Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2

beta-Arrestin2 not only plays essential roles in seven membrane-spanning receptor desensitization and internalization but also functions as a signal transducer in mitogen-activated protein kinase cascades. Here we show that the angiotensin II type 1A receptor-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HEK-293 cells is increased when the cellular level of beta-arrestin1 is down-regulated by RNA interference but is decreased or eliminated when the cellular level of beta-arrestin2 is diminished. Such reciprocal effects of down-regulated levels of beta-arrestins 1 and 2 are primarily due to differences in the ability of the two forms of beta-arrestins to directly mediate ERK activation. These results are the first to demonstrate reciprocal activity of beta-arrestin isoforms on a signaling pathway and suggest that physiological levels of beta-arrestin1 may act as "dominant-negative" inhibitors of beta-arrestin2-mediated ERK activation.     

Identification of NSF as a beta-arrestin1-binding protein. Implications for beta2-adrenergic receptor regulation

Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.     

Feedback regulation of beta-arrestin1 function by extracellular signal-regulated kinases.

The functions of beta-arrestin1 to facilitate clathrin-mediated endocytosis of the beta2-adrenergic receptor and to promote agonist-induced activation of extracellular signal-regulated kinases (ERK) are regulated by its phosphorylation/dephosphorylation at Ser-412. Cytoplasmic beta-arrestin1 is almost stoichiometrically phosphorylated at Ser-412. Dephosphorylation of beta-arrestin1 at the plasma membrane is required for targeting a signaling complex that includes the agonist-occupied receptors to the clathrin-coated pits. Here we demonstrate that beta-arrestin1 phosphorylation and function are modulated by an ERK-dependent negative feedback mechanism. ERK1 and ERK2 phosphorylate beta-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates beta-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated beta-arrestin1. Under such conditions, beta-arrestin1-mediated beta2-adrenergic receptor internalization is enhanced as is its ability to bind clathrin. In contrast, if ERK-mediated phosphorylation is increased by transfection of a constitutively active MEK1 mutant, receptor internalization is inhibited. Our results suggest that dephosphorylated beta-arrestin1 mediates endocytosis-dependent ERK activation. Following activation, ERKs phosphorylate beta-arrestin1, thereby exerting an inhibitory feedback control of its function.     

Formyl peptide receptor-mediated ERK1/2 activation occurs through G(i) and is not dependent on beta-arrestin1/2

Formyl peptide receptor (FPR) and C5a receptor (C5aR) are chemoattractant G protein-coupled receptors (GPCRs) involved in the innate immune response against bacterial infections and tissue injury. Like other GPCRs, they recruit beta-arrestin1/2 to the plasma membrane and activate the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Previous studies with several GPCRs have suggested that beta-arrestins play an important role as signal transducers by scaffolding signaling molecules such as ERK1/2. This function of the beta-arrestins was not discovered until several years after their role in desensitization and endocytosis had been reported. In this study, we investigated the role of the beta-arrestins in the activation of ERK1/2 and receptor endocytosis. We took advantage of previously described mutants of FPR that have defects in G(i) coupling or beta-arrestin recruitment. The results obtained with the mutant FPRs, as well as experiments using an inhibitor of G(i) and cells overexpressing beta-arrestin2, showed that activation of ERK1/2 takes place through G(i) and is not affected by beta-arrestins. However, overexpression of beta-arrestin2 does enhance FPR sequestration from the cell surface, suggesting a role in desensitization, as shown for many other GPCRs. Experiments with CHO C5aR cells showed similar sensitivity to the G(i) inhibitor as CHO FPR cells, suggesting that the predominant activation of ERK1/2 through G protein may be a common characteristic among chemoattractant receptors.     

Differential regulation of corticotropin releasing factor 1alpha receptor endocytosis and trafficking by beta-arrestins and Rab GTPases

The corticotropin releasing factor (CRF) type 1alpha receptor, a member of the G protein-coupled receptor (GPCR) subfamily B, is involved in the aetiology of anxiety and depressive disorders. In the present study, we examined the internalization and trafficking of the CRF1alpha receptor in both human embryonic kidney (HEK)293 cells and primary cortical neurons. We found that CRF1alpha receptor activation leads to the selective recruitment of beta-arrestin2 in both HEK293 cells and neurons. We observed distinct distribution patterns of CRF1alpha receptor and beta-arrestin2 in HEK293 cells and cortical neurons. In HEK293 cells, beta-arrestin2-green fluorescent protein (GFP) co-localized with CRF1alpha receptor in vesicles at the plasma membrane but was dissociated from the receptor in endosomes. In contrast, in primary cortical neurons, beta-arrestin2 and CRF1alpha receptor were internalized in distinct endocytic vesicles. By bioluminescence resonance energy transfer, we demonstrated that beta-arrestin2 association with CRF1alpha receptor was increased in cells transfected with G protein-coupled receptor kinase (GRK)3 and GRK6 and decreased in cells transfected with GRK2 and GRK5. In both HEK293 cells and cortical neurons, internalized CRF1alpha receptor transited from Rab5-positive early endosomes to Rab4-positive recycling endosomes and was not targeted to lysosomes. However, CRF1alpha receptor resensitization was blocked by the overexpression of wild-type, but not dominant-negative, Rab5 and Rab4 GTPases. Taken together, our results suggest that beta-arrestin trafficking differs between HEK293 cells and neurons, and that CRF1alpha receptor resensitization is regulated in an atypical manner by Rab GTPases.     

Differential desensitization, receptor phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7

Many members of the chemokine receptor family of G protein-coupled receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of receptor phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated phosphorylation of the receptor, whereas residues serine 356, 357, 364, and 365 are important for basal receptor phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the phosphorylation sites in the tail of the receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the receptor and activation of ERK1/2, and another that is impaired in these functions.     

beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2

Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.     

Intracellular mechanisms regulating corticotropin-releasing hormone receptor-2beta endocytosis and interaction with extracellularly regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling cascades

Many important physiological roles of the urocortin (UCN) family of peptides as well as CRH involve the type 2 CRH receptor (CRH-R2) and downstream activation of multiple pathways. To characterize molecular determinants of CRH-R2 functional activity, we used HEK293 cells overexpressing recombinant CRH-R2beta and investigated mechanisms involved in attenuation of CRH-R2 signaling activity and uncoupling from intracellular effectors. CRH-R2beta-mediated adenylyl cyclase activation was sensitive to homologous desensitization induced by pretreatment with either UCN-II or the weaker agonist CRH. CRH-R2beta activation induced transient beta-arrestin1 and beta-arrestin2, as well as clathrin, recruitment to the plasma membrane. Beta-arrestin2 appeared to be the main beta-arrestin subtype associated with the receptor. This was followed by CRH-R2beta endocytosis in a mechanism that exhibited distinct agonist-dependent temporal characteristics. CRH-R2beta also induced transient activation of the ERK1/2 and p38MAPK signaling cascades that peaked at 5 min and returned to basal within 20-30 min. Unlike p38MAPK, activated ERK1/2 was localized both in the cytoplasm and nucleus. Experiments employing inhibitors of receptor endocytosis showed that CRH-R2beta-MAPK interaction does not require beta-arrestin, clathrin, or receptor endocytosis. Site-directed mutagenesis studies on CRH-R2beta C terminus showed that the amino acid cassette TAAV at the end of the C terminus is important for CRH-R2beta signaling because loss of a potential phospho-acceptor site in mutant receptors containing deletion or Ala substitution of the cassette TAAV resulted in reduced ERK1/2 activation and accelerated receptor internalization. These findings provide new insights about the signaling mechanisms regulating CRH-R2beta functional activity and determining its biological responses.     

Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor

The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely G(q)/G(11)) or beta-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized beta-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t((1/2)) approximately 2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, beta-arrestin2-dependent activation was slower (peak 5-10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via beta-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and beta-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints.     

Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors

Beta-arrestins are key negative regulators and scaffolds of G protein-coupled receptor (GPCR) signalling. Beta-arrestin1 and beta-arrestin2 preferentially bind to the phosphorylated GPCRs in response to agonist stimulation, resulting in receptor internalization and desensitization. The critical roles of GPCR kinases (GRKs)-catalyzed receptor phosphorylation and interaction of beta-arrestins with the phosphorylated receptor in receptor internalization are well established. However, emerging evidence suggests that an agonist-stimulated internalization mechanism that is independent of receptor phosphorylation may also be employed in some cases, although the molecular mechanism for the phosphorylation-independent GPCR internalization is not clear. The current study investigated the role of receptor phosphorylation and the involvement of different beta-arrestin subtypes in agonist-induced delta-opioid receptor (DOR) internalization in HEK293 cells. Results from flow cytometry, fluorescence microscopy, and surface biotin labelling experiments showed that elimination of agonist-induced DOR phosphorylation by mutation GRK binding or phosphorylation sites only partially blocked agonist-induced receptor internalization, indicating the presence of an agonist-induced, GRK-independent mechanism for DOR internalization. Fluorescence and co-immunoprecipitation studies indicated that both the wild-type DOR and the phosphorylation-deficient mutant receptor could bind and recruit beta-arrestin1 and beta-arrestin2 to the plasma membrane in an agonist-stimulated manner. Furthermore, internalization of both the wild-type and phosphorylation-deficient receptors was increased by overexpression of either type of beta-arrestins and blocked by dominant-negative mutants of beta-arrestin-mediated internalization, demonstrating that both phosphorylation-dependent and -independent internalization require beta-arrestin. Moreover, double-stranded RNA-mediated interference experiments showed that either beta-arrestin1 or beta-arrestin2 subtype-specific RNAi only partially inhibited agonist-induced internalization of the wild-type DOR. However, agonist-induced internalization of the phosphorylation-deficient DOR was not affected by beta-arrestin1-specific RNAi but was blocked by RNAi against beta-arrestin2 subtype. These data indicate that endogenous beta-arrestin1 functions exclusively in the phosphorylation-dependent receptor internalization, whereas endogenous beta-arrestin2, but not beta-arrestin1, is required for the phosphorylation-independent receptor internalization. These results thus provide the first evidence of different requirement for beta-arrestin isoforms in the agonist induced phosphorylation-dependent and -independent GPCR internalization.     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.     

Insulin-induced beta-arrestin1 Ser-412 phosphorylation is a mechanism for desensitization of ERK activation by Galphai-coupled receptors

Beta-arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of beta-arrestin1 at position Ser-412 is a regulator of beta-arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in beta-arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated beta-arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with beta-arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, beta-arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased beta-arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by beta-arrestin1-dependent receptors with no effect on beta-adrenergic receptor Gs-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by Galphai-coupled receptors.     

Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination

Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein beta-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the beta(2)-adrenergic receptor (beta(2)AR) this requires ubiquitination of beta-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of beta-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g. beta(2)AR), which recycle rapidly, beta-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g. V2 vasopressin receptors), which recycle slowly, beta-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the beta(2)AR or V2 vasopressin receptor leads, respectively, to transient or stable beta-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of beta-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (beta(2)AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both beta-arrestin trafficking and beta-arrestin ubiquitination. To explore the functional consequences of beta-arrestin ubiquitination we constructed a yellow fluorescent protein-tagged beta-arrestin2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases. This "permanently ubiquitinated" beta-arrestin did not dissociate from the beta(2)AR but rather internalized with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this beta-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of beta(2)AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and beta-arrestin was increased dramatically, suggesting that deubiquitination of beta-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of beta-arrestin determines the stability of the receptor-beta-arrestin complex as well as the trafficking pattern of beta-arrestin.