G Protein-coupled Receptor Kinases Family Research Articles

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by β-arrestin recruiting GPR84 agonists

In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84- but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by β-arrestin recruitment data. The ROS production induced by a non β-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this β-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with β-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils.

Historical Perspective of the G Protein-Coupled Receptor Kinase Family.

Agonist activation of G protein-coupled receptors promotes sequential interaction of the receptor with heterotrimeric G proteins, G protein-coupled receptor kinases (GRKs), and arrestins. GRKs play a central role in mediating the switch from G protein to arrestin interaction and thereby control processes such as receptor desensitization and trafficking and arrestin-mediated signaling. In this review, I provide a historical perspective on some of the early studies that identified the family of GRKs with a primary focus on the non-visual GRKs. These studies included identification, purification, and cloning of the β-adrenergic receptor kinase in the mid- to late-1980s and subsequent cloning and characterization of additional members of the GRK family. This helped to lay the groundwork for ensuing work focused on understanding the structure and function of these important enzymes.

Chemerin-activated functions of CMKLR1 are regulated by G protein-coupled receptor kinase 6 (GRK6) and β-arrestin 2 in inflammatory macrophages

Chemerin receptor (CMKLR1) is a G protein-coupled receptor (GPCR) implicated in macrophage-mediated inflammation and in several forms of human arthritis. Analogous to other GPCR, CMKLR1 is likely regulated by G protein-coupled receptor kinase (GRK) phosphorylation of intracellular domains in an activation-dependent manner, which leads to recruitment and termination of intracellular signaling via desensitization and internalization of the receptor. The ubiquitously expressed GRK family members include GRK2, GRK3, GRK5, and GRK6, but it is unknown which GRK regulates CMKLR1 cellular and signaling functions. Our data show that activation of CMKLR1 by chemerin in primary macrophages leads to signaling and functional outcomes that are regulated by GRK6 and β-arrestin 2. We show that arrestin recruitment to CMKLR1 following chemerin stimulation is enhanced with co-expression of GRK6. Further, internalization of endogenous CMKLR1, following the addition of chemerin, is decreased in inflammatory macrophages from GRK6- and β-arrestin 2-deficient mice. These GRK6- and β-arrestin 2-deficient macrophages display increased migration toward chemerin and altered AKT and Extracellular-signal Related Kinase (ERK) signaling. Our findings show that chemerin-activated CMKLR1 regulation in inflammatory macrophages is largely GRK6 and β-arrestin mediated, which may impact innate immunity and have therapeutic implications in rheumatic disease.

Abstract 3461: PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner

Abstract Background: Our group were first to establish a link between PID1 (Phosphotyrosine Interaction Domain containing 1) and cancer, showing PID1's tumor inhibitory effect in medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor (ATRT) brain tumor cell lines and correlation of higher PID1 mRNA with longer patient survival. PID1 was first identified in 2006 and is associated with obesity-related insulin resistance and mitochondrial dysfunction. Here we present a novel interacting partner of PID1 which phosphorylates and modulates its growth suppressive functions. Methods: In silico predictions, in vitro kinase assay, phosphoproteomic analysis, co-immunoprecipitation, SDS-PAGE, western blotting, flow cytometry. Cells: CHLA-07-BSGBM pediatric glioblastoma, U87 glioblastoma and D283 medulloblastoma cell lines, HEK293FT, primary pooled mouse embryo fibroblasts (MEFs). Results: Using prediction algorithms we found that PID1 was a likely phosphorylation target of a member of the G protein-coupled receptor kinase (GRK) family. In vitro kinase assays coupled with phosphoproteomic analysis confirmed this phosphorylation. Overexpressed HA-PID1 and this GRK could be reciprocally co-immunoprecipitated in 293FT cells and endogenous GRK was co-IPd by endogenous PID1 in CHLA-07-BSGBM. A kinase-dead GRK mutant co-IP'd with PID1 similar to the wildtype GRK indicating that the GRK-PID1 interaction did not require the kinase function of this GRK. Deletion mutants of PID1 differentially bound the GRK in co-IPs in a manner that corresponded to the differential growth inhibition these PID1 deletion mutants exerted in glioblastoma and medulloblastoma cell lines. Functionally, siRNA silencing of this GRK caused apoptosis (annexin-PI positive cells and PARP-1 cleavage) in Pid1wt MEFs suggesting that this GRK mediates survival in MEFs. Apoptosis by the GRK siRNA was mitigated in Pid1ko compared to Pid1wt MEFs, suggesting that PID1 functions downstream of the GRK and negatively regulates this GRK's pro-survival signaling. Importantly, we found similar results in glioblastoma cells using gain/loss of function of the two proteins. Conclusions: We found that PID1 is phosphorylated by a member of the GRK family and interacts with it, affecting this GRK's pro-survival activity. Experiments are now uncovering the molecular ordering of PID1 and this GRK in mediating the growth-suppressive effect of PID1 in brain tumors. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Eslam Nouri Nigjeh, Yang Fu, Ebrahim Zandi, Xiaojiang Chen, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3461.

GRK2 targeted knock-down results in spontaneous hypertension, and altered vascular GPCR signaling.

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8–12 weeks of age in shGRK2 mice due to enhanced Gαq/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gαs and Gαq/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.

GRK2 Targeted Knock-down Results in Spontaneous Hypertension, and Altered Vascular GPCR Signaling

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8-12 weeks of age in shGRK2 mice due to enhanced Gαq/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gαs and Gαq/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.

Pharmacogenomics of the Heptahelical Receptor Regulators G-Protein-Coupled Receptor Kinases and Arrestins: The Known and the Unknown

Heptahelical G-protein-coupled receptors are the most diverse and therapeutically important family of receptors, playing major roles in the physiology of various organs and tissues. They couple their ligand binding to G-protein activation, which then transmits intracellular signals. G-protein signaling is terminated by phosphorylation of the receptor by the family of G-protein-coupled receptor kinases (GRKs), followed by arrestin (Arr) binding, which uncouples the phosphorylated receptor from the G-protein and subsequently targets the receptor for internalization. Moreover, Arrs can transmit signals in their own right during receptor internalization. Genetic polymorphisms in receptors, as well as in GRK and Arr family members per se, which affect regulation of receptor signaling and function, have just started being identified and characterized. The present review will discuss what is known so far in this evolving field of GRK/Arr pharmacogenomics, as well as highlight important areas likely to produce invaluable information in the future.

GRK2 Inhibition in Heart Failure: Something Old, Something New

Despite significant advances in pharmacological and clinical treatment, heart failure (HF) remains the number one killer disease in the western world. HF is a chronic and progressive clinical syndrome mainly characterized by reduction in left ventricular ejection fraction and adverse remodeling of the myocardium. One of its hallmark molecular abnormalities is elevation of cardiac G protein-coupled receptor (GPCR) kinase (GRK)-2, originally termed beta-adrenergic receptor kinase-1 (βARK1), a member of the GRK family of serine/threonine protein kinases which phosphorylate and desensitize GPCRs. Up-regulated GRK2 in the heart underlies the diminished contractile responsiveness of the heart to positive inotropes, as it abrogates the pro-contractile signaling of various important cardiac receptors: mainly β-adrenergic receptors (βARs), but also angiotensin II type 1 receptors (AT(1)Rs), etc. Thus, cardiac-specific GRK2 inhibition via various transgenic approaches is postulated to combat chronic HF symptoms by increasing cardiac function, and even be salutary in some cases by increasing survival. This has been extensively documented over the past 15 years through a vast series of preclinical studies on animals of all sizes and shapes, from small mice up to large rabbits and pigs closely resembling human physiology, and genetically manipulated to have cardiac GRK2 inhibited or deleted, transiently or permanently. However, over the past several years, it has become increasingly clear that GRK2, like other members of the GRK family, exerts additional effects that can aggravate HF, in addition to merely blunt cardiac contractility by opposing cardiac βAR G protein-mediated signaling. One of these newly discovered cardiotoxic effects of GRK2, uncovered by our laboratory, is promotion by adrenal GRK2 of sympathetic hyperactivity of the failing heart, a significant morbidity factor in HF, targeted therapeutically nowadays by the use of beta-blockers in HF pharmacotherapy. Thus, new avenues for therapeutic exploitation of GRK2 inhibition in HF treatment might be possible in the near future. The present review gives first a brief account of what has already been documented about the benefits of cardiac GRK2 genetic manipulation in HF as a positive inotropic therapy for the disease, and then goes on to discuss in detail the intriguing new possibility that has emerged of lowering GRK2 activity in the adrenal gland, which could constitute a novel sympatholytic therapy for HF that helps relieve the devastatingly cardiotoxic sympathetic overload of the failing heart.

Atypical Actions of G Protein-Coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) and β-arrestins have been known as regulators of G protein-coupled receptors. However, it has been recently reported that GRKs and β-arrestins mediate receptor-mediated cellular responses in a G proteinindependent manner. In this scheme, GRKs work as a mediator or a scaffold protein. Among 7 members of the GRK family (GRK1-GRK7), GRK2 is the most extensively studied in vitro and in vivo. GRK2 is involved in cellular migration, insulin signaling, and cardiovascular disease. GRK6 in concert with β-arrestin 2 mediates chemoattractant-stimulated chemotaxis of T and B lymphocytes. GRK5 shuttles between the cytosol and nucleus, and regulates the activities of transcription factors. GRK3 and GRK4 do not seem to have striking effects on cellular responses other than receptor regulation. GRK1 and GRK7 play specifi c roles in regulation of rhodopsin function. In this review, these newly discovered functions of GRKs are briefl y described.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

The M 3 -Muscarinic Receptor Is Phosphorylated and Regulated by Casein Kinase 2

After binding to ligands, almost all G protein-coupled receptors (GPCRs) are rapidly phosphorylated--a modification with important functional consequences, such as desensitization and internalization. Many of the kinases involved in the phosphorylation of GPCRs are members of the family of GPCR kinases (GRKs). GPCRs are often phosphorylated at multiple sites and by more than one kinase, so the regulation of GPCRs by phosphorylation is quite complex. Torrecilla et al . have extended their previous studies by examining the role of casein kinase 2 (CK2) in the phosphorylation and regulation of the M 3 -muscarinic receptor. Both knockdown of CK2 with siRNA and blocking of its activity with specific inhibitors reduced the amount of phosphorylation of the M 3 -muscarinic receptor in CHO cells, as measured by immunoprecipitation assays. The use of mutant M 3 -muscarinic receptors showed that the sites phosphorylated by CK2 were in the third intracellular loop. This region is also targeted by GRK2 and GRK6, although further analysis showed that the specific sites phosphorylated by CK2 and GRK2 or -6 did not significantly overlap. Inhibitors of CK2 reduced the phosphorylation of the M 3 -muscarinic receptor in mouse cerebellar granule (CG) neurons. GRK-mediated phosphorylation causes internalization of the M 3 -muscarinic receptor, but CK2-mediated phosphorylation had no such effect. Whereas knockdown of CK2 did not change the ability of the M 3 -muscarinic receptor to activate extracellular signal-regulated kinase 1 and 2 (ERK1/2), activation of the c-Jun pathway was increased. Analysis of phosphopeptides from digested M 3 -muscarinic receptors from either CHO cells or CG neurons revealed different patterns of phosphorylation, implying that the phosphorylation of a given receptor is cell type-specific, potentially leading to different functional outcomes. I. Torrecilla, E. J. Spragg, B. Poulin, P. J. McWilliams, S. C. Mistry, A. Blaukat, A. B. Tobin, Phosphorylation and regulation of a G protein-coupled receptor by protein kinase CK2. J. Cell Biol . 177 , 127-137 (2007). [Abstract] [Full Text]

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

G Protein-coupled Receptor (GPCR) Kinase 2 Regulates Agonist-independent Gq/11 Signaling from the Mouse Cytomegalovirus GPCR M33

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 Kinase Specificity for β-Arrestin Recruitment to the β2-Adrenergic Receptor Revealed by Fluorescence Resonance Energy Transfer

The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.

Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.

Structure/Function Analysis of α2A-Adrenergic Receptor Interaction with G Protein-coupledReceptor Kinase 2

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.

Once and Future Signaling: G Protein-Coupled Receptor Kinase Control of Neuronal Sensitivity

G protein-coupled receptors (GPCRs) are the most numerous class of cell surface receptor, and substances acting through GPCRs mediate many critical signaling events and physiological processes. GPCR sensitivity and signaling is dynamic, responding rapidly to adjust to changes in the ambient level of stimulation of target cells. One important mediator of such receptor sensitivity is the family of GPCR kinases (GRKs). Like heterotrimeric G proteins, GRKs recognize agonist-bound, activated receptors, and this recognition promotes catalytic activation of GRKs, resulting in the preferential phosphorylation of activated receptors. GRK-phosphorylated receptors are then targeted by arrestin proteins, which bind to phosphorylated receptors. Arrestin-bound receptors are uncoupled from heterotrimeric G proteins, resulting in decreased sensitivity to further receptor stimulation (desensitization). Arrestin-bound receptors are also accelerated into internalization pathways and linked to distinct arrestin-mediated signaling pathways. GRKs thus serve as gatekeepers for receptors, terminating some signaling pathways and initiating others. One major outstanding question concerning GRKs understanding the mechanisms by which any particular receptor subtype (of the 800 or so in the body) is regulated by a specific GRK(s), and the consequences of this specificity. An understanding of this regulatory specificity could allow targeting of GRK function to ameliorate diseases involving GPCR dysregulation.

Phosphorylation and Chronic Agonist Treatment Atypically Modulate GABAB Receptor Cell Surface Stability

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

Species-Specific Differences in Expression of G-Protein-Coupled Receptor Kinase (GRK) 7 and GRK1 in Mammalian Cone Photoreceptor Cells: Implications for Cone Cell Phototransduction

Desensitization plays an important role in the rapid termination of G-protein signaling pathways. This process, which involves phosphorylation by a G-protein-coupled receptor kinase (GRK) followed by arrestin binding, has been studied extensively in the rod photoreceptor cell of the mammalian retina. In contrast, less is known regarding desensitization in cone photoreceptor cells, which occurs more rapidly than in rod cells. Recently, our laboratory has cloned a novel GRK family member, GRK7, from the retina of a cone-dominant mammal, the 13-lined ground squirrel. Here we report the cloning of GRK7 from rod-dominant pig and human retinas, suggesting that this kinase plays a role in human visual signaling. Because GRK1 (rhodopsin kinase), the GRK that mediates rhodopsin desensitization in the rod cell, is reportedly expressed in both rods and cones, a detailed comparison of the localization of the two kinases is a necessary step toward determining their potential roles in cone visual signaling. Immunocytochemical analysis using antibodies selective for these two GRKs unexpectedly demonstrated species-specific differences in GRK7 and GRK1 expression in cones. In pigs and dogs, cones express only GRK7, whereas in mice and rats, we detected only GRK1 in cones. These results suggest that either GRK7 or GRK1 may participate in cone opsin desensitization, depending on the expression pattern of the kinases in different species. In contrast, GRK7 and GRK1 are coexpressed in monkey and human cones, suggesting that coordinate regulation of desensitization by both kinases may occur in primates.