Pharmacological inhibition of G protein-coupled receptor kinase 5 decreases high-fat diet-induced hepatic steatosis in mice.

G protein-coupled receptors (GPCRs) engage multiple transducers to regulate distinct physiological processes. These transducers include various G proteins subtypes, GPCR kinases (GRKs), and β-arrestins. In addition to promoting receptor desensitization, β-arrestins serve as scaffolds for signaling via non-G protein pathways. Biased signaling enables GPCRs to selectively engage specific transducers, typically through different conformational states of GPCRs. While significant focus has been placed on developing biased ligands that preferentially activate specific G proteins or β-arrestins, the strategy focused on modulating particular G protein subunits (Gα versus βγ) remains underexplored. Recently, members of the KCTD (potassium channel tetramerization domain-containing) family have emerged as critical regulators of GPCR signaling, particularly through their roles in mediating Gβγ degradation or uncoupling Gβγ from downstream effectors. This ability positions the KCTD family as potential targets for selectively modulating Gβγ signaling with minimal impact on Gα-mediated pathways. In this chapter, we introduce the KCTD family, summarize current knowledge of their role in GPCR signaling regulation, and highlight unsolved questions in existing models, along with directions for future research.

Myocardial infarction (MI) is a pathological condition associated with various cardiovascular diseases and leads to heart failure. Nuclear factor-kappa B (NF-κB) is upregulated in the infarcted heart. G protein-coupled receptor kinase 5 (GRK5) also plays a complex role in both tissue repair and maladaptive hypertrophy in cardiovascular diseases; however, its effect on NF-κB-mediated inflammation has not yet been elucidated. Thus, this study aims to investigate the effects of Amlexanox (AMX), a potential GRK5 inhibitor, in an animal model of MI by assessing its impact on GRK5-mediated NF-κB/inflammatory processes. Thirty-two male mice were randomly allocated into four groups: control, MI, (MI treated with vehicle (MI + V), and MI + AMX (AMX: 2.5 mg/100 g/day). MI was induced using ISO on days 21 and 22. The cardioprotective impacts of Amlexanox were verified by evaluating cardiac injury, inflammatory biomarker concentrations, and histopathological alterations in cardiomyocytes. MI induction was confirmed by increases in heart weight/body weight ratio (HW/BW) (p < 0.001), troponin (p < 0.001), creatine kinase (p < 0.001), and LDH (p < 0.001). Treatment with AMX resulted in a significant reduction in cardiac injury biomarkers (p < 0.001) and IL-6 (p < 0.05). The protein level of NF-κB(p65) and NF-κB(p105) was significantly increased in cardiac myocytes of the MI group. Treatment with AMX led to a significant decrease in NF-κB(p65) and (p105) expression (p < 0.01 and p < 0.001, respectively), and GRK5 and MEF2α protein levels were also upregulated. In conclusion, AMX shows potential cardioprotective effects by modulating the GRK5/MEF2-mediated NF-κB inflammatory signaling pathway.

Background Ambient fine particulate matter (PM2.5) is a major environmental risk factor for hypertension, yet the renal sodium-handling mechanisms remain incompletely understood. This study investigates the role of G protein-coupled receptor kinase 4 (GRK4) and its downstream signalling axis in PM2.5-induced hypertensive pathogenesis. Methods Male Sprague-Dawley rats were exposed to PM2.5 (10 or 40 mg/kg) via intratracheal instillation for 12 weeks. Hemodynamic parameters, renal function, and molecular alterations were analysed using immunohistochemistry, Western blot, qPCR, and co-immunoprecipitation. GRK4 expression was manipulated via lentiviral vectors to validate its role in blood pressure regulation. Results PM2.5 exposure induced dose-dependent hypertension, renal dysfunction, and sodium retention. Mechanistically, PM2.5 upregulated renal GRK4 expression through promoter hypomethylation, enhancing its interaction with Nedd4L (a ubiquitin ligase). Phosphorylated Nedd4L (p-Nedd4L) reduced epithelial sodium channel (ENaC) ubiquitination, leading to ENaC accumulation and sodium reabsorption. GRK4 overexpression exacerbated hypertension and sodium retention, whereas GRK4 knockdown attenuated these effects. Conclusion This study identifies a novel signalling axis—GRK4/Nedd4L/ENaC—in PM2.5-induced hypertension, highlighting epigenetic and post-translational regulatory mechanisms. These findings provide mechanistic insights into environmentally mediated hypertensive pathogenesis and suggest potential therapeutic targets for PM2.5-related cardiovascular diseases.

GRK4 R65L causes salt-sensitive hypertension by augmenting renal Hao2-mediated oxidative stress via increasing the phosphorylation of TPI1 and promoting H3K27ac expression.

Activation of most G protein-coupled receptors (GPCRs) initiates several branches of signaling. The consequences of some of these can be therapeutically beneficial, whereas others may cause unwanted side effects. Therefore, search for ligands biasing receptor signaling toward desired directions and away from undesired ones is increasingly popular. Currently, the field is focusing on G protein versus arrestin dichotomy. Arrestin binding to most GPCRs critically depends on receptor phosphorylation by GPCR kinases (GRKs). Existing data suggest that to bias signaling for or against arrestin-mediated branches one needs to enhance or reduce, respectively, the propensity of the receptor to recruit GRKs and become phosphorylated. As GRKs are gatekeepers for arrestin-mediated signaling, rational design of biased ligands should be based on the comparison of receptor complexes with G proteins with the structures of GPCRs bound to GRKs rather than arrestins. The same GPCR may engage distinct GRK subtypes differently, establishing an additional layer of bias. SIGNIFICANCE STATEMENT: Because arrestin binding to G protein-coupled receptors (GPCRs) requires prior receptor phosphorylation, GPCR-GPCR kinase complexes, not GPCR complexes with arrestins, should be compared to GPCR-G protein complexes to guide the design of G protein- and arrestin-biased ligands.

Activation of GPR81 protects against myocardial ischemia-reperfusion injury under aberrant glucose metabolism condition by modulating VILIP-1/GRK2/GLUT4 pathway.

Sympathetic stimulation produces beneficial changes in cardiac function through β-adrenergic receptor (βAR) production of cAMP and subsequent alteration of electrical and mechanical activity. Long term activation of cAMP production also contributes to cardiac remodeling and detrimental changes associated with heart failure. However, sympathetic responses are mediated by the endogenous neurotransmitter norepinephrine (NE), which is also a potent α 1 -adrenergic receptor (α 1 AR) agonist, and α 1 AR activation can produce significant effects on the heart as well. What is less clear is how α 1 - and β-adrenergic responses interact with one another. Previous studies have demonstrated that α 1 AR activation can inhibit β-adrenergic regulation of electrical and mechanical activity of cardiac myocytes, although the signaling mechanisms involved were not previously known. In the present study, we used FRET-based biosensors in adult rat ventricular myocytes to demonstrate that this crosstalk effect involves inhibition of cAMP production by nuclear α 1A ARs acting on βARs found on the plasma membrane. Furthermore, we established that this inside-out signaling mechanism involves a mitogen-activated protein kinase (MAPK) pathway that uncouples βARs from downstream signaling in a G protein coupled receptor kinase (GRK)/arrestin-dependent manner. These results reveal a novel, non-canonical signaling mechanism contributing to α 1 AR responses in the heart, and that this effect limits βAR production of cAMP by NE. This mechanism may contribute to the cardioprotective effect previously ascribed to α 1A AR activation. These findings also clearly demonstrate the importance of considering the contributions of α 1 and βARs together when studying the influence of the sympathetic nervous system on the heart.

Background: G Protein-Coupled Receptor Kinase 2 (GRK2) contributes to cardiac dysfunction through β-Adrenoceptor (βAR) desensitization following its upregulation. Inhibition of GRK2 has been shown to improve cardiac function. Nitric oxide (NO), through the formation of S-nitrosothiols (SNOs), serves as an endogenous inhibitor of GRK2 activity. β3-Adrenoceptors (β3ARs), in contrast, resist GRK2 and are upregulated in cardiac pathologies. Activation of β3ARs can confer cardioprotection via NO signaling. Hypothesis: We hypothesized that β3AR activation confers cardioprotection through NO-dependent inhibition of GRK2, and that this protection is lost when GRK2 is resistant to S-nitrosylation. Furthermore, we proposed that proteomic profiling would reveal molecular pathways underlying this mechanism. Methods: Male C57BL/6J wild-type (WT) and GRK2-C340S knock-in (KI) mice, which carry a Cys340Ser mutation making GRK2 resistant to S-nitrosylation, underwent 40 minutes of left anterior descending coronary artery occlusion followed by 24 hours of reperfusion under isoflurane anesthesia. The β3AR agonist CL 316,243 was given at reperfusion onset. Cardiac function was evaluated by echocardiography (ejection fraction, EF; fractional shortening, FS), and infarct size was assessed by TTC staining. Heart tissues were subjected to mass spectrometry-based quantitative proteomics and pathway analysis. Results: In WT mice, CL 316,243 significantly improved EF and FS compared to vehicle-treated controls. These effects were abolished in GRK2-C340S mice, indicating that GRK2 S-nitrosylation is necessary for β3AR-mediated cardioprotection. WT mice also had significantly smaller infarct size compared to KI mice. Proteomic analysis revealed 397 significantly differentially expressed proteins (137 downregulated, 260 upregulated) in CL-treated KI mice versus WT. Additionally, enriched pathway analysis showed an upregulation of the innate immune response (52-associated proteins) and a downregulation of lipolysis regulation (19-associated proteins). Conclusion: β3AR stimulation protects the heart after ischemia/reperfusion through NO-dependent S-nitrosylation of GRK2, which is essential for this effect. However, proteomic analysis revealed that CL-treated KI mice were enriched in novel pathways, including innate immunity and lipid metabolism, proposing a potential mechanism for the loss of protection and, therefore, implicating the β3AR–NO–GRK2 axis as a promising therapeutic target.

Mechanistic insights into how g protein-coupled receptor kinases (GRKs) regulate thrombopoiesis

G protein-coupled receptors (GPCRs) display bias towards either G proteins or GPCR kinase (GRK)-mediated β-arrestin signaling depending on the agonist stabilized receptor conformation and cellular context. The cellular location of GPCRs particularly within plasma membrane microdomains such as lipid rafts contributes to biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits biased signaling in response to thrombin and activated protein C (APC). APC-induced β-arrestin-2 (βarr2) biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural protein of caveolae and regulates signaling through protein-protein interactions. The mechanisms by which Cav1 contributes to APC/PAR1-induced βarr2 biased signaling is not known. Here we report that APC-activated PAR1 modulates Cav1 phosphorylation via a βarr2- and c-Src-dependent pathway. APC also regulates the dynamics of endogenous PAR1-Cav1 and GRK5-Cav1 co-localization examined by single molecule super-resolution stochastic optical reconstruction microscopy imaging in human cultured endothelial cells. We further demonstrate that GRK5 interacts with Cav1 in intact cells through an N-terminus aromatic-rich consensus Cav1 binding motif. Unlike wildtype GRK5, a GRK5 mutant defective in Cav1 binding localized predominantly to the cytoplasm rather than the plasma membrane and failed to promote βarr2 recruitment to APC-activated PAR1. These studies suggest that Cav1 itself contributes to the regulation of APC-activated PAR1 βarr2 biased signaling likely through multiple mechanisms that may converge on GRK5.

Identification of a regulatory sequence within the third intracellular loop that governs β-arrestin binding to the muscarinic M5 receptor.

Aim: Baicalin and ginsenoside Rb1 show the ability to promote adipocyte browning, but their effects, especially combined treatment, and the related mechanisms under pathological conditions are less known. The study investigated the regulation of browning markers by baicalin and Rb1 under lipid overload and explored the potential implication of a serine/threonine protein kinase G protein-coupled receptor kinase 2 (GRK2). Methods: The 3T3-L1 cells under palmitic acid (PA) stimulation and male ICR mice on a high-fat diet (HFD) challenge were used to evaluate the effects of drugs. Results: GRK2 silencing and overexpression inversely regulated the protein abundance of PGC-1α and UCP-1 in 3T3-L1 adipocytes. Baicalin, Rb1, and their combination decreased the PA-induced elevation of GRK2 while increasing the thermogenetic markers at the protein and mRNA levels. In vivo, the tested drugs restored the expression of thermogenetic and mitochondrial biogenetic markers in the inguinal white adipose tissue (WAT) of HFD-fed mice. Consistently, the drug-treated mice displayed an improved metabolic profile. The baicalin-Rb1 combination showed a more potent effect in some examinations, and its effect was comparable to that of GRK2 inhibitor paroxetine or AMP-activated protein kinase activator metformin. Conclusions: Baicalin and Rb1, alone or in combination, improved the browning of adipocytes during differentiation and prevented the whitening shift of WAT on an HFD, which was associated with the downregulation of GRK2. The study expands the understanding of the anti-obesity effects of baicalin and Rb1 and the potential of Scutellariae Radix-Ginseng Radix et Rhizoma compatibility for treating obesity-associated metabolic diseases.

β-Arrestins (βarr1 and βarr2) are key transducers of G protein-coupled receptor (GPCR) signaling, orchestrating both shared and isoform-specific intracellular pathways. Phosphorylation of the receptor C-terminal tail by GPCR kinases encodes regulatory "barcodes" that modulate β-arrestin conformations and interactions with downstream effectors. However, how distinct phosphorylation patterns shape β-arrestin structure and function remains poorly understood. In this study, we integrate all-atom molecular dynamics simulations with machine learning, including graph neural networks, to systematically characterize the barcode-specific conformational landscape of β-arrestins bound to the phosphorylated vasopressin receptor 2 tail (V2Rpp). We find that V2Rpp engages βarr1 more stably than βarr2, mediated by isoform-specific residue contacts that trigger distinct allosteric responses. These include differential interdomain rotations and rearrangements in key structural motifs, potentially facilitating selective effector protein engagement. Furthermore, we identify critical residue networks that transmit phosphorylation signals to effector-binding interfaces in a barcode- and isoform-specific manner. Notably, βarr1 exhibits stronger allosteric coupling between V2Rpp and c-edge loop 2 compared to βarr2, which is consistent with its enhanced membrane association. Together, these findings advance our understanding of the molecular mechanisms by which β-arrestins interpret GPCR phosphorylation signatures, offering a framework that could aid in the design of pathway-selective therapeutics.

Vascular smooth muscle cell (VSMC) phenotypic modulation is responsible for the pathogenesis of hyper-muscularized arterial diseases. Recent studies have highlighted the critical role of epigenetic regulation in VSMC fate. However, the mechanisms underlying the precise regulation of the epigenetic machinery in VSMC remain unclear. Using mouse aortic smooth muscle cells, carotid artery injury mouse model, and human atherosclerosis data sets, we identified GRK2 (G-protein-coupled receptor kinase 2) as a novel epigenetic regulator governing VSMC fate. GRK2 expression was found to be elevated in dedifferentiated VSMCs. Pharmacological or genetic silencing of GRK2 inhibited VSMC phenotypic switching. Mechanistic investigations demonstrated that GRK2 modulated VSMC phenotype via DNMT1 (DNA methyltransferase 1)-mediated DNA methylation. GRK2 phosphorylated DNMT1, stabilizing it by modulating its ubiquitination. Hypermethylated VSMC exhibited reduced expression of contractile-associated proteins. Inhibition of DNMT1 abolished the effects of GRK2 overexpression on VSMC phenotype, indicating a DNMT1-mediated mechanism. Our findings revealed that the GRK2-DNMT1 signaling axis is a critical regulator in VSMC phenotypic switching and present a potential therapeutic target for vascular remodeling.

Omentin-1 attenuates salt-sensitive hypertension via GRK4/AT1R downregulation mediated by the ROS/c-Myc pathway.

Herein, the design, synthesis, and evaluation of small molecules, drug‐like G protein‐coupled receptor kinase 5 (GRK5) inhibitors are reported. GRK5 has become an important drug development target against heart failure and cancer. GRK6, a close homolog of GRK5, is considered as a possible therapeutic target for multiple myeloma. A series of drug‐like GRK5 inhibitors that form noncovalent interactions in the GRK5 active site are designed. In the design of these molecules, pyrroloindoline basic scaffold of sunitinib, an FDA‐approved drug, is utilized and various N‐heterocyclic carboxamides in the active site are incorporated. Several inhibitors exhibit low nanomolar GRK5 inhibitory activity and high selectivity over GRK2. Several compounds also display very potent activity and selectivity for GRK6. A high‐resolution X‐ray crystal structure of one of these small molecule inhibitors in complex with GRK5 is determined. The structure provides important molecular insights regarding ligand‐binding site interactions, GRK5 inhibition, and selectivity against GRK2.

Activation primes GPCRs for versatile coupling.

Trans‐Coumaryl acetate (T‐CA) is formed by the esterification of coumarin with acetic acid and belongs to the reprogramming products of aromatic amino acid and fatty acid metabolism. Currently, the impact of T‐CA on the progression of septic acute kidney injury (SAKI) and its underlying mechanisms are not clear. A lipopolysaccharide (LPS)‐treated HK‐2 cell injury model was constructed, and a mouse SAKI model was constructed using a cecum ligation and puncture method. The impacts of T‐CA on HK‐2 cell survival and cytotoxicity were examined using a Cell Counting Kit‐8 assay and lactate dehydrogenase kit. Inflammatory factors, Superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS), adenosine 5′‐triphosphate (ATP), and mitochondrial membrane potential levels were measured using different kits. Apoptosis was identified using Hoechst 33258 and Terminal Deoxynucleotidyl Transferase mediated dUTP Nick‐End Labeling (TUNEL) staining. Changes in renal histopathological injury and indicator protein expression in SAKI mice were observed by transmission electron microscopy and pathological staining. Western blot was used to assess the levels of G protein‐coupled receptor kinase 5 (GRK5)/NF‐κB/nuclear factor erythroid‐2 related factor 2 (Nrf2) pathway, apoptosis and mitochondrial damage‐related proteins. T‐CA (2.5–20 μM) treatment for 24 h did not negatively impact HK‐2 cell viability. In vitro, T‐CA attenuated LPS‐induced HK‐2 cell injury while reducing cell mortality, inflammatory factor levels and oxidative stress injury. In vivo, intraperitoneal injection of 40 mg/kg of T‐CA attenuated renal histopathological damage and apoptosis in SAKI mice. Additionally, T‐CA reduced mitochondrial damage, MDA and ROS levels, and increased SOD, GSH, and ATP levels. T‐CA down‐regulated GRK5 protein, hindered NF‐κB activation and activated Nrf2 pathway, and NF‐κB activator Phorbol 12‐myristate 13‐acetate (PMA), Nrf2 inhibitor ML385 treatment and overexpression of GRK5 weakened the protective effect of T‐CA in SAKI model. T‐CA has the potential to improve SAKI by inhibiting mitochondrial dysfunction, increase cell viability and ameliorate renal injury through the GRK5/NF‐κB/Nrf2 pathway in SAKI models.

Background/Objectives: G protein-coupled receptors (GPCRs) can promote ligand-biased signaling, yet the mechanisms that promote bias are not well understood. We have shown that C-C Chemokine Ligand 19 (CCL19) and CCL21 promote ligand-biased internalization and signaling of C-C Chemokine Receptor 7 (CCR7) in T cells. The roles of GPCR kinases (GRKs) in regulating biased CCR7 internalization and biased signaling in T cells are unclear. GRK2 is a serine/threonine kinase that phosphorylates GPCRs in response to ligand binding and is recruited to the plasma membrane via its C-terminal pleckstrin homology domain to phosphatidylinositol 4,5-bisphosphate (PIP2). Methods: Human embryonic kidney cells (HEK293) transfected to express wild-type and mutant GRK2 and human CCR7, human T cell lines harboring heterozygous deletions of GRK2, and naïve primary T cells from GRK2 heterozygous (GRK2+/−) or GRK2f/f CD4-Cre mice were used to examine the effects of GRK2 on ligand-induced CCR7 signaling in T cells. We used flow cytometry to assay the effect of GRK2 on CCR7 internalization, Fluorescence Resonance Energy Transfer (FRET) to define the effect of GRK2 on CCR7 activation of Gαi isoforms and transwell migration assays to examine the effect of GRK2 on chemotaxis. Since chemotaxis via CCR7 is mediated by phospholipase Cγ1 (PLCγ1), Western blot assays were used to measure the effect of GRK2 during downstream signaling via phosphorylation of PLCγ1. Results: We found that following CCL19 binding, GRK2 promoted kinase-dependent CCR7 recruitment of arrestin-3, rapid CCR7 internalization and Gαi3 recruitment to CCR7. In contrast, following binding of CCL21 to CCR7, GRK2 slowed CCR7 internalization, induced recruitment of Gαi2 to the activated receptor, and promoted chemotaxis. Since we have shown that CCL21 promotes chemotaxis via PLCγ1, we examined the effect of GRK2 on PLCγ1 activation and found that GRK2 had no effect on CCL21-mediated PLCγ1 phosphorylation. Conclusions: GRK2 promotes differential signaling downstream of CCR7 activation by CCL19 and CCL21 and provides a model for biased signaling downstream of a GPCR driven by GRK2.