GRK2 Protein Levels Research Articles

GRK2-mediated AKT activation controls cell cycle progression and G2 checkpoint in a p53-dependent manner

Cell cycle checkpoints, activated by stressful events, halt the cell cycle progression, and prevent the transmission of damaged DNA. These checkpoints prompt cell repair but also trigger cell death if damage persists. Decision-making between these responses is multifactorial and context-dependent, with the tumor suppressor p53 playing a central role. In many tumor cells, p53 alterations lead to G1/S checkpoint loss and the weakening of the G2 checkpoint, rendering cell viability dependent on the strength of the latter through mechanisms not fully characterized. Cells with a strong pro-survival drive can evade cell death despite substantial DNA lesions. Deciphering the integration of survival pathways with p53-dependent and -independent mechanisms governing the G2/M transition is crucial for understanding G2 arrest functionality and predicting tumor cell response to chemotherapy. The serine/threonine kinase GRK2 emerges as a signaling node in cell cycle modulation. In cycling cells, but not in G2 checkpoint-arrested cells, GRK2 protein levels decline during G2/M transition through a process triggered by CDK2-dependent phosphorylation of GRK2 at the S670 residue and Mdm2 ubiquitination. We report now that this downmodulation in G2 prevents the unscheduled activation of the PI3K/AKT pathway, allowing cells to progress into mitosis. Conversely, higher GRK2 levels lead to tyrosine phosphorylation by the kinase c-Abl, promoting the direct association of GRK2 with the p85 regulatory subunit of PI3K and AKT activation in a GRK2 catalytic-independent manner. Hyperactivation of AKT is conditioned by p53’s scaffolding function, triggering FOXO3a phosphorylation, impaired Cyclin B1 accumulation, and CDK1 activation, causing a G2/M transition delay. Upon G2 checkpoint activation, GRK2 potentiates early arrest independently of p53 through AKT activation. However, its ability to overcome the G2 checkpoint in viable conditions depends on p53. Our results suggest that integrating the GRK2/PI3K/AKT axis with non-canonical functions of p53 might confer a survival advantage to tumor cells.

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.

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 11977: Dapagliflozin Downregulates Adrenal G Protein-Coupled Receptor-Kinase-2 to Exert Sympatholysis in Heart Failure

Introduction: Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor exerting several cardiorenal protective effects independently of blood glucose regulation. Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity, especially in heart failure (HF). GRK2 severely dysregulates adrenal sympatho-inhibitory α 2 -adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Hypothesis: SGLT2 inhibition with dapagliflozin lowers SNS hyperactivity in part by opposing adrenal GRK2 actions. Methods: We used the rat pheochromocytoma PC12 cell line expressing human α 2A AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Results: 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, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2 AR 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). GRK2 and TH downregulation were also observed in PC12 cells treated with 5 μM dapagliflozin for 24 hours. Importantly, α 2 AR-dependent inhibition of CA secretion was markedly enhanced 24 hrs post-dapagliflozin application. This was due to reduced GRK2-dependent receptor desensitization induced by dapagliflozin, since this effect was abolished in PC12 cells overexpressing GRK2. Conclusions: Dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, thus reducing CA biosynthesis, and GRK2, thus reducing α 2 AR desensitization to lower CA secretion. This adrenal-mediated sympatholysis may be part of the increasingly apparent cardiovascular benefits of SGLT2 inhibitors in human HF irrespective of diabetic status.

Dapagliflozin Exerts Adrenal Sympatholysis via G protein-Coupled Receptor Kinase-2 & Tyrosine Hydroxylase Downregulation

Abstract The sodium-glucose co-transporter (SGLT)-2 inhibitor dapagliflozin was recently reported to reduce renal tyrosine hydroxylase (TH) and norepinephrine levels, lowering blood pressure and preventing endothelial dysfunction, in a murine model of neurogenic hypertension. This suggested that dapagliflozin may combat sympathetic nervous system (SNS) hyperactivity, which is known to accompany and aggravate chronic heart failure (CHF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and SNS hyperactivity, especially in CHF. GRK2 severely dysregulates adrenal sympatho-inhibitory α 2-adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Therefore, we hypothesized herein that SGLT2 inhibition with dapagliflozin may lower SNS hyperactivity in the adrenal gland by antagonizing GRK2 actions on α 2ARs in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α 2AAR, as well as freshly isolated adrenal glands from adult male Sprague-Dawley rats treated with dapagliflozin in vivo. We measured circulating norepinephrine (NE) in vivo via RIA, GRK2 & TH expression levels via real-time PCR and immunoblotting, adrenal α 2AR density (Bmax) via saturation radioligand binding with [methyl-3H]-rauwolscine, and G protein activation via the GTPγS assay. 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 TH mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2AR 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 and TH downregulation) were completely recapitulated in PC12 α 2AAR-expressing cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α 2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced at 24 hrs post-dapagliflozin application in PC12 cells. This was the result of reduced GRK2-dependent receptor desensitization, since dapagliflozin lacked this effect in cells co-transfected with a GRK2-encoding adenovirus to acutely overexpress GRK2. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, which reduces CA biosynthesis, and GRK2, which reduces α 2AR desensitization in favor of enhanced inhibition of CA secretion.

Sex Differences in High Fat Diet-Induced Metabolic Alterations Correlate with Changes in the Modulation of GRK2 Levels.

A differential sex-related sensitivity has been reported in obesity and insulin resistance-related cardio-metabolic diseases, with a lower incidence of these pathologies being observed in young females when compared to age-matched males. However, such relative protection is lost with age. The mechanisms underlying such sex and age-related changes in the susceptibility to diabetes and obesity are not fully understood. Herein, we report that the relative protection that is displayed by young female mice, as compared to male littermates, against some of the metabolic alterations that are induced by feeding a high fat diet (HFD), correlates with a lower upregulation of the protein levels of G protein-coupled receptor kinase (GRK2), which is a key regulator of both insulin and G protein-coupled receptor signaling, in the liver and adipose tissue. Interestingly, when the HFD is initiated in middle-aged (32 weeks) female mice, these animals are no longer protected and display a more overt obese and insulin-resistant phenotype, along with a more evident increase in the GRK2 protein levels in metabolically relevant tissues in such conditions. Our data suggest that GRK2 dosage might be involved in the sex and age-biased sensitivity to insulin resistance-related pathologies.

Low GRK2 Underlies Hyperalgesic Priming by Glial Cell-Derived Neurotrophic Factor.

Background: We recently identified the balance between the level of G protein coupled receptor kinase 2 (GRK2) and Epac1 in nociceptors as a key factor in the transition from acute to chronic pain that occurs in mice ‘primed’ by an inflammatory stimulus. Here, we examined the contribution of GRK2 and Epac-signaling to growth factor-induced hyperalgesic priming.Methods: Mice were primed by intraplantar injection with glial cell-derived neurotrophic factor (GDNF). Mechanical allodynia in response to PGE2 was followed over time in primed and non-primed animals. GRK2 protein levels in dorsal root ganglion (DRG) neurons were quantified by immunohistochemistry. The effect of herpes simplex virus (HSV)-GRK2 amplicons to restore GRK2 levels or of an Epac inhibitor on PGE2 allodynia in primed mice was examined.Results: Glial cell-derived neurotrophic factor-induced hyperalgesia disappeared within 12 days. The hyperalgesic response to a subsequent intraplantar injection of PGE2 was prolonged from <24 h in control mice to more than 72 h in GDNF-primed mice. In male and female primed mice, PGE2 hyperalgesia was inhibited by oral administration of the Epac inhibitor ESI-09, while the drug had no effect in control mice. Mice primed with GDNF had reduced levels of GRK2 in IB4(+) small DRG neurons, but normal GRK2 levels in IB4(-) DRG neurons. Intraplantar administration of HSV-GRK2 amplicons to increase GRK2 protein levels prevented the prolongation of PGE2-induced hyperalgesia in GDNF-primed mice.Conclusion: Low GRK2 in nociceptors is critical to develop a primed state in response to GDNF and leads to engagement of Epac signaling and transition to chronic PGE2-induced hyperalgesia. Increasing GRK2 protein or inhibiting Epac signaling may represent new avenues for preventing transition to a chronic pain state.

G protein Coupled Receptor Kinase 2 Levels Regulate Differential Internalization of C-C Chemokine Receptor 7 (CCR7) in Primary Mouse T Cells

Abstract G protein-coupled receptor (GPCR) signaling can be regulated by phosphorylation via GPCR kinases (GRKs) and recruitment of b-arrestins. The C-C chemokine receptor 7 (CCR7) GPCR, has two ligands, CCL19 and CCL21 that promote chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct signaling pathways are activated in T cells depending upon which ligand is bound to the CCR7. It remains unclear to what extent GRKs contribute to differential internalization of ligand-bound CCR7 and subsequent signaling that controls T cell migration. We used naïve primary T cells from wild type C57 BL/6 mice (WT) or syngeneic mice heterozygous for the GRK2 gene that express 50% GRK2 protein levels compared to WT mice. T cells were incubated with 40 nM of CCL19 or CCL21 for up to 8 minutes. Incubation with CCL19, but not CCL21, led to CCR7 internalization; however, both ligands facilitated rapid, sustained CCR7 internalization in GRK2+/− T cells. The reduced GRK2 levels in the heterozygote T cells had no significant effect on migration to CCL19 or CCL21 compared to WT T cells. Titration transfection experiments in HEK293T cells or CRISPR-Cas9 knockdown of GRK2 in CEM T cells suggested that GRK2 is required for ligand-mediated CCR7 internalization, while higher GRK2 levels inhibit the internalization process. CCR7 internalization was blocked following transfection of a kinase dead GRK2 mutant indicating that GRK2 phosphorylation is required for CCR7 internalization. Immunomicroscopy suggested arrestin-3 formed clusters, independent of the ligand used. Overall, these data suggest that cellular concentration of GRK2 plays an important role in the internalization of ligand/CCR7.

Abstract 254: MicroRNA Regulation of G Protein-Coupled Receptor Kinase 2 After Cardiac Injury

The development and progression of heart failure (HF) involves many processes that occur after cardiac stress and/or injury. Compensatory neuro-hormonal activation and chronic stimulation of the sympathetic nervous system (SNS) is accompanied by the pathological increase in G protein-coupled receptor kinase (GRK) 2 expression. Data indicates that elevated GRK2 activity participates in the progression of HF after cardiac injury such as a myocardial infarction (MI) and this includes chronic β 1 -adrenergic receptor (β 1 AR) desensitization. The molecular transcriptional or translational mechanism of GRK2 upregulation in the heart is largely unknown. MicroRNAs (miRNAs) have been discovered as critical regulatory molecules in the heart as their expression profiles become dysregulated after cardiac injury. Further, miRNAs can play key roles in orchestrating many phenotypic changes observed in HF. Previous studies have shown that components of the βAR pathway, including the β 1 AR, are targeted by miRNAs whose levels are aberrant during cardiac injury. This study was started to determine if GRK2 may also be targeted by a miRNA that goes down after injury, participating in the up-regulation of this pathological kinase. MiRNA profiles of 2 week post-MI C57BL/6 mice were examined through miRNA microarray analysis. MiRNAs that were down-regulated after MI and had seed sequence complementarity to GRK2’s 3’UTR were selected as candidates for further testing. MiR-378a-5p (miR-378a) was chosen as a suitable candidate and transfection of a miR-378a mimic into neonatal rat ventricular myocytes was able to significantly reduce GRK2 protein levels compared to a negative control, while mRNA levels seem to be unaffected. This suggests miR-378a may work primarily through translational inhibition rather than transcriptional inhibition. Using a 3’UTR reporter system, we found that miR-378a was able to directly bind to the 3’UTR of GRK2 and inhibit reporter activity. These data support the hypothesis that GRK2 is regulated by a miRNA in the heart and provides a possible mechanism for GRK2 post-transcriptional regulation . In vivo studies are currently in progress to determine if miR-378a may be a novel therapeutic strategy to decrease GRK2 levels in the failing heart.

P2Y2 receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX-2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y(2) mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y(2) and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction.

G protein–coupled receptor kinase 2 (GRK2) modulation and cell cycle progression

Cell cycle progression requires changes in the activity or levels of a variety of key signaling proteins. G protein-coupled receptor kinase 2 (GRK2) plays a central role in G protein-coupled receptor regulation. Recent research is uncovering its involvement in additional cellular functions, but the potential role of GRK2 in the cell cycle has not been addressed. We report that GRK2 protein levels are transiently down-regulated during the G2/M transition by a mechanism involving CDK2-mediated phosphorylation of GRK2 at Serine670, which triggers binding to the prolyl-isomerase Pin1 and subsequent degradation. Prevention of GRK2 phosphorylation at S670 impedes normal GRK2 down-regulation and markedly delays cell cycle progression. Interestingly, we find that endogenous GRK2 down-regulation is prevented on activation of the G2/M checkpoint by doxorubicin and that stabilized GRK2 levels in such conditions inversely correlate with the p53 response and the induction of apoptosis, suggesting that GRK2 participates in the regulatory network controlling cell cycle arrest and survival in such conditions.

Normalization of GRK2 protein and mRNA measures in patients with depression predict response to antidepressants

G-protein-coupled receptor kinases (GRKs) interfere in receptor-G-protein coupling leading to desensitization of G-protein-mediated receptor signalling. G-protein-coupled receptor signalling and its desensitization were previously implicated in the pathophysiology, diagnosis and treatment of mood disorders. The present study aimed to evaluate alterations in GRK2 protein and mRNA levels in mononuclear leukocytes (MNL) of untreated patients with major depression and the effects and time-course of antidepressant treatments on these alterations. Repeated GRK2 protein and mRNA measurements were carried in MNL of 24 patients with major depression. Each patient was examined while untreated and after 1, 2, 3 and 4 wk of antidepressant treatment; 24 healthy subjects were also studied. GRK2 protein and mRNA levels were evaluated through immunoblot analyses using monoclonal antibodies against GRK2 and reverse transcriptase-polymerase chain reaction, respectively. GRK2 protein and mRNA levels in MNL of untreated patients with major depression were significantly lower than the measures characterizing healthy subjects. The decreased GRK2 protein and mRNA levels were alleviated by antidepressant treatment. Normalization of GRK2 measures preceded, and, thus, could predict clinical improvement by 1-2 wk. These findings support the implication of GRK2 in the pathophysiology of major depression and in the mechanism underlying antidepressant-induced receptor down-regulation and therapeutic effects. GRK2 measurements in patients with depression may potentially serve for biochemical diagnostic purposes and for monitoring and predicting response to antidepressants.

Toll-like receptors differentially regulate GPCR kinases and arrestins in primary macrophages

G-protein coupled receptor kinases (GRKs) and arrestins (ARRs) are ubiquitously distributed crucial signaling proteins that are critical in the regulation of responsiveness of G-protein coupled receptors (GPCRs). Toll-like receptors (TLRs) (class of pattern recognition receptors) play a vital role in macrophage biology and innate immunity. Because GPCR responsiveness is regulated in part by the expression levels of GRKs/ARRs, the focus of this work was to uncover potential cross-talk mechanisms between TLRs and GPCRs via regulation of GRK/ARR expression in primary mouse macrophages. We demonstrate here that activation of TLR2 and 4 (but not TLR3 and 7) significantly decrease ARR2 but not ARR3 protein levels in macrophages. Compared to this, activation of TLR2, 4, and 7 (but not TLR3) significantly decrease GRK5 and 6 protein levels. Surprisingly, GRK2 protein levels are markedly increased by TLR2, 3, 4 and 7. Mechanistically, expression of ARR2 and GRK5 are regulated at transcriptional as well as post-translational levels. Downregulation of GRK6 by LPS is regulated primarily at the post-translational level. TLR4-induced GRK2 level, however, is both transcriptionally and post-transcriptionally regulated. Our results demonstrate previously unknown crucial regulatory mechanisms that alter ARR/GRK expression levels in macrophages that might modify many, if not all, GPCR-mediated innate immune responses.

Lymphocyte Levels of GRK2 (βARK1) Mirror Changes in the LVAD-Supported Failing Human Heart: Lower GRK2 Associated With Improved β-Adrenergic Signaling After Mechanical Unloading

Background In human heart failure, increased expression of G protein–coupled receptor kinases (GRKs) causes the loss of β-adrenergic receptor (βAR) signaling and function. Mechanical unloading with a left ventricular assist device (LVAD) promotes reverse remodeling, which includes restoration of βAR responsiveness. We tested the hypothesis that LVAD support of the failing human heart alters the expression and activity of GRKs and we sought to determine whether changes in myocardial GRKs could be tracked in lymphocytes. Methods and Results Paired samples of human LV tissue (n = 12) and blood were obtained at the time of LVAD implantation (heart failure) and subsequent cardiac transplantation (LVAD). βAR signaling was quantified by receptor density and adenylyl cyclase activity. Immunoblotting and real-time reverse transcription polymerase chain reaction were used to measure GRK2 and GRK5 protein and mRNA levels. Rhodopsin phosphorylation was used to assess total GRK activity. Consistent with reverse remodeling, βAR density and signaling were restored to nonfailing levels after LVAD support. GRK2 protein levels were significantly reduced 55% after LVAD support and GRK2 mRNA was similarly reduced. In contrast, GRK5 protein and mRNA levels were unchanged. Total myocardial GRK activity was reduced similar to the drop in GRK2 expression. In lymphocytes, GRK2 protein levels were decreased after LVAD support and there was a significant positive correlation between myocardial and lymphocyte GRK2 levels in both heart failure and LVAD samples. Conclusion The changes in myocardial GRK2 expression and activity that are mirrored in lymphocytes provide a possible mechanism for the restoration of βAR signaling and reverse remodeling after mechanical unloading in the failing heart. Moreover, lymphocytes may provide a surrogate marker of myocardial GRK2 in these patients.

Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4

Chemokine receptors belong to the family of G-protein-coupled receptors (GPCR). Phosphorylation of GPCR by GPCR kinases (GRKs) is considered to play an important role in desensitization of these receptors. We have recently shown in patients with rheumatoid arthritis that the level of GRK2 in lymphocytes is reduced by approximately 50%. However, the physiological relevance of reduced GRK2 levels in lymphocytes is not known. Here, we investigated whether reduced GRK2 expression changes the chemotactic response of T cells to the chemokines CCL3, CCL4, and CCL5. Activated T cells from GRK2+/- mice, which have a 50% reduction in GRK2 protein levels, showed a significant 40% increase in chemotaxis toward the CCR5 ligand CCL4. In addition, chemotaxis toward the CCR1 and CCR5 ligands CCL3 and CCL5 was also increased. Binding of CCL4 to activated T cells from GRK2+/- and wild-type (WT) mice was similar, but agonist-induced CCR5 phosphorylation was attenuated in GRK2+/- cells. Moreover, the calcium response and phosphorylation of protein kinase B and extracellular-regulated kinase in response to CCL4 were significantly increased in GRK2+/- T cells, showing that signaling is increased when the level of GRK2 is reduced. GRK2+/- and WT cells do become refractory to restimulation with CCL4. In conclusion, a 50% decrease in T cell GRK2 expression results in increased responsiveness to CCL3, CCL4, and CCL5, suggesting that the 50% reduction in lymphocyte GRK2 level as observed during inflammation can have functional consequences for the response of these cells to chemokines.

Chronic opioid antagonist treatment dose-dependently regulates μ-opioid receptors and trafficking proteins in vivo

Chronic opioid antagonist treatment increases the density of μ-opioid receptors (μOR) in many model systems. In previous studies, naltrexone treatment produced an increase in μOR density accompanied by decreases in GRK-2 and DYN-2 protein abundance. To examine the relationship between changes in receptor density and proteins involved in receptor trafficking, the dose-dependent effect of chronic naloxone infusion was determined. Dose-dependent antagonism of morphine analgesia was also examined. Mice were infused with naloxone (0.1, 1.0, 5.0 mg/kg/day sc) for 7 days via osmotic pump. Controls were treated with placebo pellets. On the 7th day, morphine dose–response studies were determined using the tail flick. Other mice were sacrificed at the end of the treatment and spinal cords were collected for determination of μOR density and GRK-2 and DYN-2 protein abundance. Naloxone infusion dose-dependently increased spinal μOR density with no change in affinity. The increases in μ-receptor density were proportional to dose-dependent decreases in GRK-2 and DYN-2 protein levels. Furthermore, naloxone dose-dependently antagonized morphine. These data suggest that opioid antagonist-induced μOR up-regulation in mouse spinal cord is associated with regulation of proteins involved in receptor trafficking and support suggestions that opioid antagonist-induced receptor up-regulation is due to reduced constitutive internalization of opioid receptors.

Oxidative stress decreases G protein-coupled receptor kinase 2 in lymphocytes via a calpain-dependent mechanism.

G protein-coupled receptor kinase (GRK) 2 plays a crucial role in regulating the extent of desensitization and resensitization of G protein-coupled receptors (GPCRs). We have shown that the expression level of GRK2 in lymphocytes decreases during inflammatory diseases such as arthritis. Reactive oxygen species play an important role in a variety of inflammatory conditions, including arthritis. We demonstrate herein that oxidative stress, induced by exposure of lymphocytes to H(2)O(2), results in a 50% reduction in GRK2 protein levels and GRK activity with no changes in mRNA expression. Treatment of lymphocytes with the tyrosine kinase inhibitor genistein partially reverses the effect of H(2)O(2) on GRK2 levels, although we did not detect direct tyrosine phosphorylation of GRK2. Inhibition of the nonproteasomal protease calpain by calpeptin can prevent the H(2)O(2)-induced GRK2 decrease. In vitro experiments confirm that GRK2 is partially digested by m-calpain in a calcium-dependent way. Functionally, H(2)O(2)-induced decrease in GRK2 levels is associated with an ~70% decrease in agonist-induced beta(2)-adrenergic receptor sequestration. We describe oxidative stress as a novel mechanism for regulation of the intracellular level of GRK2 during inflammatory processes. Moreover, our data demonstrate that oxidative stress may change the functioning of GPCRs via calpain-dependent regulation of GRK2 levels.

Expression and activity of g protein-coupled receptor kinases in differentiated thyroid carcinoma.

Most of the TSH effects on the proliferation and differentiation of thyroid cells are mediated by cAMP via an adenylyl cyclase-activating Gs protein. TSH receptor responsiveness in cell cultures, is regulated by G protein-coupled receptor kinase (GRK) 2 and 5. To determine whether an alteration in activity and expression of GRKs might be associated with variable levels of TSH receptor desensitization in vivo, we studied human thyroid tissues including 21 normal tissues and 18 differentiated carcinomas. GRK activity was assayed by rhodopsin phosphorylation, and GRK protein and mRNA expressions assessed by immunoblotting and real-time quantitative RT-PCR, respectively. GRK2 and GRK5 were found as the predominant isoforms in the human thyroid. GRK5 protein expression was significantly decreased in differentiated thyroid carcinoma (P < 0.02) and paralleled a decrease in GRK mRNA expression (P < 0.02). In contrast, no difference in protein and mRNA levels of GRK2 were observed between normal and cancerous thyroid tissues. Although GRK2 protein levels correlated with GRK activities, we demonstrated a significant increase in GRK activity in differentiated thyroid carcinoma (P < 0.02). Less TSH receptor desensitization occurred in differentiated carcinoma than in normal thyroid tissue, as judged by TSH-stimulated cAMP response in human thyroid cells in primary culture. In conclusion, this study indicates that GRK2 activity and GRK5 expression have opposite regulations in cancer cells. Furthermore, the decrease in GRK5 expression may underlie the reduction in homologous desensitization of the TSH receptor in differentiated thyroid carcinoma, contributing to explain the increased cAMP levels in these tumors.

Effect of hypothyroidism on G protein-coupled receptor kinase 2 expression levels in rat liver, lung, and heart.

GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of beta-adrenergic and other G protein-coupled receptors and plays an important role in their desensitization and modulation. Alterations in thyroid hormone levels have been reported to lead to important changes in adrenergic receptor responsiveness and signaling in a variety of tissues. In this context, we have explored the effects of experimental hypothyroidism on GRK2 protein levels in rat heart, lung, and liver using a specific antibody. Hypothyroid animals show significant up-regulation ( approximately 50% increase compared with controls) in GRK2 levels in heart and lung at 60 days after birth, whereas a 50% reduction is detected in the liver at this stage. These alterations are selective, as beta-adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or beta-arrestin-1, display a different pattern of expression changes in the hypothyroid animals. The reported changes in GRK2 levels and in the receptor/kinase ratio predict alterations in adrenergic receptor desensitization and signal transduction efficacy consistent with those observed in thyroid disorders, thus suggesting a relevant role for the modulation of GRK2 expression in this physiopathological condition.

Decreased expression and activity of G-protein-coupled receptor kinases in peripheral blood mononuclear cells of patients with rheumatoid arthritis.

Beta2-Adrenergic and chemokine receptor antagonists delay the onset and reduce the severity of joint injury in rheumatoid arthritis. beta2-Adrenergic and chemokine receptors belong to the G-protein-coupled receptor family whose responsiveness is turned off by the G-protein-coupled receptor kinase family (GRK-1 to 6). GRKs phosphorylate receptors in an agonist-dependent manner resulting in receptor/G-protein uncoupling via subsequent binding of arrestin proteins. We assessed the activity of GRKs in lymphocytes of rheumatoid arthritis (RA) patients by rhodopsin phosphorylation. We found a significant decrease in GRK activity in RA subjects that is mirrored by a decrease in GRK-2 protein expression. Moreover, GRK-6 protein expression is reduced in RA patients whereas GRK-5 protein levels were unchanged. In search of an underlying mechanism, we demonstrated that proinflammatory cytokines induce a decrease in GRK-2 protein levels in leukocytes from healthy donors. Since proinflammatory cytokines are abundantly expressed in RA, it may provide an explanation for the decrease in GRK-2 expression and activity in patients. No changes in beta2-adrenergic receptor number and Kd were detected. However, RA patients showed a significantly increased cAMP production and inhibition of TNF-alpha production by beta2-adrenergic stimulation, suggesting that reduced GRK activity is associated with increased sensitivity to beta2-adrenergic activation.