Regulatory effects of GRK2 on GPCRs and possible use as a drug target

G protein-coupled receptor kinase 2(GRK2),as a key Ser/Thr protein kinase,belong to the member of the G protein-coupled receptor kinase(GRK)family.The C-terminus of GRK2 including a plekstrin homology domain and the N-terminus of GRK2 including the RGS homology domain with binding sites for several proteins and lipids such as G protein-coupled receptors(GPCRs),G protein,phospholipase C,phosphatidylinositol 4,5-bisphosphate,extracellular signal-regulated kinase,protein kinase A and Gβγ,which can regulate the activity of GRK2.GRK2 can regulate GPCR desensitization and internalization by phosphorylating the GPCR,promoting the affinity of binding to arrestins,and uncoupling the receptors from G proteins,which play an important role in maintaining the balance between the receptors and signal transduction.Previous studies have indicated that cardiac GRK2overexpression can promote the phosphorylation ofβ-adrenergic receptor(βAR)leading toβAR desensitization and internalization,which play a pivotal role in inducing heart failure(HF)-related dysfunction and myocyte death.GRK2,as a regulator of cell function,is overexpression in hypertension.Overexpression GRK2 can inhibit Akt/e NOS signaling pathway and decreased the production and activation of e NOS leading to endothelial dysfunction.Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast-like synoviocytes.In this review,we mainly discussed the evidence for the association between GRK2 overexpression and various diseases,which suggests that GRK2 may be an effective drug target for preventing and treating heart failure,hypertension and inflammatory disease.     

Involvement of intramolecular interactions in the regulation of G protein-coupled receptor kinase 2

The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein betagamma subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as Gbetagamma subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.     

Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases

A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.     

Regulatory mechanisms underlying GKR2 levels in U937 cells: evidence for GRK3 involvement

G protein-coupled receptors represent the most diverse group of proteins involved in transmembrane signalling, that participate in the regulation of a wide range of physicochemical messengers through the interaction with heterotrimeric G proteins. In addition, GPCRs stimulation also triggers a negative feedback mechanism, known as desensitization that prevents the potentially harmful effects caused by persistent receptor stimulation. In this adaptative response, G protein-coupled receptor kinases (GRKs) play a key role and alterations in their function are related to diverse pathophysiological situations. Based on the scarce knowledge about the regulation of GRK2 by other kinases of the same family, the aim of the present work was to investigate the regulation of GRK2 levels in systems where other GRKs are diminished by antisense technique. Present findings show that in U937 cells GRK2 levels are regulated by GRK3 and not by GRK6 through a mechanism involving InsP upregulation. This work reports a novel GRK3-mediated GRK2 regulatory mechanism and further suggests that GRK2 may also act as a compensatory kinase tending to counterbalance the reduction in GRK3 levels. This study provides the first evidence for the existence of GRKs cross-regulation.     

Selective regulation of Gq signaling by G protein-coupled receptor kinase 2: direct interaction of kinase N terminus with activated galphaq

In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of Galphas- and Galphaq-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate Galphaq signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine(2C), which is coupled to Galphaq. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with Galphaq, direct activation of Galphaq signaling (by AlF(4)(-)) was desensitized by GRK2-Nter, indicating an effect at the Galpha-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the Galphaq subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with Galphaq (only when activated) but not with Galphas and Galphao. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to Galphaq and to regulate its signaling.     

G蛋白偶联受体激酶活性调控及其在恶性肿瘤中的作用

G蛋白偶联受体(GPCR),是一类重要的细胞表面受体。G蛋白偶联受体激酶(GRK)属于丝氨酸/苏氨酸蛋白激酶家族,其亚型广泛存在与各种组织,能够特异性地使活化的GPCR发生磷酸化及脱敏,从而终止GPCR介导的信号转导通路。新的研究还发现,GRK不仅作用于GPCR,也可以通过使非GPCR磷酸化或通过非磷酸化作用参与信号转导。GRK不仅能够调节GPCR和非GPCR,其自身活性也可受到多种因素的调节。本文结合GRK的多种功能作用和GRK活性调控,对GRK在脑、内分泌、生殖系统、消化系统及黑色素肿瘤中的作用做简要综述。     

GRK2: multiple roles beyond G protein-coupled receptor desensitization

G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or interacting directly with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).     

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts

In recent years, new strategies in cancer therapy have been developed targeting key signaling molecules in the receptor tyrosine kinase signal transduction pathway. In contrast, most therapeutical concepts to manipulate G protein-coupled receptors (GPCR)-mediated disorders are still limited to the use of receptor-specific agonists or antagonists. Visible progress in the understanding of GPCR signaling complexity, especially the detection of several families of highly target- and cell-specific regulator proteins of GPCRs, G proteins, and effector components may open new horizons to develop novel therapeutical concepts targeting GPCR signaling elements. Thus, this review will focus on different molecular levels that may be of particular interest in terms of new drug development such as: (i) GPCR subtypes, allosteric binding sites, dimerization and constitutive activity, the use of RAMPs (receptor-activity-modifying proteins) and RASSLs (receptor activated solely by synthetic ligands); (ii) AGS (activators of G protein signaling) and RGS (regulators of G protein signaling) proteins which modify G protein activity; (iii) the high diversity of isozymes involved in the generation, signal transmission, and degradation of second messenger molecules.     

Nerve growth factor stimulation of p42/p44 mitogen-activated protein kinase in PC12 cells: role of G(i/o), G protein-coupled receptor kinase 2, beta-arrestin I, and endocytic processing.

In this study, we have shown that nerve growth factor (NGF)-dependent activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in PC12 cells can be partially blocked by pertussis toxin (which inactivates the G proteins G(i/o)). This suggests that the Trk A receptor may use a G protein-coupled receptor pathway to signal to p42/p44 MAPK. This was supported by data showing that the NGF-dependent activation of p42/p44 MAPK is potentiated in cells transfected with G protein-coupled receptor kinase 2 (GRK2) or beta-arrestin I. Moreover, GRK2 is constitutively bound with the Trk A receptor, whereas NGF stimulates the pertussis toxin-sensitive binding of beta-arrestin I to the TrkA receptor-GRK2 complex. Both GRK2 and beta-arrestin I are involved in clathrin-mediated endocytic signaling to p42/p44 MAPK. Indeed, inhibitors of clathrin-mediated endocytosis (e.g., monodansylcadaverine, concanavalin A, and hyperosmolar sucrose) reduced the NGF-dependent activation of p42/p44 MAPK. Finally, we have found that the G protein-coupled receptor-dependent component regulating p42/p44 MAPK is required for NGF-induced differentiation of PC12 cells. Thus, NGF-dependent inhibition of DNA synthesis was partially blocked by PD098059 (inhibitor of MAPK kinase-1 activation) and pertussis toxin. Our findings are the first to show that the Trk A receptor uses a classic G protein-coupled receptor-signaling pathway to promote differentiation of PC12 cells.     

Structural mechanism of GPCR-arrestin interaction: recent breakthroughs

G protein-coupled receptors (GPCRs) are a major membrane receptor family with important physiological and pathological functions. In the classical signaling pathway, ligand-activated GPCRs couple to G proteins, thereby inducing G protein-dependent signaling pathways and phosphorylation by G protein-coupled receptor kinases (GRKs). This leads to an interaction with arrestins, which results in GPCR desensitization. Recently, non-classical GPCR signaling pathways, mediated by GPCR-bound arrestins, have been identified. Consequently, arrestins play important roles in GPCR signaling not only with respect to desensitization but also in relation to G protein-independent signal transduction. These findings have led to efforts to develop functionally biased (i.e. signal transduction biased) GPCR-targeting drugs. One of these efforts is aimed at understanding the structural mechanism of functionally biased GPCR signaling, which includes understanding the G protein-selectivity or arrestin-selectivity of GPCRs. This goal has not yet been achieved; however, great progress has been made during the last 3 years toward understanding the structural mechanism of GPCR-mediated arrestin activation. This review will discuss the recent breakthroughs in the conformational understanding of GPCR-arrestin interaction.     

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

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

Selective regulation of G protein-coupled receptor-mediated signaling by G protein-coupled receptor kinase 2 in FRTL-5 cells: analysis of thyrotropin, alpha(1B)-adrenergic, and A(1) adenosine receptor-mediated responses.

G protein-coupled receptor kinases (GRKs) play a key role in the process of receptor homologous desensitization. In the present study, we address the question of whether a variety of receptors coupled to different G protein subtypes and naturally expressed on the same cell are selectively regulated by GRK2. The signaling stimulated by thyrotropin (TSH), alpha(1B)-adrenergic, and A(1) adenosine receptors was studied in FRTL-5 cells permanently transfected to overexpress GRK2 and GRK2-K220R, a kinase dead GRK dominant negative mutant. In FRTL-5 overexpressing GRK2, TSH-induced cyclic AMP response was attenuated, indicating that TSH receptor is desensitized by this kinase. Consistently, FRTL-5 cells overexpressing GRK2-K220R show increased TSH-induced cyclic AMP response, demonstrating that this receptor is under tonic control by GRK. Unlike TSH receptor, alpha(1B)-adrenergic receptor response was unaffected in FRTL-5 overexpressing GRK2 and GRK2-K220R. When A(1) adenosine receptors were stimulated, G(ialpha)-mediated cyclic AMP inhibition was totally unaffected by overexpression of either GRK2 or GRK2-K220R. By contrast, G(betagamma)-mediated response (activation of mitogen-activated protein kinases) was efficiently desensitized by GRK2 but was unaffected by GRK2-K220R overexpression. The present study documents that overexpression of GRK2 results in a selective regulation of different G protein-coupled receptors expressed on the same cell and that this kinase can regulate preferentially only one of the different pathways activated by the same receptor. The preferential regulation of the A(1) adenosine receptor-stimulated mitogen-activated protein kinases by GRK2 indicates that this kinase can have additional regulatory effects on G(betagamma)-stimulated pathways, possibly through direct binding and regulation of the receptor-G(betagamma) complex.     

GRK2 as a therapeutic target for heart failure

Introduction: G protein-coupled receptor (GPCR) kinase-2 (GRK2) is a regulator of GPCRs, in particular β-adrenergic receptors (ARs), and as demonstrated by decades of investigation, it has a pivotal role in the development and progression of cardiovascular disease, like heart failure (HF). Indeed elevated levels and activity of this kinase are able to promote the dysfunction of both cardiac and adrenal α- and β-ARs and to dysregulate other protective signaling pathway, such as sphingosine 1-phospate and insulin. Moreover, recent discoveries suggest that GRK2 can signal independently from GPCRs, in a ‘non-canonical’ manner, via interaction with non-GPCR molecule or via its mitochondrial localization.

Areas covered: Based on this premise, GRK2 inhibition or its genetic deletion has been tested in several disparate animal models of cardiovascular disease, showing to protect the heart from adverse remodeling and dysfunction.

Expert opinion: HF is one of the leading cause of death worldwide with enormous health care costs. For this reason, the identification of new therapeutic targets like GRK2 and strategies such as its inhibition represents a new hope in the fight against HF development and progression. Herein, we will update the readers about the ‘state-of-art’ of GRK2 inhibition as a potent therapeutic strategy in HF.     

Enhanced expression of G protein-coupled receptor kinase 2 selectively increases the sensitivity of A2A adenosine receptors to agonist-induced desensitization

1. G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor (GPCR) responses. We have previously shown that stable expression of a dominant negative mutant G protein- coupled receptor kinase 2 (GRK2) construct in NG108-15 mouse neuroblastoma x rat glioma cells suppresses the agonist-induced desensitization of A_2A and A_2B adenosine receptor-stimulated adenylyl cyclase activity (Mundell et al., 1997). To further determine the role of GRK2 in agonist-induced desensitization of these adenosine receptors, we stably overexpressed wild type GRK2 in NG108-15 cells.
2. In homogenates prepared from cells overexpressing GRK2, the acute stimulation of adenylyl cyclase by activation of A_2A and A_2B adenosine receptors was markedly reduced, but could be reversed by pretreating the cells with AD (adenosine deaminase), to remove extracellular adenosine from the medium. On the other hand, acute stimulation of adenylyl cyclase by secretin, iloprost, NaF and forskolin was the same in GRK2 overexpressing cells and plasmid-transfected control cells.
3. Cells overexpressing GRK2 were more sensitive to adenosine receptor agonist-induced desensitization than plasmid-transfected control cells. This effect was selective since the agonist sensitivity of desensitization for secretin and IP-prostanoid receptor-stimulated adenylyl cyclase activity was not affected by GRK2 overexpression.
4. These results further implicate GRK2 as the likely mechanism by which A₂ adenosine receptors undergo short-term desensitization in NG108-15 cells, and indicate that even when overexpressed, GRK2 retains its substrate specificity for native receptors in intact cells. Furthermore, the susceptibility of GPCRs to desensitization appears to depend on the level of GRK expression, such that in cells that express high levels of GRK2, low agonist concentrations may be sufficient to trigger GRK-mediated desensitization.

     

Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.     

Molecular mechanism of selectivity among G protein-coupled receptor kinase 2 inhibitors

G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-Gβγ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-Gβγ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.     

Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac beta-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, alpha(1)-adrenergic, and beta(1)-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC(50) for GRK3, 5 +/- 0.7 pmol/mg of protein; EC(50) for GRK3ct, 2 +/- 0.2 pmol/mg of protein). The alpha(1)-adrenergic receptor was also a preferred substrate of GRK3 (IC(50),7 +/- 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and alpha(1)-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced beta(1)-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at beta(1)-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling beta(1)-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and alpha(1)-adrenergic receptors.     

Desensitization of endogenously expressed delta-opioid receptors: no evidence for involvement of G protein-coupled receptor kinase 2.

The involvement of G protein-coupled receptor kinase 2 (GRK2) in the agonist-induced desensitization of delta-opioid receptor-mediated inhibition of cAMP formation in NG108-15 mouse neuroblastomaxrat glioma hybrid cells was investigated. Pretreatment of wild-type cells with the delta-opioid receptor agonist [D-Pen(2,5)]-enkephalin (DPDPE; 100 nM) for as little as 5 min produced marked desensitization of subsequent DPDPE-mediated inhibition of iloprost (300 nM)-stimulated cAMP formation. In NG108-15 cells stably overexpressing wild-type GRK2 or dominant negative mutant GRK2 (DNM GRK2), the DPDPE-induced desensitization of cAMP inhibition was the same as in plasmid-transfected control cells. Pretreatment of wild-type cells with the inhibitors of receptor internalization, concanavalin A (0.25 mg ml(-1)) or hypertonic sucrose (0.4 M), also failed to inhibit DPDPE-mediated desensitization. Finally, in NG108-15 cells stably overexpressing G protein-coupled receptor kinase 6 (GRK6), DPDPE-induced desensitization was significantly increased as compared to plasmid-transfected control cells. These results indicate that GRK2 is unlikely to mediate the desensitization of endogenous delta-opioid receptors in NG108-15 cells, but that other GRKs, such as GRK6, may be more important.     

Effects of chlorpyrifos oxon on M2 muscarinic receptor internalization in different cell types

The muscarinic M2 receptor is a member of the G protein-coupled receptor (GPCR) superfamily. Agonist activation of GPCR leads to their phosphorylation, desensitization, internalization, and subsequent endocytic recycling or lysosomal degradation. Agonist-induced phosphorylation of M2 receptors is mediated by G-protein receptor kinase 2 (GRK2). The active metabolite of the organophosphorus insecticide chlorpyrifos, i.e., chlorpyrifos oxon (CPO), inhibited agonist-induced phosphorylation of human recombinant M2 receptors by GRK2 in vitro in a concentration-dependent manner. In both intact HEL 299 cells (human embryonic lung fibroblasts expressing M2 receptors) and CHO-M2 cells (stably expressing M2 receptors), the muscarinic agonist carbachol (100 microM) led to receptor internalization as determined by reduced specific binding to the membrane-impermeable radioligand [(3)H]-N-methylscopolamine (NMS). CPO alone (100 microM) exerted no significant effect on NMS binding in either HEL 299 or CHO-M2 cells. In HEL 299 cells, CPO did not influence carbachol-induced internalization, whereas in CHO-M2 cells CPO blocked internalization. In primary striatal neurons, M2 receptors appeared widely and diffusely distributed. Exposure to either carbachol or CPO led to apparent receptor internalization with an increased percent of cells exhibiting punctate domains of immunostaining, while combined exposure to both carbachol and CPO led to a significantly higher percent of cells exhibiting this appearance. The data suggest that CPO may differentially influence agonist-stimulated M2 receptor internalization in a cell-dependent manner.