G Protein-coupled Receptor Kinases Phosphorylation Research Articles

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

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

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

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

Grk7 but not Grk1 undergoes cAMP-dependent phosphorylation in zebrafish cone photoreceptors and mediates cone photoresponse recovery to elevated cAMP

In the vertebrate retina, phosphorylation of photoactivated visual pigments in rods and cones by G protein-coupled receptor kinases (GRKs) is essential for sustained visual function. Previous invitro analysis demonstrated that GRK1 and GRK7 are phosphorylated by PKA, resulting in a reduced capacity to phosphorylate rhodopsin. Invivo observations revealed that GRK phosphorylation occurs in the dark and is cAMP dependent. In many vertebrates, including humans and zebrafish, GRK1 is expressed in both rods and cones while GRK7 is expressed only in cones. However, mice express only GRK1 in both rods and cones and lack GRK7. We recently generated a mutation in Grk1 that deletes the phosphorylation site, Ser21. This mutant demonstrated delayed dark adaptation in mouse rods but not in cones invivo, suggesting GRK1 may serve a different role depending upon the photoreceptor cell type in which it is expressed. Here, zebrafish were selected to evaluate the role of cAMP-dependent GRK phosphorylation in cone photoreceptor recovery. Electroretinogram analyses of larvae treated with forskolin show that elevated intracellular cAMP significantly decreases recovery of the cone photoresponse, which is mediated by Grk7a rather than Grk1b. Using a cone-specific dominant negative PKA transgene, we show for the first time that PKA is required for Grk7a phosphorylation invivo. Lastly, immunoblot analyses of rod grk1a-/- and cone grk1b-/- zebrafish and Nrl-/- mouse show that cone-expressed Grk1 does not undergo cAMP-dependent phosphorylation invivo. These results provide a better understanding of the function of Grk phosphorylation relative to cone adaptation and recovery.

Targeting Adiponectin Receptor 1 Phosphorylation Against Ischemic Heart Failure.

Despite significantly reduced acute myocardial infarction (MI) mortality in recent years, ischemic heart failure continues to escalate. Therapeutic interventions effectively reversing pathological remodeling are an urgent unmet medical need. We recently demonstrated that AdipoR1 (APN [adiponectin] receptor 1) phosphorylation by GRK2 (G-protein-coupled receptor kinase 2) contributes to maladaptive remodeling in the ischemic heart. The current study clarified the underlying mechanisms leading to AdipoR1 phosphorylative desensitization and investigated whether blocking AdipoR1 phosphorylation may restore its protective signaling, reversing post-MI remodeling. Specific sites and underlying molecular mechanisms responsible for AdipoR1 phosphorylative desensitization were investigated in vitro (neonatal and adult cardiomyocytes). The effects of AdipoR1 phosphorylation inhibition upon APN post-MI remodeling and heart failure progression were investigated in vivo. Among 4 previously identified sites sensitive to GRK2 phosphorylation, alanine substitution of Ser205 (AdipoR1S205A), but not other 3 sites, rescued GRK2-suppressed AdipoR1 functions, restoring APN-induced cell salvage kinase activation and reducing oxidative cell death. The molecular investigation followed by functional determination demonstrated that AdipoR1 phosphorylation promoted clathrin-dependent (not caveolae) endocytosis and lysosomal-mediated (not proteasome) degradation, reducing AdipoR1 protein level and suppressing AdipoR1-mediated cytoprotective action. GRK2-induced AdipoR1 endocytosis and degradation were blocked by AdipoR1S205A overexpression. Moreover, AdipoR1S205E (pseudophosphorylation) phenocopied GRK2 effects, promoted AdipoR1 endocytosis and degradation, and inhibited AdipoR1 biological function. Most importantly, AdipoR1 function was preserved during heart failure development in AdipoR1-KO (AdipoR1 knockout) mice reexpressing hAdipoR1S205A. APN administration in the failing heart reversed post-MI remodeling and improved cardiac function. However, reexpressing hAdipoR1WT in AdipoR1-KO mice failed to restore APN cardioprotection. Ser205 is responsible for AdipoR1 phosphorylative desensitization in the failing heart. Blockade of AdipoR1 phosphorylation followed by pharmacological APN administration is a novel therapy effective in reversing post-MI remodeling and mitigating heart failure progression.

The ROS/GRK2/HIF-1α/NLRP3 Pathway Mediates Pyroptosis of Fibroblast-Like Synoviocytes and the Regulation of Monomer Derivatives of Paeoniflorin.

Hypoxia is an important factor in the development of synovitis in rheumatoid arthritis (RA). The previous study of the research group found that monomeric derivatives of paeoniflorin (MDP) can alleviate joint inflammation in adjuvant-induced arthritis (AA) rats by inhibiting macrophage pyroptosis. This study revealed increased levels of hypoxia-inducible factor- (HIF-) 1α and N-terminal p30 fragment of GSDMD (GSDMD-N) in fibroblast-like synoviocytes (FLS) of RA patients and AA rats, while MDP significantly inhibited their expression. Subsequently, FLS were exposed to a hypoxic environment or treated with cobalt ion in vitro. Western blot and immunofluorescence analysis showed increased expression of G protein-coupled receptor kinase 2 (GRK2), HIF-1α, nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), ASC, caspase-1, cleaved-caspase-1, and GSDMD-N. Electron microscopy revealed FLS pyroptosis after exposure in hypoxia. Next, corresponding shRNAs were transferred into FLS to knock down hypoxia-inducible factor- (HIF-) 1α, and in turn, NLRP3 and western blot results confirmed the same. The enhanced level of GSDMD was reversed under hypoxia by inhibiting NLRP3 expression. Knockdown and overexpression of GRK2 in FLS revealed GRK2 to be a positive regulator of HIF-1α. Levels of GRK2 and HIF-1α were inhibited by eliminating excess reactive oxygen species (ROS). Furthermore, MDP reduced FLS pyroptosis through targeted inhibition of GRK2 phosphorylation. According to these findings, hypoxia induces FLS pyroptosis through the ROS/GRK2/HIF-1α/NLRP3 pathway, while MDP regulates this pathway to reduce FLS pyroptosis.

Phosphorylation of the D 1 Dopamine Receptor by G Protein‐Coupled Receptor Kinases: phosphorylation site identification and linkage to functional effects

Dopamine (DA) receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward-related behaviors, as well as cardiovascular and renal physiology. Multiple diseases are linked to dysregulated dopaminergic functioning including Parkinson's disease, schizophrenia, substance use disorder, and hypertension. DARs are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs (D1R and D5R) increase cAMP, while the D2-like DARs (D2R, D3R, D4R) decrease cAMP. All DARs also recruit β-arrestin which activates separate signaling cascades and also initiates receptor desensitization and internalization. Generally, agonist activation of GPCRs, including DARs, rapidly leads to receptor desensitization and a return to basal levels of signaling. This occurs even in the continued presence of agonist, ensuring homeostasis. This desensitization process is intimately linked with receptor phosphorylation. The D1R is highly phosphorylated, with 32 intracellular serine and threonine residues, and is known to be phosphorylated by several kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs). Previous studies by our lab indicate that the D1R is phosphorylated on its third intracellular loop (ICL3) and C-terminus in a hierarchical fashion, in that phosphorylation must first occur on the C-terminus before the ICL3 can be phosphorylated. Using systematic mutational analyses, we previously identified the PKC-mediated D1R phosphorylation sites. We have now extended these studies to completely identify the DA-induced, GRK-mediated phosphorylation sites on the D1R. We found that GRK-mediated phosphorylation involves several serine and threonine residues on the C-terminus and ICL3. Mutation of these residues to alanine or valine, respectively, abolishes DA-induced D1R phosphorylation and severely impairs β-arrestin recruitment, but causes little effect on G protein-mediated signaling. Our results indicate that a large fraction of DA-induced D1R phosphorylation occurs on residues T360 and S362 in the proximal C-terminus, and that these residues are also responsible for the majority of DA-induced β-arrestin recruitment to the D1R. Using HEK cells that have had their endogenous GRKs knocked out via CRISPR, we found that DA-induced β-arrestin recruitment to the D1R is severely impaired. However, β-arrestin recruitment can be restored by expressing exogenous GRKs in these cells, further suggesting that DA-induced β-arrestin recruitment to the D1R is highly dependent on GRK phosphorylation. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs can add layers of regulatory fine-tuning through differential effects on D1R signaling or trafficking outcomes.

GPCR Kinase 2 (GRK2) Phosphorylation of the α 2A ‐Adrenergic Receptor in Intact Cells

The α2A-adrenergic receptor (α2AAR) is activated by the catecholamine norepinephrine and plays a many roles including neuromodulation, vasoconstriction in the cardiovascular system, regulation of insulin secretion and platelet aggregation. Platelet α2AAR serves as an excellent substrate for agonist-dependent phosphorylation by G protein-coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α2AAR is structurally distinct from the well-characterized beta2-adrenergic receptor (β2AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phosphor-acceptors. When overexpressed with GRK2, the receptor is phosphorylated at all 4 phospho-sites. Our long-term goal is to understand how GRK2 interacts with and is activated by GPCRs. We have mapped residues on GRK2 that are required for phosphorylation of the β2AR and recruitment to α2AAR in intact cells. We wish to assay phosphorylation of α2AAR in intact cells and because no commercially available α2AAR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that detects α2AAR phosphorylation at serines 296-299 was generated and characterized. This antibody detects an epinephrine-stimulated signal that is increased two-fold by transfection of GRK2 in COS-7 cells. We also found that the α2AAR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α2AAR is a good substrate for an endogenous COS-7 cell kinase. Mutagenesis of the receptor has allowed us to measure agonist- and GRK-dependent phosphorylation of α2AAR. We are now in a position to characterize GRK2 phosphorylation of this receptor.

Modulation of CXCR4-Mediated Gi1 Activation by EGF Receptor and GRK2.

The CXCL12 chemokine receptor CXCR4 belongs to the GPCR superfamily and is often overexpressed in cancer, being involved in tumor progression and metastasis. How CXCR4 signaling integrates with other relevant oncogenic transduction pathways and the role of GPCR regulatory mechanisms in such contexts are not well-understood. Recent data indicate concurrent upregulation in certain tumors of CXCR4, EGF receptor (EGFR), and G protein-coupled receptor kinase 2 (GRK2), a signaling node functionally linked to both receptor types. We have investigated in a model system the effect of the EGFR and GRK2 status on CXCL12/CXCR4-mediated activation of Gi, the earliest step downstream of receptor activation. We find that overexpressed and activated EGFR reduces CXCR4-mediated Gi1 activation and that GRK2 phosphorylation at tyrosine residues is required to exert its inhibitory actions on CXCR4–Gi stimulation, suggesting a shared path of modulation. Our data point to a role for GRK2 in the crosstalk of the CXCR4 and EGFR signal transduction pathways in pathological contexts characterized by concurrent overactivation of these proteins.

GPCR Kinase 2 (GRK2) Phosphosphorylation of the Alpha 2A Adrenergic Receptor in Intact Cells

The α2A‐adrenergic receptor (α2A‐AR) is activated by the catecholamine norepinephrine and plays a variety of roles including neuromodulation, vasoconstriction in the cardiovascular system, regulation of insulin secretion and platelet aggregation. Platelet α2A‐AR serves as an excellent substrate for agonist‐dependent phosphorylation by G protein‐coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α2A‐AR is structurally distinct from the well‐characterized β2‐adrenergic receptor (β2AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phospho‐acceptors. When overexpressed with GRK2, the receptor is phosphorylated at all 4 phospho‐sites. Our long‐term goal is to understand how GRK2 interacts with and is activated by GPCRs. We have mapped residues on GRK2 that are required for phosphorylation of β2AR and recruitment to α2A‐AR in intact cells. We wish to assay phosphorylation of α2A‐AR in intact cells and because no commercially available α2A‐AR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that recognizes α2A‐AR phosphorylation at serines 296–299 was generated and characterized. This antibody detects an epinephrine‐stimulated signal that is increased two‐fold by transfection of GRK2 in COS‐7 cells. We also found that the α2A‐AR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α2A‐AR is a good substrate for an endogenous COS‐7 cell kinase. Mutagenesis of the receptor has allowed us to measure agonist‐ and GRK‐dependent phosphorylation of α2A‐AR. We are now in a position to characterize GRK2 phosphorylation of this receptor.Support or Funding InformationThis work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).

Generating Phosphorylation Site Antibodies to Study α2A‐Adrenergic Receptor Phosphorylation by G Protein‐coupled Receptor Kinase 2 in Intact Cells

G protein‐coupled receptors (GPCRs) are transmembrane proteins which are activated by the binding of agonist to create a cell signal response. GPCRs are regulated through phosphorylation by G protein‐coupled receptor kinases. We focus on G protein‐coupled receptor kinase 2 (GRK2) and have previously characterized phosphorylation of the β2‐adrenergic receptor (β2‐AR) by this kinase. Currently, we are trying to determine if the same mutations shown to effect β2‐AR phosphorylation are also important for phosphorylation of the α2A‐adrenergic receptor (α2A‐AR). The α2A‐AR plays a role in regulating insulin, neurotransmitter secretion, and is a pharmaceutical target for pain relief. It differs from β2‐AR in that α2A‐AR has a larger third intracellular loop, containing four consecutive serine residues. These residues can be phosphorylated by GRK2 in the presence of agonist, causing a desensitization response. One way to detect receptor phosphorylation is to use antibodies that specifically recognize phosphorylated receptor. Since there is no commercially available antibody that can detect phosphorylation of α2A‐AR, we developed an antibody, RB617, which detects the phosphorylation of two serine residues. Using this antibody, we have developed methodologies to study GRK2 phosphorylation of α2A‐AR in intact COS‐7 cells. We tested the dose‐response of α2A‐AR to epinephrine and determined the EC50 for GRK2‐dependent phosphorylation to be 35 nM. In the future, we plan to assess the effect of GRK2 mutations previously shown to affect β2‐AR phosphorylation to better understand the role of GRK2 in α2A‐AR desensitization.Support or Funding InformationThis work was funded by NSF MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).

LDK378 inhibits the recruitment of myeloid-derived suppressor cells to spleen via the p38-GRK2-CCR2 pathway in mice with sepsis.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that are persistently increased in abundance and associated with adverse clinical outcomes in sepsis. Here, we investigated the therapeutic potential of an anaplastic lymphoma kinase inhibitor, LDK378, in cecal ligation and puncture (CLP)-induced polymicrobial sepsis and examined its effects on the recruitment of MDSCs. LDK378 significantly improved the survival of CLP-induced polymicrobial septic mice, which was paralleled by reduced organ injury, decreased release of inflammatory cytokines and decreased recruitment of MDSCs to the spleen. Importantly, LDK378 inhibited the migration of MDSCs to the spleen by blocking the CLP-mediated upregulation of CC chemokine receptor 2 (CCR2), a chemokine receptor critical for the recruitment of MDSCs. Mechanistically, LDK378 treatment blocked the CLP-induced CCR2 upregulation of MDSCs via partially inhibiting the phosphorylation of p38 and G-protein-coupled receptor kinase-2 (GRK2) in bone marrow MDSCs of septic mice. Furthermore, invitro experiments also showed that lipopolysaccharide (LPS)-induced migration of MDSCs was similarly owing to the activation of GRK2 and upregulation of CCR2 by LPS, whereas the treatment with LDK378 partially blocked the LPS-induced phosphorylation of p38 and GRK2 and decreased the expression of CCR2 on the cell surface, therefore leading to the suppression of MDSC migration. Together, these findings unravel a novel function of LDK378 in the host response to infection and suggest that LDK378 could be a potential therapeutic agent for sepsis.

Degradation of GRK2 and AKT is an early and detrimental event in myocardial ischemia/reperfusion.

BackgroundIdentification of signaling pathways altered at early stages after cardiac ischemia/reperfusion (I/R) is crucial to develop timely therapies aimed at reducing I/R injury. The expression of G protein-coupled receptor kinase 2 (GRK2), a key signaling hub, is up-regulated in the long-term in patients and in experimental models of heart failure. However, whether GRK2 levels change at early time points following myocardial I/R and its functional impact during this period remain to be established.MethodsWe have investigated the temporal changes of GRK2 expression and their potential relationships with the cardioprotective AKT pathway in isolated rat hearts and porcine preclinical models of I/R.FindingsContrary to the maladaptive up-regulation of GRK2 reported at later times after myocardial infarction, successive GRK2 phosphorylation at specific sites during ischemia and early reperfusion elicits GRK2 degradation by the proteasome and calpains, respectively, thus keeping GRK2 levels low during early I/R in rat hearts. Concurrently, I/R promotes decay of the prolyl-isomerase Pin1, a positive regulator of AKT stability, and a marked loss of total AKT protein, resulting in an overall decreased activity of this pro-survival pathway. A similar pattern of concomitant down-modulation of GRK2/AKT/Pin1 protein levels in early I/R was observed in pig hearts. Calpain and proteasome inhibition prevents GRK2/Pin1/AKT degradation, restores bulk AKT pathway activity and attenuates myocardial I/R injury in isolated rat hearts.InterpretationPreventing transient degradation of GRK2 and AKT during early I/R might improve the potential of endogenous cardioprotection mechanisms and of conditioning strategies.

Abstract 635: β 2 -adrenergic Stimulation Compartmentalizes β 1 Signaling Into Nanoscale Local Domains by Targeting the C-terminus of β 1 -adrenoceptors

β-adrenergic receptors (βARs) are prototypical G-protein coupled receptors that play a pivotal role in sympathetic regulation. In heart cells, β 1 R signaling mediates a global cAMP response, regulating both L-type Ca 2+ channels in the sarcolemma/T-tubules (SL/TTs) and ryanodine receptors in the sarcoplasmic reticulum (SR). In contrast, β 2 AR mediates local cAMP signaling with little effect on the function of SR proteins. Accumulating evidence in pathological and transgenic models has suggested a functional complexity of β 2 AR beyond its regulation of L-type Ca 2+ influx. So we investigated the signaling relationship between β 1 ARs and β 2 ARs. Using whole-cell patch clamp combined with confocal calcium imaging, we simultaneously measured L-type Ca 2+ currents and Ca 2+ transients in adult cardiomyocytes. β 1 AR stimulation with norepinephrine or isoproterenol increased both Ca 2+ currents and Ca 2+ transients. β 2 AR stimulation with Zinterol or Salbutamol only increased Ca 2+ currents (onside compartmentalization) and surprisingly inhibited the ability of β 1 AR in upregulating Ca 2+ transients (offside compartmentalization). Further evidence showed that β 2 AR activation recruited G-protein coupled receptor kinase 2 (GRK2) to cell membrane, which phosphorylated the ‘SSES’ serine cluster on β 1 AR carboxyl terminal. Phosphorylated β 1 AR coupled to β-arrestin1 and further recruited phosphodiesterase-4, leading to localized cAMP degradation. Immunocytochemical and biochemical analysis showed the β 2 AR-mediated offside compartmentalization was confined to the nanoscale domain. A knock-in rat model harboring mutations of the three serine residues in ‘SSES’ cluster of the β 1 AR C-terminus, a component of the putative β-arrestin1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by β 2 AR activation. This finding reveals a fundamental “negative feed-forward” mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient β 1 AR response of sympathetic excitation, providing many potential novel targets for drug discovery against cardiovascular diseases.

Beta1-Adrenergic Receptor Regulation Revisited.

The actions and regulation of cardiomyocyte β1ARs differ in several respects from the properties described for the prototypical β2AR subtype; a mechanism to explain the unique properties of the β1AR subtype has never been obvious. This viewpoint summarizes recent studies that identify a novel signaling paradigm for the β1AR, implicating the N-terminus as a molecular determinant of β1AR responsiveness.

GRK2 mediates TCR-induced transactivation of CXCR4 and TCR–CXCR4 complex formation that drives PI3Kγ/PREX1 signaling and T cell cytokine secretion

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.

Estrogen Regulation of GRK2 Inactivates Kappa Opioid Receptor Signaling Mediating Analgesia, But Not Aversion.

Activation of κ opioid receptors (KORs) produces analgesia and aversion via distinct intracellular signaling pathways, but whether G protein-biased KOR agonists can be designed to have clinical utility will depend on a better understanding of the signaling mechanisms involved. We found that KOR activation produced conditioned place aversion and potentiated CPP for cocaine in male and female C57BL/6N mice. Consistent with this, males and females both showed arrestin-mediated increases in phospho-p38 MAPK following KOR activation. Unlike in males, however, KOR activation had inconsistent analgesic effects in females and KOR increased Gβγ-mediated ERK phosphorylation in males, but not females. KOR desensitization was not responsible for the lack of response in females because neither Grk3 nor Pdyn gene knock-out enhanced analgesia. Instead, responsiveness was estrous cycle dependent because KOR analgesia was evident during low estrogen phases of the cycle and in ovariectomized (OVX) females. Estradiol treatment of OVX females suppressed KOR-mediated analgesia, demonstrating that estradiol was sufficient to blunt Gβγ-mediated KOR signals. G protein-coupled receptor kinase 2 (GRK2) is known to regulate ERK activation, and we found that the inhibitory, phosphorylated form of GRK2 was significantly higher in intact females. GRK2/3 inhibition by CMPD101 increased KOR stimulation of phospho-ERK in females, decreased sex differences in KOR-mediated inhibition of dopamine release, and enhanced mu opioid receptor and KOR-mediated analgesia in females. In OVX females, estradiol increased the association between GRK2 and Gβγ. These studies suggest that estradiol, through increased phosphorylation of GRK2 and possible sequestration of Gβγ by GRK2, blunts G protein-mediated signals.SIGNIFICANCE STATEMENT Chronic pain disorders are more prevalent in females than males, but opioid receptor agonists show inconsistent analgesic efficacy in females. κ opioid receptor (KOR) agonists have been tested in clinical trials for treating pain disorders based on their analgesic properties and low addictive potential. However, the molecular mechanisms underlying sex differences in KOR actions were previously unknown. Our studies identify an intracellular mechanism involving estradiol regulation of G protein-coupled receptor kinase 2 that is responsible for sexually dimorphic analgesic responses following opioid receptor activation. Understanding this mechanism will be critical for developing effective nonaddictive opioid analgesics for use in women and characterizing sexually dimorphic effects in other inhibitory G protein-coupled receptor signaling responses.

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D3 receptor (D3R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D3R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D3R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D3R, which can be extended to the crosstalk between other RTKs and GPCRs.

GRK2-S670A Mice reveal cardioprotection post ischemia-reperfusion

Background: β-adrenergic receptors (βARs) are critical regulators of cardiac contractility whose function are drastically impaired during heart failure (HF). Activated βARs are regulated via phosphorylation by G-protein coupled receptor kinase-2 (GRK2) and subsequent interaction with β-arrestin. Animal and human studies have shown that GRK2 is elevated in HF pathogenesis. In addition to GRK2 being involved in pathological βAR our lab has uncovered non-canonical actions of this kinase in HF development. One such novel action of GRK2 is within mitochondria where we have shown increased GRK2 localization after oxidative stress. Our lab has shown that in the heart, phosphorylation of GRK2 via Map kinase at residue Ser670 promotes binding to Hsp90 and further enhances translocation of GRK2 to the mitochondria after ischemia. Methods: To determine the ultimate role of this pathway in the post-ischemic heart we have developed and generated novel GRK2-S670A knock-in mice where all endogenous GRK2 cannot be regulated by phosphorylation at this residue. Baseline characterization of these mice show a minimal phenotype, however we have found significant differences after ischemia-reperfusion (I/R) injury. Results: Compared to control mice (WT) expressing wild-type GRK2, GRK2-S670A mice have significantly less infarction size and improved cardiac function post-24h I/R. GRK2-S670A showed improved ejection fraction post-IR (53.56%+/- 2.08 S670A vs 42.38%+/- 1.6 WT) and smaller infarcted areas (13.67%+/- 1.976 S670A vs 20.78%+/- 3.579 WT) when compared to control mice. Further, we found that mitochondrial GRK2 protein levels were lower in the GRK2-S670A mice at the area at risk post I/R when compared to WT mice (0.8103 +/- 0.1458 S670A vs 1.610 +/- 0.1983 WT). Conclusion: Overall, our data suggest that phosphorylation at Ser670 of GRK2 promotes translocation to the mitochondria leading to detrimental effects including cell death. This mechanism suggests a novel way to develop pharmacological interventions to aid in the treatment of HF.

Identification of a signaling cascade that maintains constitutive δ-opioid receptor incompetence in peripheral sensory neurons

μ-Opioid receptor (MOR) agonists are often used to treat severe pain but can result in adverse side effects. To circumvent systemic side effects, targeting peripheral opioid receptors is an attractive alternative treatment for severe pain. Activation of the δ-opioid receptor (DOR) produces similar analgesia with reduced side effects. However, until primed by inflammation, peripheral DOR is analgesically incompetent, raising interest in the mechanism. We recently identified a novel role for G-protein-coupled receptor kinase 2 (GRK2) that renders DOR analgesically incompetent at the plasma membrane. However, the mechanism that maintains constitutive GRK2 association with DOR is unknown. Protein kinase A (PKA) phosphorylation of GRK2 at Ser-685 targets it to the plasma membrane. Protein kinase A-anchoring protein 79/150 (AKAP), residing at the plasma membrane in neurons, scaffolds PKA to target proteins to mediate downstream signal. Therefore, we sought to determine whether GRK2-mediated DOR desensitization is directed by PKA via AKAP scaffolding. Membrane fractions from cultured rat sensory neurons following AKAP siRNA transfection and from AKAP-knock-out mice had less PKA activity, GRK2 Ser-685 phosphorylation, and GRK2 plasma membrane targeting than controls. Site-directed mutagenesis revealed that GRK2 Ser-685 phosphorylation drives the association of GRK2 with plasma membrane-associated DOR. Moreover, overexpression studies with AKAP mutants indicated that impaired AKAP-mediated PKA scaffolding significantly reduces DOR-GRK2 association at the plasma membrane and consequently increases DOR activity in sensory neurons without a priming event. These findings suggest that AKAP scaffolds PKA to increase plasma membrane targeting and phosphorylation of GRK2 to maintain DOR analgesic incompetence in peripheral sensory neurons.

Caveolin 1 and G-Protein–Coupled Receptor Kinase-2 Coregulate Endothelial Nitric Oxide Synthase Activity in Sinusoidal Endothelial Cells

Liver injury leads to a vasculopathy in which post-translational modifications of endothelial nitric oxide synthase (eNOS) lead to impaired nitric oxide synthesis. We hypothesized that caveolin 1 (CAV1), a well-known eNOS interactor, regulates eNOS activity in sinusoidal endothelial cells (SECs) via its interaction with G-protein-coupled receptor kinase-2 (GRK2) that also post-translationally modifies eNOS. Liver injury with portal hypertension was established using bile duct ligation in rats. CAV1 function was modified using a CAV1 scaffolding domain construct and cDNAs encoding wild-type CAV1, and CAV1 phosphorylation was increased in injured SECs, resulting in increased GRK2-CAV1 interaction and decreased eNOS activity. In injured SECs, endothelin-1 blocked CAV1 phosphorylation induced by CAV1 scaffolding domain, indicating that CAV1 interaction with GRK2 is inversely regulated by endothelin-1 and CAV1 scaffolding domain after liver injury. In addition, after transduction with DNA encoding wild-type CAV1 into SECs isolated from Cav1-deficient mice, GRK2 association with CAV1 was evident, whereas transduction with a dominant negative CAV1 mutated at tyrosine 14 reduced the interaction. Finally, isoproterenol-induced GRK2 phosphorylation enhanced CAV1-GRK2 interaction and reduced eNOS activity. Our data suggest a novel mechanism and model in which CAV1 phosphorylation facilitates CAV1 scaffolding and GRK2-CAV1 interaction, thus clustering eNOS within a complex that inhibits eNOS activity. This process takes place in injured, but not in normal, SECs.