KCTD Family: Emerging Regulators of GPCR Biased Signaling.

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (alpha, beta, gamma, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. alpha-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the alpha and beta isoforms, whereas GRK5 prefers alpha-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gbetagamma subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of β-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.

THE 2014 FASEB SCIENCE Research Conference (SRC) on G Protein-Coupled Receptor Kinases: From Molecules to Diseases focused on recent advances in understanding the biochemistry of G protein-coupled receptor kinases (GRKs), their physiologic functions, and roles in pathologic conditions. GRKs are key regulators that, together with arrestins, determine the rate and extent of homologous desensitization of G protein-coupled receptors (GPCRs). GPCRs mediate responses to hormones and neurotransmitters inmultiple tissues and cell types and are targetedby many drugs. Therefore, proteins regulating GPCR signaling are critical for a variety of diseases and represent important therapeutic targets. Recent seminal discoveries attracted attention to novel aspects of GRKbiology, such as regulation of non-GPCR receptors and control of GPCR signaling in a phosphorylation-independent manner. This meeting brought together researchers studying varying aspects of GRK biology in this rapidly expanding field, from structural biologists to physiologists and physicianscientists.Thediscussionsbroadenedourunderstandingof fundamental functions of these exciting proteins and facilitated collaborations. The interdisciplinary nature of the meeting stems from appreciation that only joint effort of investigators studying the GRK action in different model systems will overcome remaining challenges in the GRK field andwill allow researchers to fully exploit the potential of GRKs as therapeutic targets. GPCRs are the largest superfamily of signaling proteins, with several hundred subtypes in all mammalian species. These receptors share a 7-transmembrane span structure and transmit the signal of agonist binding to the cell surface receptor in uniform manner via activation of intracellular heterotrimeric G proteins, as the name implies. The GPCR family occupies a position of extraordinary importance in physiology and medicine, not only because GPCRs control every aspect of physiological function but also because GPCRs are targeted by a large percentage of clinically used drugs. It is not surprising that GPCR signaling is under strict control viamultiplemechanisms.One Attendees of the FASEB Science Research Conference: G Protein-Coupled Receptor Kinases: From Molecules to Diseases (June 8–13, 2014) in Steamboat Springs, CO, USA.

Editor's evaluation: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

Decision letter: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

G Protein-Coupled Receptors (GPCRs) are the largest and most diverse family of cell receptors in the human genome and account for ~30% of all FDA-approved drugs. GPCR signaling is mediated by various effectors, including G proteins, β-arrestins, and GPCR kinases (GRKs). Some ligands preferentially activate G protein- or β-arrestin-dependent signaling pathways, a phenomenon known as biased agonism. One proposed mechanism for GPCR biased signaling—the phosphorylation barcode—hypothesizes that unique phosphorylation patterns of a GPCR induce differential signaling pathways, thereby producing highly specific cellular outputs. To study the phosphorylation barcode hypothesis as a mechanism of biased agonism, we explored the differential recruitment of GRKs 2, 3, 5, and 6 to CXCR3, a chemokine receptor which binds three endogenous biased ligands, CXCL9, CXCL10, and CXCL11. We hypothesized there to be differential recruitment of the GRKs to CXCR3 depending on the endogenous ligand used for stimulation. We first used a split nano-luciferase assay in HEK293T cells to examine the recruitment of GRKs 2, 3, 5, and 6 to the wild type receptor and a variety of phosphorylation deficient mutants following stimulation with the endogenous ligand. An intramolecular biosensor assay known as FlAsh-BRET—fluorescent arsenical hairpin-bioluminescence resonance energy transfer—was also used to monitor β-arrestin2 conformational changes in GRK 2, 3, 5, 6 knockout HEK293 cells following rescue of each individual GRK and stimulation with the endogenous ligands. The split luciferase assay showed evidence of ligand bias across CXCR3′s endogenous ligands. Whereas GRKs 2 and 3 were recruited to the receptor, GRKs 5 and 6 were shown to not actively recruit. GRK recruitment at the phosphorylation mutants and the WT receptor revealed evidence of ligand bias in both the magnitude and location (C-terminus vs. receptor core) of GRK recruitment to the receptor for GRKs 2 and 3. The FlAsh-BRET assay to monitor β-arrestin2 conformation changes revealed biased activation of β-arrestin2, which was differentially directed by the individual GRKs, including GRK5 and GRK6. We demonstrate the endogenous ligands of CXCR3 lead to differential recruitment of the GRKs which promote diverse β-arrestin2 conformations. Our work provides supporting evidence that an overall biased cellular response involves the complex interaction between a GPCR ligand, phosphorylated receptor, GRKs, and other effectors. Further exploration of the biased properties of chemokine ligands and the roles of the GRKs and phosphorylation patterns of CXCR3 may underscore the therapeutic promise of developing highly specific biased agonists to treat a variety of disorders involving chemokine receptors and the broader GPCR family.

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.

G Protein–Coupled Receptor Kinases in Cardiovascular Disease: Why “Where” Matters

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.

G protein‐coupled receptor (GPCR) signaling is tightly regulated by GPCR kinases (GRKs) and β‐arrestins 1 & 2 (βarr1/2). Following agonist stimulation, GRKs and βarr1/2 are important for returning cells to their physiological resting states by acting in tandem to promote receptor desensitization by reducing the ability of receptors to couple with G proteins and by targeting active GPCRs for endocytosis. In addition, GRKs and arrestins are also known to instigate non‐canonical signaling by scaffolding and activating various effector molecules. Although these functions of βarr1/2 are generalizable to all GPCRs, they can diverge dependent upon the ligand activating the same GPCR and the dominant function can vary between GPCRs. Although inherent properties of the GPCR likely specify these discrete functions, the determinants remain poorly understood. To address this, we are using the chemokine receptor CXCR5 as a model GPCR. CXCR5, and its endogenous ligand, CXCL13, have been linked to diseases ranging from cancer, chronic inflammation, and cardiovascular disease, among others, and yet very little is known about the regulation of CXCR5 signaling. To investigate this, we used CRISPR/Cas9 gene edited βarr1/2 knockout and matched parental HEK293 cells in bioluminescence resonance energy transfer (BRET), receptor trafficking, and signaling assays. Our data show a dose‐dependent increase in the BRET response following CXCL13 stimulation between βarr1/2‐GFP and CXCR5‐Rluc8, suggesting that βarr1/2 is recruited to activated CXCR5 at the plasma membrane. A selective inhibitor of GRK2/3, but not a non‐selective PKC inhibitor, reduced βarr1/2 recruitment to CXCR5 following CXCL13 stimulation. Mutation of potential phosphorylation sites within the carboxy‐terminus of CXCR5 did not impact CXCL13‐stimulated endocytosis, although βarr1/2 recruitment was impaired. Remarkably, CXCL13‐stimulated CXCR5 endocytosis was not impacted in βarr1/2 knockout HEK293 cells, while CXCL13‐stimulated ERK‐1/2 phosphorylation was reduced compared to parental HEK293 cells. Based on these data we hypothesize that CXCR5 signaling is regulated through a complex process that involves GRKs andβ‐arrestins via conventional and non‐conventional mechanisms.To explore this further, in future experiments we will identify the receptor determinants mediating GRK‐phosphorylation and βarr1/2 recruitment to CXCR5 and their contributions to desensitization, endocytosis, and signaling. Overall, we expect to learn how receptor phosphorylation contributes to the divergent functions of βarr1/2 via CXCR5. This research will help illuminate novel mechanisms governing GPCR regulation by GRKs and arrestins, which may translate into better therapeutics for diseases involving GPCR signaling.

G protein-coupled receptor kinase 2 is essential to enable vasoconstrictor-mediated arterial smooth muscle proliferation