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.     

Agonist-stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells: agonist-stimulated endocytosis is beta-arrestin1 isoform-specific.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that contribute to the regulation of integrative brain functions such as cognition, motor control, and neural development. Metabotropic glutamate receptors are members of a unique class of GPCRs (class III) that include the calcium sensing and gamma-aminobutyric acid type B receptors. Although mGluRs bear little sequence homology to well-characterized members of the GPCR superfamily, both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) contribute to mGluR desensitization. Therefore, in the present study, we examined whether beta-arrestins, regulators of GPCR desensitization and endocytosis, are required for mGluR1a desensitization and internalization in human embryonic kidney (HEK) 293 cells. Unlike what has been reported for other GPCRs, we find that in response to agonist stimulation, mGluR1a internalization is selectively mediated by beta-arrestin1 in HEK 293 cells. However, even though beta-arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither beta-arrestin1 nor beta-arrestin2 seems to contribute to mGluR1a desensitization in HEK 293 cells. We also observed extensive tonic mGluR1a internalization via clathrin-coated vesicles in the absence of agonist. The tonic internalization of mGluR1a is insensitive to antagonist treatment, dominant-negative mutants of GRK2, beta-arrestin1, and dynamin as well as treatments that disrupt caveolae, but is blocked by hypertonic sucrose and concanavalin A treatment. Internalized mGluR1a is colocalized with clathrin, transferrin receptor, beta2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with beta-arrestin in response to agonist, the agonist-independent internalization of mGluR1a involves the beta-arrestin-independent targeting of mGluR1a to clathrin-coated vesicles.     

G蛋白偶联受体激酶和arrestins在受体调节中的作用

Ｇ 蛋白偶联受体（ Ｇｐｒｏｔｅｉｎ ｃｏｕｐｌｅｄ ｒｅｃｅｐｔｏｒ ， Ｇ Ｐ Ｃ Ｒｓ）是一大家族，介导许多激素的信号转导。在激动剂的持续作用下， Ｇ Ｐ Ｃ Ｒｓ 可发生对激动剂的敏感性下降，即受体减敏，现认为这一过程主要由 Ｇ 蛋白偶联受体激酶（ Ｇｐｒｏｔｅｉｎ ｃｏｕ ｐｌｅｄ ｒｅｃｅｐｔｏｒ ｋｉｎａｓｅｓ ， Ｇ Ｒ Ｋｓ） 和ａｒｒｅｓｔｉｎｓ 两大蛋白家族介导： Ｇ Ｒ Ｋｓ 先结合并磷酸化被激动剂占领的受体，然后ａｒｒｅｓｔｉｎｓ与磷酸化的受体结合，阻止受体与 Ｇ 蛋白发生作用，导致受体功能减退。近来发现， Ｇ Ｒ Ｋｓ 和ａｒｒｅｓｔｉｎｓ 还参与受体的内陷机制，而受体的复敏又与内陷密切相关     

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).     

Novel roles for arrestins in G protein-coupled receptor biology and drug discovery

The G protein-coupled receptors (GPCRs) are the largest family of membrane proteins and represent some of the most important pharmaceutical targets. These receptors, encoded by several hundred genes, are activated by a wide variety of endogenous and synthetic ligands. The study of the signal transduction pathways activated by these receptors and the associated mechanisms controlling biological responses have been pivotal in identifying key intracellular molecules for regulating receptor responsiveness. The beta-arrestin proteins, which were initially discovered due to their role in GPCR desensitization, serve equally important roles in regulating internalization and alternative signaling events. This review focuses on the different functions of beta-arrestins to demonstrate how these proteins can help to identify new ligands for GPCRs and how they can serve as a platform for drug discovery.     

Phosphorylation-independent attenuation of GPCR signalling

The uncoupling of G-protein-coupled receptors (GPCRs) from their cognate heterotrimeric G proteins provides an essential physiological 'feedback' mechanism that protects against both acute and chronic overstimulation of receptors. The primary mechanism by which GPCR activity is regulated is the feedback phosphorylation of activated GPCRs by kinases that are dependent on second messengers, GPCR kinases (GRKs) and arrestins. It has recently become apparent, however, that GRK2-mediated regulation of GPCR responsiveness also involves a phosphorylation-independent component that requires both heterotrimeric G-protein alpha-subunit interactions and GPCR binding. Moreover, in addition to GRK2, a growing number of GPCR-interacting proteins might contribute to the phosphorylation-independent G-protein uncoupling of GPCRs. Here, new information about the mechanisms underlying this phosphorylation-independent regulation of receptor and G-protein coupling is reviewed.     

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.     

The D1 dopamine receptor is constitutively phosphorylated by G protein-coupled receptor kinase 4

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist-activated GPCRs, initiating their homologous desensitization. In this article, we present data showing that GRK4 constitutively phosphorylates the D1 receptor in the absence of agonist activation. This constitutive phosphorylation is mediated exclusively by the alpha isoform of GRK4; the beta, gamma, and delta isoforms are ineffective in this regard. Mutational analysis reveals that the constitutive phosphorylation mediated by GRK4alpha is restricted to the distal region of the carboxyl terminus of the receptor, specifically to residues Thr428 and Ser431. Phosphorylation of the D1 receptor by GRK4alpha results in a decrease in cAMP accumulation, an increase in receptor internalization, and a decrease in total receptor number--all of which are abolished in a D1 receptor mutant containing T428V and S431A. The increase in internalized D1 receptors induced by GRK4alpha phosphorylation is due to enhanced receptor internalization rather than retarded trafficking of newly synthesized receptors to the cell surface. The constitutive phosphorylation of the D1 receptor by GRK4alpha does not alter agonist-induced desensitization of the receptor because dopamine pretreatment produced a similar decrease in cAMP accumulation in control cells versus cells expressing GRK4alpha. These observations shift the attenuation of D1 receptor signaling from a purely agonist-driven process to one that is additionally modulated by the complement of kinases that are coexpressed in the same cell. Furthermore, our data provide direct evidence that, in contrast to current dogma, GRKs can (at least in some instances) constitutively phosphorylate GPCRs in the absence of agonist activation resulting in constitutive desensitization.     

Agonist-selective mechanisms of GPCR desensitization

The widely accepted model of G protein-coupled receptor (GPCR) regulation describes a system where the agonist-activated receptors couple to G proteins to induce a cellular response, and are subsequently phosphorylated by a family of kinases called the G protein-coupled receptor kinases (GRKs). The GRK-phosphorylated receptor then acts as a substrate for the binding of a family of proteins called arrestins, which uncouple the receptor and G protein so desensitizing the agonist-induced response. Other kinases, principally the second messenger-dependent protein kinases, are also known to play a role in the desensitization of many GPCR responses. It is now clear that there are subtle and complex interactions between GRKs and second messenger-dependent protein kinases in the regulation of GPCR function. Functional selectivity describes the ability of agonists to stabilize different active conformations of the same GPCR. With regard to desensitization, distinct agonist-activated conformations of a GPCR could undergo different molecular mechanisms of desensitization. An example of this is the mu opioid receptor (MOPr), where the agonists morphine and [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) induce desensitization of the MOPr by different mechanisms, largely protein kinase C (PKC)- or GRK-dependent, respectively. This can be best explained by supposing that these two agonists stabilize distinct conformations of the MOPr, which are nevertheless able to couple to the relevant G-proteins and produce similar responses, yet are sufficiently different to trigger different regulatory processes. There is evidence that other GPCRs also undergo agonist-selective desensitization, but the full therapeutic consequences of this phenomenon await further detailed study.     

与类风湿关节炎相关的G蛋白偶联受体及治疗药物作用靶点

很多胞外信号直接或间接通过G蛋白偶联受体向胞内传输。多种G蛋白偶联受体包括趋化因子受体、前列腺素(prostaglandins,PGs)受体、β2-肾上腺素受体(β2-adrener-gicreceptor)、致炎肽P物质(proinflammatorypeptidesub-stanceP,SP)受体、蛋白酶活化受体2(protease-activatedre-ceptor2,PAR-2)等在免疫应答调节中起至关重要的作用。该文综述了与类风湿关节炎相关的G蛋白偶联受体(Gpro-tein-coupledreceptors,GPCRs)信号转导的一些蛋白作用,包括G蛋白偶联受体激酶、视紫红质抑制蛋白(arrestin)、G蛋白信号转导调节因子、G蛋白偶联致炎受体等。作用于这些信号或其转导过程的药物正成为类风湿关节炎治疗的新策略之一。     

Histamine H2 receptor trafficking: role of arrestin, dynamin, and clathrin in histamine H2 receptor internalization

Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 (a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with beta-arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs.     

Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.     

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.     

Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid.

Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.     

Differences in motilin receptor desensitization after stimulation with motilin or motilides are due to alternative receptor trafficking

BACKGROUNDS & AIMS: The motilin receptor (MTLR) is an important therapeutic target for treatment of hypomotility disorders. The negative outcome in clinical trials with the motilin agonist, ABT-229, indicated that desensitization may limit the therapeutic usefulness of motilides. We therefore compared the mechanisms involved in the intracellular trafficking of the MTLR after stimulation with motilin, erythromycin-A (EM-A) or ABT-229. METHODS: Desensitization was studied by measuring changes in Ca2+ rises and by receptor binding studies in CHO cells co-expressing the Ca2+ indicator apoaequorin and the MTLR, C-terminally tagged with EGFP. Receptor phosphorylation was studied by immunoprecipitation. MTLR-EGFP trafficking to organelles and translocation of beta-arrestins were visualized by fluorescence microscopy. RESULTS: Agonist-induced desensitization of the MTLR was due to receptor internalization with potencies (p-int50) in the order of: ABT-229 (8.3)>motilin (7.86)>EM-A (4.77) but with no differences in the internalization kinetics (t(1/2): approximately 25 min). The percentage cell surface receptor loss was more profound after exposure to ABT-229 (88+/-1%) than to motilin (63+/-10%) or EM-A (34+/-2%). For motilin and EM-A MTLR phosphorylation probably occurs via G protein-coupled receptor kinases while for ABT-229 phosphorylation was also protein kinase C dependent. All agonists translocated cytosolic beta-arrestin-2 with greater affinity to the plasma membrane than beta-arrestin-1. After internalization the MTLR co-localized with transferrin but not with cathepsin D. After stimulation with motilin and EM-A the t(1/2) for MTLR resensitization was 3h and 1h, respectively but amounted 26h for ABT-229. CONCLUSION: Our results suggest that the resensitization kinetics determine the desensitization properties of the motilin agonists.     

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.     

Desensitization and trafficking of μ-opioid receptors in locus ceruleus neurons: modulation by kinases

The phosphorylation of μ-opioid receptors (MOPRs) by G protein-coupled receptor kinases (GRKs), followed by arrestin binding, is thought to be a key pathway leading to desensitization and internalization. The present study used the combination of intracellular and whole-cell recordings from rats and mice, as well as live cell imaging of Flag-tagged MOPRs from mouse locus ceruleus neurons, to examine the role of protein kinases in acute desensitization and receptor trafficking. Inhibition of GRKs by using heparin or GRK2-mutant mice did not block desensitization or alter the rate of recovery from desensitization. The nonselective kinase inhibitor staurosporine did not reduce the extent of [Met(5)]enkephalin (ME)-induced desensitization but increased the rate of recovery from desensitization. In the presence of staurosporine, ME-activated FlagMOPRs were internalized but did not traffic away from the plasma membrane. The increased rate of recovery from desensitization correlated with the enhancement in the recycling of receptors to the plasma membrane. ME-induced MOPR desensitization persisted and the trafficking of receptors was modified after inhibition of protein kinases. The results suggest that desensitization of MOPRs may be an early step after agonist binding that is modulated by but is not dependent on kinase activity.     

Relative opioid efficacy is determined by the complements of the G protein-coupled receptor desensitization machinery

G protein-coupled receptor regulation by G protein-coupled receptor kinases and beta-arrestins can lead to desensitization and subsequent internalization of the receptor. In in vitro and cellular systems, beta-arrestins do not seem to play a major role in regulating micro opioid receptor (microOR) responsiveness. Removal of the betaarrestin2 (betaarr2) gene in mice leads paradoxically to enhanced and prolonged microOR-mediated antinociception. The betaarr2 knockout (betaarr2-KO) mice also fail to develop morphine antinociceptive tolerance in the hot-plate test, further indicating that the betaarr2 protein plays an essential role in microOR regulation in vivo. In this study, the contribution of betaarr2 to the regulation of the microOR was examined in both human embryonic kidney 293 cells and in betaarr2-KO mice after treatment with several opiate agonists. A green fluorescent protein tagged betaarr2 was used to assess receptor-betaarr2 interactions in living cells. Opiate agonists that induced robust betaarr2-green fluorescent protein translocation produced similar analgesia profiles in wild-type and betaarr2-KO mice, whereas those that do not promote robust betaarr2 recruitment, such as morphine and heroin, produce enhanced analgesia in vivo. In this report, we present a rationale to explain the seemingly paradoxical relationship between beta-arrestins and microOR regulation wherein morphine-like agonists fail to promote efficient internalization and resensitization of the receptor.     

Signal transduction pathways of G protein-coupled receptors and their cross-talk with receptor tyrosine kinases: lessons from bradykinin signaling

G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.     

The effect of inhibitors of receptor internalization on the desensitization and resensitization of three Gs-coupled receptor responses

1. Many G protein-coupled receptors (GPCRs) are known to internalize following agonist exposure, however the relative importance of this mechanism for the desensitization and resensitization of different GPCRs is unclear. In the present study, we have pretreated NG108-15 cells with hypertonic sucrose or concanavalin A (con A), to investigate the effects of these inhibitors of internalization on the agonist-induced desensitization and subsequent resensitization of three G_s-coupled receptor responses.
2. Incubation of cells with sucrose or con A did not affect subsequent acute agonist stimulation of the A_2A adenosine receptor or the agonist-induced desensitization of this receptor response. However, the resensitization of the A_2A adenosine receptor response following agonist removal was abolished in the presence of sucrose or con A.
3. Sucrose or con A treatment affected neither the desensitization nor resensitization of IP-prostanoid receptor responsiveness. On the other hand con A but not sucrose reduced the agonist-induced desensitization of secretin receptor responsiveness. However, secretin receptor responsiveness did not resensitize within the time period studied whether or not inhibitors of internalization were present.
4. These results indicate that receptor internalization appears to subserve different functions for different GPCRs.