Overexpression Of GRK2 Research Articles

Adrenal G protein-coupled Receptor Kinase (GRK)-2 Downregulation And Regulator of G protein Signaling (RGS)-4 Upregulation Mediate The Sympatholytic Effect Of Dapagliflozin

Dapagliflozin is a sodium/­glucose co-­transporter (SGLT)-­2 inhibitor with beneficial cardiovascular effects independent of its anti­diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering, beneficial in chronic human heart failure (HF). Adrenal G protein­-coupled receptor kinase (GRK)-­2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-­inhibitory α2­-adrenergic receptors (ARs). On the other hand, we recently reported that Regulator of G protein Signaling (RGS)-­4 inhibits free fatty acid receptor (FFAR)-­3-stimulated NE release from sympathetic neurons, while it is also known to inhibit adrenal CA secretion induced by acetylcholine. Thus, we hypothesized herein that dapagliflozin may lower SNS hyperactivity by enhancing adrenal α2AR and RGS4 functions. We used the rat pheochromocytoma PC12 cell line expressing human α2A­AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217±67 pg/ml, n=6), compared to vehicle-­treated rats (363±77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-­treated vs. vehicle­-treated adrenal glands. In contrast, RGS4 mRNA and protein levels were increased by dapagliflozin ­treatment. Adrenal α2AR density was higher in dapagliflozin- ­vs. vehicle-­treated rats (51.3±7.3 vs. 26.1±8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR signaling towards inhibition of CA secretion was markedly enhanced, while the cholinergic agonist carbachol-induced CA secretion was reduced post-­dapagliflozin treatment of PC12 cells. GRK2 overexpression and RGS4 siRNA-mediated knockdown partially reversed dapagliflozin‘s effect on CA secretion, confirming the involvement of GRK2 and RGS4 in dapagliflozin‘s sympatholytic effects in PC12 cells. In conclusion, dapagliflozin exerts adrenal sympatholysis via: a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization (i.e., enhances α2AR sympatho-inhibitory signaling); and b) upregulation of RGS4, which directly opposes cholinergic-stimulated CA secretion. 1) NIH/NHLBI #HL155718-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of G protein-coupled receptor kinases in GLP-1R β-arrestin recruitment and internalisation

The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require β-arrestins. While differences in GLP-1R trafficking and β-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and β-arrestin recruitment profiles using cells where endogenous β-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical β-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R β-arrestin 1 and β-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with β-arrestin 1 recruitment more sensitive to GRK knockout than β-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated β-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Abstract P3152: Diabetic Upregulation Of Grk2 Impairs Adipocyte Exosome Surface Adiponectin-mediated Cardioprotection And Exacerbates Ischemic Heart Failure

Background: Type 2 diabetes mellitus (DM) significantly exacerbates ischemic heart failure (IHF) by incompletely understood mechanisms. We recently reported that DM switches adipocyte-derived exosome (Exo) from cardioprotective signaling activators in nondiabetes (ND) to vehicles carrying cytotoxic molecules from DM adipocyte to heart. However, it remains unclear how DM triggers this switch and whether blocking it can protect against DM exacerbation of IHF. Methods and Results: ND or DM (12 weeks of high-fat diet) mice were subjected to MI/R (90 minutes/4 weeks) and treated with Exo (isolated from ND epidydimal fat pads and intramyocardially injected immediately before reperfusion). Exo administration significantly attenuated IHF in ND but not in DM mice. In vitro mechanistic and in vivo concept-prove experiments were conducted to determine how Exo protects against IHF and how DM blocks this protection. In cardiomyocytes (CM) from ND mice, Exo rapidly activated multiple injury salvage kinases (ISKs, including ERK, AMPK, and ACC) and attenuated cell death, suggesting that Exo-surface molecules mediate Exo cardioprotection. Exo-Flow (a method specifically detects Exo surface molecules) experiments demonstrated that adipocyte-derived Exo carries high levels of adiponectin (APN) on their surface. APN antibody-neutralized Exo or APNKO mice-derived Exo neither activated ISKs nor attenuated IHF. Exo failed to activate ISKs and protect IHF in AdipoR1KO mice. These results demonstrated that the adipocyte Exo surface-localized APN and CM-expressed AdipoR1 are essential in protective communication between adipocyte and heart. GRK2, a kinase that phosphorylates AdipoR1 at S205 and blocks APN cardioprotection, was dramatically upregulated in DM hearts. GRK2 overexpression blocked Exo-induced ISKs activation and cellular protection in WT CM but not in AdipoR1 S205A knock-in CM. Finally, Exo administration in AAV9-mediated CM-specific AdipoR1 S205A knock-in mice significantly attenuated diabetic IHF, while it failed to protect in AdipoR1 S205E mice. Conclusions: This study sheds light on the mechanisms underlying DM exacerbation of IHF and highlights the potential of targeting the APN/AdipoR1/GRK2 axis to protect against this condition.

ZBTB16 eases lipopolysaccharide‑elicited inflammation, apoptosis and degradation of extracellular matrix in chondrocytes during osteoarthritis by suppressing GRK2 transcription.

Osteoarthritis (OA) is a chronic degenerative disease of the bone that is a major contributor of disability in the elderly population. Zinc finger and BTB domain-containing 16 (ZBTB16) is a transcription factor that has been previously revealed to be impaired in human OA tissues. The present study was designed to elaborate the potential impact of ZBTB16 on OA and to possibly assess any latent regulatory mechanism. ZBTB16 expression in human OA tissues was examined using the Gene Expression Series (GSE) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE169077) whereas ZBTB16 expression in chondrocytes was examined using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Cell viability was examined using a Cell Counting Kit-8 assay. A TUNEL assay and western blotting were used to assess cell apoptosis and apoptosis-related markers, including Bcl-2, Bax and cleaved caspase-3. The levels and expression of inflammatory factors, including TNF-α, IL-1β and IL-6, were determined by ELISA and western blotting. RT-qPCR and western blotting were also used to analyze the expression levels of extracellular matrix (ECM)-degrading enzymes, including MMP-13, a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5, aggrecan and collagen type II α1. After the potential binding of ZBTB16 with the G protein coupled receptor kinase type 2 (GRK2) promoter was predicted using the Cistrome DB database, GRK2 expression was confirmed by RT-qPCR and western blotting. Chromatin immunoprecipitation and luciferase reporter assays were then used to determine the potential interaction between ZBTB16 and the GRK2 promoter. Following GRK2 overexpression in ZBTB16-overexpressing chondrocytes by co-transfection of GRK2 and ZBTB16 overexpression plasmids, the aforementioned functional experiments were performed again. ZBTB16 expression was found to be reduced in human OA tissues compared with in normal cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. ZBTB16 overexpression increased cell viability whilst decreasing apoptosis, inflammation and ECM degradation by LPS-treated chondrocytes. In addition, GRK2 expression was found to be increased in LPS-stimulated chondrocytes. ZBTB16 successfully bound to the GRK2 promoter, which negatively modulated GRK2 expression. GRK2 upregulation reversed the effects of ZBTB16 overexpression on the viability, apoptosis, inflammation and ECM degradation by LPS-challenged chondrocytes. In conclusion, these data suggest that ZBTB16 may inhibit the development of OA through the transcriptional inactivation of GRK2.

Dynamic Desensitization of Gaq Signaling and Gaq‐dependent GPCR Crosstalk by GRKs

G protein‐coupled receptor (GPCR) signaling is subject to many regulatory mechanisms that occur over a range of time scales. Compared to analysis of slow, trafficking‐based mechanisms relatively little attention has been paid to rapid mechanisms that take place on the seconds time scale. In our study, we utilized live cell calcium imaging to investigate the acute effects of a subfamily of GRKs, GRK2 and 3 on both Gq signaling and Gi/s‐Gq crosstalk. We first focused on group 1 metabotropic glutamate receptors (mGluRs) and found that overexpression of GRK2 and 3 drives an acute desensitization of Gq‐dependent signaling. This desensitization effect occurs on the seconds time scale, is rapidly reversible, and depends on direct interaction between GRK and Gq but is independent of its kinase activity. We find that this mode of rapid desensitization is generalizable across Gq‐coupled GPCRs and is insensitive to the widely used GRK2/3 kinase inhibitor CMPD101. Finally, we studied whether the extent and timing of crosstalk between Gi and Gs‐coupled receptors with Gq‐coupled receptors is also regulated by GRK2. We find that GRK2 dramatically reduces the level of functional crosstalk and accelerated the timing of desensitization of calcium responses to Gi or Gs‐coupled receptor activation. This regulatory effect of GRK on crosstalk is dependent on its interaction with both Gq and G beta gamma, but is independent of its kinase activity as well. Together this work reveals potent forms of GPCR signaling regulation with major implications across physiological contexts.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice

BackgroundG protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia.MethodsThe mice model of inflammatory pain was built by subcutaneous injection of Complete Freund’s Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and downregulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR.ResultsIntraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain.ConclusionThe restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Abstract 10526: Surface Located Adiponectin On Adipocytes-derived Exosomes Mediates Adipocytes/cardiomyocytes Communication And Contributes To Cardioprotection

Introduction and Hypothesis: Increasing evidence emphasizes the roles of adipocyte-cardiomyocyte (ADp-CM) communication plays in health and diabetes. We recently reported that diabetes switched ADp-derived Exosomes (ADp-Exo) from carrying cardioprotective molecules to delivering cardiotoxic molecules to CM. However, the molecular mechanisms mediating ADp-Exo cardioprotection and its alteration by diabetes remain unknown. Methods and Results: ADp-Exo was isolated from epididymal white adipocytes of WT mice. Western Blot demonstrated high level of adiponectin (APN), a potent cardioprotective adipokine, present in ADp-Exo. More interestingly, APN was detected on the surface of ADp-Exo by flow cytometry using Exo-flow kit (System Biosciences). To determine whether surface-located APN on ADp-Exo protect heart, WT ADp-Exo were intramyocardially injected. Mice were subjected to 90 minutes coronary occlusion, followed by 4 weeks of reperfusion. Cardiac function was evaluated by echocardiograph weekly. WT ADp-Exo significantly reduced MI/R injury in vivo , as evidenced by improved LVEF, radial and longitudinal myocardial Strain and SR (vs vehicle group, p<0.05). Importantly, pre-incubation of ADp-Exo with an APN neutralization antibody blocked ADp-Exo cardioprotection. To determine the role of CM AdipoR1, ex vivo and in vitro experiments were performed. WT ADp-Exo activated AdipoR1 pathway (increased phosphorylation of ACC, ERK and AMPK) in CM and protected against cell injury and death (MTT and LDH assay). However, surface APN blocked WT ADp-Exo or APN -/- ADp-Exo failed to active this signaling and protection. WT ADp-Exo failed to activate cell salvage kinases in AdipoR1 -/- CM. Finally, overexpression of GRK2, a pathogenic molecule that we reported to cause AdipoR1 S205 phosphorylation, blocked WT ADp-Exo cardioprotection. AAV9-mediated overexpression of a phosphorylation resistant AdipoR1 (AdipoR1 S205A ) restored ADp-Exo protective action. Conclusions: We demonstrate for the first time that ADp-Exo surface located APN mediates ADp-CM communication via CM AdipoR1 pathway, promoting cardioprotection. This protective system is blocked by GRK2-induced AdipoR1 phosphorylation in diabetic heart, promoting MI/R injury.

Spinal Neuronal GRK2 Contributes to Preventive Effect by Electroacupuncture on Cisplatin-Induced Peripheral Neuropathy in Mice.

BACKGROUND:The main symptoms of chemotherapy-induced peripheral neuropathy (CIPN) include pain and numbness. Neuronal G protein–coupled receptor kinase 2 (GRK2) plays an important role in various pain models. Cisplatin treatment can induce the activation of proinflammatory microglia in spinal cord. The purpose of this study was to investigate the role of spinal neuronal GRK2 in cisplatin-induced CIPN and in the prevention of CIPN by electroacupuncture (EA).METHODS:The pain and sensory deficit behaviors of mice were examined by von Frey test and adhesive removal test. The expression of neuronal GRK2 in the spinal cord is regulated by intraspinal injection of adeno-associated virus (AAV) containing neuron-specific promoters. The protein levels of GRK2, triggering receptor expressed on myeloid cells 2 (TREM2), and DNAX-activating protein of 12 kDa (DAP12) in spinal dorsal horn were detected by Western blot, the density of intraepidermal nerve fibers (IENFs) was detected by immunofluorescence, and microglia activation were evaluated by real-time polymerase chain reaction (PCR).RESULTS:In this study, cisplatin treatment led to the decrease of GRK2 expression in the dorsal horn of spinal cord. Overexpression of neuronal GRK2 in spinal cord by intraspinal injection of an AAV vector expressing GRK2 with human synapsin (hSyn) promotor significantly inhibited the loss of IENFs and alleviated the mechanical pain and sensory deficits induced by cisplatin. Real-time PCR analysis showed that the overexpression of neuronal GRK2 significantly inhibited the messenger RNA (mRNA) upregulation of proinflammatory cytokine interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), and M1 microglia marker cluster of differentiation (CD)16 induced by cisplatin. Furthermore, the TREM2 and DAP12, which has been demonstrated to play a role in microglia activation and in the development of CIPN, were also downregulated by overexpression of neuronal GRK2 in this study. Interestingly, preventive treatment with EA completely mimics the effect of overexpression of neuronal GRK2 in the spinal cord in this mouse model of cisplatin-induced CIPN. EA increased GRK2 level in spinal dorsal horn after cisplatin treatment. Intraspinal injection of AAV vector specifically downregulated neuronal GRK2, completely reversed the regulatory effect of EA on CIPN and microglia activation. All these indicated that the neuronal GRK2 mediated microglial activation contributed to the process of CIPN.CONCLUSIONS:Neuronal GRK2 in the spinal cord contributed to the preventive effect of EA on CIPN. The neuronal GRK2 may be a potential target for CIPN intervention.

Enhanced insulin‐responsive AS160 elicits cardioprotection during metabolic stress

Heart disease is the leading cause of death in the United States, and cardiovascular mortality rates positively correlate with the presence of diabetes and metabolic syndrome. There is a consensus that abdominal obesity is a major driver in the pathogenesis of metabolic syndrome, with severe adverse effects on cardiovascular risk factors. In the midst of a growing recognition of the effect of adipose tissue dysfunction and adipokine imbalance on cardiovascular disease, a less-well understood mechanism is the effect of cardiac metabolism and function on systemic metabolic homeostasis. There is increasing recognition that signals produced by the heart can affect distant organ function; however, little is known regarding mechanistic links. While elevated GRK2 activity and expression occur early in cardiovascular disease and contribute to progression, ongoing studies show cardiac restricted expression of a short, amino terminal fragment of GRK2 (βARKnt) preserves cardiac structure and function during chronic pressure overload. Surprisingly, these studies revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male βARKnt mice at baseline and following cardiac stress. In contrast, cardiac overexpression of GRK2 produces an overall lean phenotype, while the peptide inhibitor of GRK2 (βARKct) enhances the obesogenic phenotype. Proteomic analysis to identify βARKnt binding partners that may underlie the improved cardiovascular and metabolic phenotype uncovered a selective functional interaction of both endogenous GRK2 and βARKnt with AKT substrate of 160kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in βARKnt mice, cardiomyocyte insulin sensitivity is improved during stress, without metabolic switching. These mice demonstrate an ~23% increase in phosphorylated Akt, ~75% increase in phosphorylated and total AS160, and upregulation of the mTORC1 pathway. Seahorse of dissociated adult cardiomyocytes and Oroboros of fresh, permeabilized cardiac tissue exhibited enhanced spare respiratory activity and ATP production in the presence of glucose and palmitate. Unlike control mice that develop insulin resistance in response to high fat diet stress, the βARKnt mice demonstrate enhanced glucose tolerance and improved insulin sensitivity (glucose tolerance test area under curve (AUC) is 2958 vs 2354; insulin tolerance AUC is 1245 vs 991.7 at 14 weeks), increased energy expenditure, and enhanced cardiac function at 4 and 14 weeks. These data suggest that the enhanced AS160-mediated signaling in the βARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism. Ongoing studies seek to elucidate the underlying mechanisms of these beneficial effects, to gain insights into development of new therapeutic strategies for metabolic syndrome and obesity-related cardiovascular disease.

Downregulation of GRK5 hampers the migration of breast cancer cells

Sunitinib is a multispecific kinase inhibitor and one of its targets is the kinase GRK5, which is regulating a multitude of G protein-coupled receptors (GPCRs). In this study we demonstrate that a decreased GRK5 expression induced by knock-down experiments or sunitinib treatment hampers the migration of cancer cell lines. A proteomic analysis revealed many pathways related to cell migration which were down regulated upon the GRK5 knock-down. Furthermore, we found in MDA-MB-231 breast cancer cells that the inhibition of migration is mediated by the GPCR gastrin releasing peptide receptor (GRPR) leading to a reduced expression of migration regulating downstream targets like CDC42 and ROCK1. An in silico Kaplan Meier analysis revealed that GRK5 and GRPR overexpression reduces the distant metastasis free survival in triple-negative breast cancer (TNBC) patients. Thus, we suggest a novel anti-migratory effect of impaired GRK5 expression which induces a negative feedback loop on GRPR signalling.

GRK2 promotes growth of medulloblastoma cells and protects them from chemotherapy-induced apoptosis

G-protein coupled receptor kinase 2 (GRK2; ADRBK1, BARK1) is most known as a regulator of G-protein coupled receptors. However, GRK2 also has other functions. Medulloblastomas are the most common malignant brain cancers in children. GRK2 has not been implicated in medulloblastoma biology. Here we report that GRK2 knockdown slowed cell growth, diminished proliferation, and enhanced cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines UW228-2 and Daoy. Reciprocally, GRK2 overexpression attenuated apoptosis induced by these chemotherapy drugs. Cisplatin and etoposide increased phosphorylation of AKT (S473) and GRK2 knockdown mitigated this increase. Cisplatin and etoposide attenuated ERK phosphorylation, but GRK2 knockdown did not alter this effect. Wildtype GRK2 reversed the increase in cisplatin- and etoposide-induced apoptosis caused by GRK2 knockdown. GRK2-K220R (kinase dead) and GRK2-S670A (unphosphorylated, constitutively active) conferred protection from cisplatin that was similar to wildtype GRK2, suggesting that this protection may be mediated though a kinase-independent activity of GRK2. These data demonstrate that GRK2 contributes to proliferation and survival of these medulloblastoma cell lines and to their protection from cisplatin- and etoposide-induced apoptosis.

Biased agonism of clinically approved μ-opioid receptor agonists and TRV130 is not controlled by binding and signaling kinetics

Binding and signaling kinetics have previously proven important in validation of biased agonism at GPCRs. Here we provide a comprehensive kinetic pharmacological comparison of clinically relevant μ-opioid receptor agonists, including the novel biased agonist oliceridine (TRV130) which is in clinical trial for pain management. We demonstrate that the bias profile observed for the selected agonists is not time-dependent and that agonists with dramatic differences in their binding kinetic properties can display the same degree of bias. Binding kinetics analyses demonstrate that buprenorphine has 18-fold higher receptor residence time than oliceridine. This is thus the largest pharmacodynamic difference between the clinically approved drug buprenorphine and the clinical candidate oliceridine, since their bias profiles are similar. Further, we provide the first pharmacological characterization of (S)-TRV130 demonstrating that it has a similar pharmacological profile as the (R)-form, oliceridine, but displays 90-fold lower potency than the (R)-form. This difference is driven by a significantly slower association rate. Finally, we show that the selected agonists are differentially affected by G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) expression. GRK2 and GRK5 overexpression greatly increased μ-opioid receptor internalization induced by morphine, but only had modest effects on buprenorphine and oliceridine-induced internalization. Overall, our data reveal that the clinically available drug buprenorphine displays a similar pharmacological bias profile in vitro compared to the clinical candidate drug oliceridine and that this bias is independent of binding kinetics suggesting a mechanism driven by receptor-conformations.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Abstract 2053: Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma

Abstract Background: G protein receptor kinase-2 (GRK2; ADRBK1) belongs to the family of G protein-coupled receptor (GPCR) kinases (GRKs). GRK2 phosphorylates GPCR and non-GPCR targets including cell surface receptors, cytoskeletal and mitochondrial proteins and transcription factors. It also functions in embryonic development, heart function, metabolism, and some cancer models. Here we hypothesized that targeting GRK2 will sensitizes cancer cells to therapy. We tested this in medulloblastoma, the most common malignant brain tumor in children. Methods: siRNA transfections, shRNA viral infections, SDS-PAGE, western blotting, flow cytometry. Cells: UW228 and DAOY medulloblastoma. Results: We found that GRK2 inhibition promotes anti-tumor effects of chemotherapeutic drugs cisplatin and etoposide in UW228 and DAOY medulloblastoma cell lines. GRK2 silencing increased apoptosis induced by cisplatin and etoposide and GRK2 overexpression decreased apoptosis induced by these drugs in UW228 and DAOY. Time dependent studies revealed that GRK2 overexpression delayed cisplatin- and etoposide-induced apoptosis measured by the induction of Annexin V positive population and by PARP-1 cleavage. GRK2 mutants, GRK2 K220R (kinase deficient mutant) and GRK2 S670A (cannot be phosphorylated) were similar to wildtype GRK2 in delaying the cisplatin and etoposide induced apoptosis. GRK2 knockdown antagonized serum induced AKT and ERK phosphorylation in both lines whereas GRK2 overexpression promoted phosphorylation, suggesting that GRK2 is important in medulloblastoma survival pathways. Conclusions: These results demonstrate that targeting GRK2 enhances cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines and suggests that GRK2 inhibitors may enhance responsiveness of medulloblastoma to chemotherapy. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Gregory M. Shackleford, Anat Erdreich-Epstein. Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2053.

Alteration of myocardial GRK2 produces a global metabolic phenotype.

A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. In recent years, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Here, we describe the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgβARKct) display an enhanced obesogenic phenotype when fed a high fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD fed TgβARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics we found that cardiac GRK2 signaling altered myocardial BCAA and endocannabinoid metabolism and modulated circulating BCAA and endocannabinoid metabolite profiles on a HFD, and one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. Taken together, these results suggest that metabolic changes in the heart due to GRK2 signaling on a HFD control whole body metabolism.

Insulin Resistance-Related Proteins Are Overexpressed in Patients and Rats Treated With Olanzapine and Are Reverted by Pueraria in the Rat Model.

Olanzapine, a commonly used second-generation antipsychotic, causes severe metabolic adverse effects, such as elevated blood glucose and insulin resistance (IR). Previous studies have proposed that overexpression of CD36, GGPPS, PTP-1B, GRK2, and adipose triglyceride lipase may contribute to the development of metabolic syndrome, and Pueraria could eliminate the metabolic adverse effects. The study aimed to investigate the association between olanzapine-associated IR and IR-related proteins (IRRPs) and determine the role of Pueraria in protection against the metabolic adverse effects of olanzapine. The expression levels of IRRPs were examined in schizophrenia patients and rat models with long-term olanzapine treatment. The efficacy of Pueraria on anti-IR by reducing the expression of IRRPs was comprehensively evaluated. Our study demonstrated that in schizophrenia patients chronically treated with olanzapine, the expression levels of IRRPs in patients with a high IR index significantly increased, and these phenomena were further confirmed in a rat model. The expression levels of IRRPs were reduced significantly in Pueraria-treated IR rat models. The body weight, blood glucose, and IR index were restored to levels similar to those of normal controls. The IRRPs are closely related to IR induced by olanzapine, and Pueraria could interfere with olanzapine-associated IR and revert overexpressed IRRPs. These findings suggest that IRRPs are key players in olanzapine-associated IR and that Pueraria has potential as a clinical drug to prevent the metabolic adverse effects of olanzapine, further improving compliance of schizophrenia patients.

Cannabinoid CB1 and CB2 Receptor-Mediated Arrestin Translocation: Species, Subtype, and Agonist-Dependence.

Arrestin translocation and signaling have come to the fore of the G protein-coupled receptor molecular pharmacology field. Some receptor–arrestin interactions are relatively well understood and considered responsible for specific therapeutic or adverse outcomes. Coupling of arrestins with cannabinoid receptors 1 (CB1) and 2 (CB2) has been reported, though the majority of studies have not systematically characterized the differential ligand dependence of this activity. In addition, many prior studies have utilized bovine (rather than human) arrestins, and the most widely applied assays require reporter-tagged receptors, which prevent meaningful comparison between receptor types. We have employed a bioluminescence resonance energy transfer (BRET) method that does not require the use of tagged receptors and thereby allows comparisons of arrestin translocation between receptor types, as well as with cells lacking the receptor of interest – an important control. The ability of a selection of CB1 and CB2 agonists to stimulate cell surface translocation of human and bovine β-arrestin-1 and -2 was assessed. We find that some CB1 ligands induce moderate β-arrestin-2 translocation in comparison with vasopressin V2 receptor (a robust arrestin recruiter); however, CB1 coupling with β-arrestin-1 and CB2 with either arrestin elicited low relative efficacies. A range of efficacies between ligands was evident for both receptors and arrestins. Endocannabinoid 2-arachidonoylglycerol stood out as a high efficacy ligand for translocation of β-arrestin-2 via CB1. Δ9-tetrahydrocannabinol was generally unable to elicit translocation of either arrestin subtype via CB1 or CB2; however, control experiments revealed translocation in cells not expressing CB1/CB2, which may assist in explaining some discrepancy with the literature. Overexpression of GRK2 had modest influence on CB1/CB2-induced arrestin translocation. Results with bovine and human arrestins were largely analogous, but a few instances of inconsistent rank order potencies/efficacies between bovine and human arrestins raise the possibility that subtle differences in receptor conformation stabilized by these ligands manifest in disparate affinities for the two arrestin species, with important potential consequences for interpretation in ligand bias studies. As well as contributing important information regarding CB1/CB2 ligand-dependent arrestin coupling, our study raises a number of points for consideration in the design and interpretation of arrestin recruitment assays.

GRK5‐mediated inhibitory phosphorylation is essential for inverse agonism at the cardiac mineralocorticoid receptor

BackgroundIn the heart, aldosterone binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. GRK2 and GRK5 are the most abundant cardiac G protein‐coupled receptor (GPCR)‐kinases (GRKs). Both phosphorylate GPCRs but also non‐GPCR substrates. We recently showed that GRK5 blocks the cardio‐toxic MR‐dependent effects of aldosterone in the heart by directly phosphorylating the cardiac MR and inhibiting its transcriptional activity. MR antagonist (MRA) drugs are beneifical in human CHF by blocking the cardiac MR. Novel non‐steroidal MRAs, such as finerenone, may provide better cardio‐protection against aldosterone's cardio‐toxic actions than the classic steroidal MRAs, such as sprironolactone and eplerenone. Indeed, finerenone was very recently shown to be a more potent and efficacious inverse agonist at the MR than eplerenone in terms of cardiac fibrosis/adverse remodeling attenuation. Thus, in the present study, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5‐dependent cardiac MR phosphorylation and subsequent blockade.MethodsWe used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). We measured MR phosphorylation via phospho‐serine immunoblotting & transcriptional activity via the luciferase reporter assay.ResultsGRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Contrary to eplerenone, finerenone alone potently and efficiently suppresses MR transcriptional activity in H9c2 cardiac myocytes. GRK5‐dependent phosphorylation is necessary for the robust inverse agonism displayed by finerenone at the cardiac MR, since CRISPR‐mediated GRK5 gene deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Conversely, this non‐steroidal MRA fully suppresses cardiac MR transcriptional activity upon GRK5 overexpression in H9c2 cardiomyocytes. Eplerenone alone cannot suppress cardiac MR basal activity upon GRK5 overexpression or knockout. Finally, finerenone blocks aldosterone's effects more efficiently than eplerenone in control and GRK5‐overexpressing NRVMs.ConclusionsFinerenone appears a more efficacious cardiac MR blocker than eplerenone, partly via GRK5‐dependent cardiac MR inhibition, which eplerenone does not induce. This non‐canonical effect of GRK5 on the MR is essential for the efficient blockade of aldosterone's deleterious actions in the heart.Support or Funding Information1) American Heart Association 2) NSU President's Faculty Research & Development GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract WP549: GRK5-Mediated Inhibitory Phosphorylation of the Mineralocorticoid Receptor Protects Against Aldosterone`s Stroke-Promoting Vascular Effects

Introduction: Stroke is one of the leading causes of death in the Western world. Mineralocorticoid receptor (MR) antagonism is a potential therapy for ischemic and hemorrhagic stroke patients, since aldosterone exerts deleterious effects in the cerebral vasculature via the MR. Prominent among these is activation of epidermal growth factor (EGF)-dependent vessel wall remodeling. GRK2 and GRK5 are the most abundant G protein-coupled receptor (GPCR)-kinases (GRKs) in the heart and vessels and both phosphorylate GPCRs but also non-GPCR substrates at Ser/Thr residues. The human MR gets phosphorylated at various Ser residues, including Ser-843 in the ligand binding domain (LBD) and Ser-601 in the N-terminal domain (NTD). These Ser phosphorylations occur in the cytoplasm and result in MR inhibition via direct transcriptional activity repression or cytosolic retention, respectively. Hypothesis: GRK5 phosphorylates and inhibits the MR in the heart and vessels. Methods: We used rat cardiomyocytes (H9c2) and aortic smooth muscle cells (ASMCs). We checked for GRK interactions with the MR via co-immunoprecipitation. We measured MR phosphorylation via phospho-Ser immunoblotting and MR transcriptional activity via the luciferase reporter assay. We also measured EGF and EGF receptor (EGFR) expression via real time-PCR. Results: GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. Beta2-adrenoceptor activation stimulates this non-canonical effect of GRK5. The GRK5-phosphorylated MR is incapable of activating gene transcription, since aldosterone-induced MR transcriptional activity is markedly suppressed upon GRK5 overexpression in both cardiomyocytes and ASMCs. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac and ASMC aldosterone-dependent MR transcriptional activity. Finally, beta2-adrenoceptor-stimulated GRK5 phosphorylates and inhibits the MR in ASMCs, thereby suppressing aldosterone-induced EGF and EGFR mRNA expression. Conclusions: GRK5 blocks the toxic MR-dependent effects of aldosterone in the heart and vascular smooth muscles. Thus, GRK5 stimulation (e.g. via beta2-adrenoceptor activation) may protect against the stroke-promoting effects of aldosterone in the cerebral vasculature.

GRK5 promotes tumor progression in renal cell carcinoma.

GRK5 is a multifunctional protein that is able to move within the cell in response to various stimuli to regulate key intracellular signaling from receptor activation, on plasmamembrane, to gene transcription, in the nucleus. Thus, GRK5 is involved in the development and progression of several pathological conditions including cancer. Here, we report an important tumor-promoting role for GRK5 in renal cell carcinoma (RCC). We investigated the expression pattern, clinical significance, and function of GRK5 in RCC. By using quantitative real-time polymerase chain reaction (qRT-PCR) and tissue microarray (TMA) immunohistochemistry (IHC), we first demonstrated that compared with paired adjacent nontumor (NT) tissues, RCC tissues presented with higher GRK5 expression. Moreover, we found that GRK5 upregulation was associated with poor clinical outcomes in RCC patients. In vitro, we found that GRK5 knockdown reduced viability, invasive ability, migratory ability, and decreased proportion of cells in S phase, with concomitant increase in G1 phase in RCC cell lines, while GRK5 overexpression promoted tumor cell proliferation, cell invasion, migration and increased proportion of cells in S phase, with concomitant decrease in G1 phase. Collectively, our findings describe the tumour-promoting role of GRK5 in RCC and thus provide molecular evidence for new therapeutic options in RCC.