR3 scavenging. Despite robust β-arrestin recruitment by ACKR3, knockouts of β-arrestins had little impact on CXCL12 scavenging by the receptor; nevertheless, GRK phosphorylation is essential. Employing GRK knockout cells, we discovered that GRK5 is the dominant kinase for ACKR3 phosphorylation and primary driver of chemokine scavenging in HEK293 cells. In comparison the responses elicited by GRK2 were much more limited. Because GRK2 requires free Gβγ released by heterotrimeric G protein activation, we hypothesized that the muted GRK2 impact may be due to the lack of G protein coupling by ACKR3. Indeed, addition of Gβγ significantly enhanced GRK2-mediated ACKR3 phosphorylation and scavenging of CXCL12. Additionally, co-activation of CXCR4 produced sufficient Gβγ to promote GRK2 phosphorylation of ACKR3. These results suggest a mechanism for ACKR3 to ‘sense’ CXCR4 activation through GRK2 activity. Likewise, this would allow CXCR4 to influence CXCL12 scavenging as well as its own signaling by promoting β-arrestin recruitment to ACKR3.

The dual-function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct mechanisms.

C-C chemokine receptor 2 (CCR2) is a dual-function receptor. Similar to other G protein-coupled chemokine receptors, it promotes monocyte infiltration into tissues in response to the chemokine CCL2, and, like atypical chemokine receptors (ACKRs), it scavenges chemokine from the extracellular environment. CCR2 therefore mediates CCL2-dependent signaling as a G protein-coupled receptor (GPCR) and also limits CCL2 signaling as a scavenger receptor. We investigated the mechanisms underlying CCR2 scavenging, including the involvement of intracellular proteins typically associated with GPCR signaling and internalization. Using CRISPR knockout cell lines, we showed that CCR2 scavenged by constitutively internalizing to remove CCL2 from the extracellular space and recycling back to the cell surface for further rounds of ligand sequestration. This process occurred independently of G proteins, GPCR kinases (GRKs), β-arrestins, and clathrin, which is distinct from other "professional" chemokine scavenger receptors that couple to GRKs, β-arrestins, or both. These findings set the stage for understanding the molecular regulators that determine CCR2 scavenging and may have implications for drug development targeting this therapeutically important receptor.

Dopamine-induced arrestin recruitment and desensitization of the dopamine D4 receptor is regulated by G protein-coupled receptor kinase-2.

The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.

Kinase Activity Is Not Required for G Protein-Coupled Receptor Kinase 4 Restraining mTOR Signaling during Cilia and Kidney Development.

G protein-coupled receptor kinase 4 (GRK4) regulates renal sodium and water reabsorption. Although GRK4 variants with elevated kinase activity have been associated with salt-sensitive or essential hypertension, this association has been inconsistent among different study populations. In addition, studies elucidating how GRK4 may modulate cellular signaling are sparse. In an analysis of how GRK4 affects the developing kidney, the authors found that GRK4 modulates mammalian target of rapamycin (mTOR) signaling. Loss of GRK4 in embryonic zebrafish causes kidney dysfunction and glomerular cysts. Moreover, GRK4 depletion in zebrafish and cellular mammalian models results in elongated cilia. Rescue experiments suggest that hypertension in carriers of GRK4 variants may not be explained solely by kinase hyperactivity; instead, elevated mTOR signaling may be the underlying cause. G protein-coupled receptor kinase 4 (GRK4) is considered a central regulator of blood pressure through phosphorylation of renal dopaminergic receptors and subsequent modulation of sodium excretion. Several nonsynonymous genetic variants of GRK4 have been only partially linked to hypertension, although these variants demonstrate elevated kinase activity. However, some evidence suggests that function of GRK4 variants may involve more than regulation of dopaminergic receptors alone. Little is known about the effects of GRK4 on cellular signaling, and it is also unclear whether or how altered GRK4 function might affect kidney development. To better understand the effect of GRK4 variants on the functionality of GRK4 and GRK4's actions in cellular signaling during kidney development, we studied zebrafish, human cells, and a murine kidney spheroid model. Zebrafish depleted of Grk4 develop impaired glomerular filtration, generalized edema, glomerular cysts, pronephric dilatation, and expansion of kidney cilia. In human fibroblasts and in a kidney spheroid model, GRK4 knockdown produced elongated primary cilia. Reconstitution with human wild-type GRK4 partially rescues these phenotypes. We found that kinase activity is dispensable because kinase-dead GRK4 (altered GRK4 that cannot result in phosphorylation of the targeted protein) prevented cyst formation and restored normal ciliogenesis in all tested models. Hypertension-associated genetic variants of GRK4 fail to rescue any of the observed phenotypes, suggesting a receptor-independent mechanism. Instead, we discovered unrestrained mammalian target of rapamycin signaling as an underlying cause. These findings identify GRK4 as novel regulator of cilia and of kidney development independent of GRK4's kinase function and provide evidence that the GRK4 variants believed to act as hyperactive kinases are dysfunctional for normal ciliogenesis.

GRK5-mediated inflammation and fibrosis exert cardioprotective effects during the acute phase of myocardial infarction.

During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G-protein coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation-related genes through nuclear factor-κB activation, leading to an increase in the expression levels of fibrosis-related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5-mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared to that in wild-type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.