beta -Arrestins regulate protease-activated receptor-1 desensitization but not internalization or Down-regulation.

The widely expressed beta-arrestin isoforms 1 and 2 bind phosphorylated G protein-coupled receptors (GPCRs) and mediate desensitization and internalization. Phosphorylation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, is important for desensitization and internalization, however, the role of beta-arrestins in signaling and trafficking of PAR1 remains unknown. To assess beta-arrestin function we examined signaling and trafficking of PAR1 in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin (betaarr) knockouts. Desensitization of PAR1 signaling was markedly impaired in MEFs lacking both betaarr1 and betaarr2 isoforms compared with wild-type cells. Strikingly, in cells lacking only betaarr1 PAR1 desensitization was also significantly impaired compared with betaarr2-lacking or wild-type cells. In wild-type MEFs, activated PAR1 was internalized through a dynamin- and clathrin-dependent pathway and degraded. Surprisingly, in cells lacking both betaarr1 and betaarr2 activated PAR1 was similarly internalized through a dynamin- and clathrin-dependent pathway and degraded, whereas the beta(2)-adrenergic receptor (beta(2)-AR) failed to internalize. A PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation failed to internalize in both wild-type and beta-arrestin knockout cells. Thus, PAR1 appears to utilize a distinct phosphorylation-dependent but beta-arrestin-independent pathway for internalization through clathrin-coated pits. Together, these findings strongly suggest that the individual beta-arrestin isoforms can differentially regulate GPCR desensitization and further reveal a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins.     

Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.

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

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.     

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.     

Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.     

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.     

Human substance P receptor lacking the C-terminal domain remains competent to desensitize and internalize

Substance P receptor (SPR) and its naturally occurring splice-variant, lacking the C-terminal tail, are found in brain and spinal cord. Whether C-terminally truncated SPR desensitizes like full-length SPR is controversial. We used a multivaried approach to determine whether human SPR (hSPR) and a C-terminally truncated mutant, hSPRDelta325, differ in their desensitization and internalization. In HEK-293 cells expressing either hSPRDelta325 or hSPR, SP-induced desensitization of the two receptors was similar when measured by inositol triphosphate accumulation or by transient translocation of coexpressed PKCbetaII-GFP to the plasma membrane. Moreover, translocation of beta-arrestin 1 or 2-GFP (betaarr1-GFP or betaarr2-GFP) to the plasma membrane, and receptor internalization were also similar. However, hSPR and hSPRDelta325 differ in their phosphorylation and in their ability to form beta-arrestin-containing endocytic vesicles. Unlike hSPR, hSPRDelta325 is not phosphorylated to a detectable level in intact HEK293 cells, and whereas hSPR forms vesicles containing either betaarr1-GFP or betaarr2-GFP, hSPRDelta325 does not form any vesicles with betaarr1-GFP, and forms fewer vesicles with betaarr2-GFP. We conclude that truncated hSPR undergoes agonist-dependent desensitization and internalization without detectable receptor phosphorylation.     

beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis

beta-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which beta-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the beta(2)-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous beta-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, beta-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including beta-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.     

Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors

Beta-arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. Beta-arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT1AR), but not beta2-adrenergic receptor (beta2AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a beta2AR bearing the AT1AR tail acquired the capacity to stimulate beta-arrestin redistribution to endosomes, whereas this property was lost for an AT1AR bearing the beta2AR tail. Using beta2AR/AT1AR chimeras, we tested whether the beta2AR and AT1AR carboxyl-terminal tails, in part via their association with beta-arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that beta-arrestin formed a stable complex with the AT1AR tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the beta2AR chimera bearing the AT1AR tail was observed in the absence of agonist and was inhibited by a dominant-negative beta-arrestin1 mutant. Agonist-independent AT1AR internalization was also observed after beta-arrestin2 overexpression. After internalization, the beta2AR, but not the AT1AR, was dephosphorylated and recycled back to the cell surface. However, the AT1AR tail prevented beta2AR dephosphorylation and recycling. In contrast, although the beta2AR-tail promoted AT1AR recycling, the chimeric receptor remained both phosphorylated and desensitized, suggesting that receptor dephosphorylation is not a property common to all receptors. In summary, we show that the carboxyl-terminal tails of GPCRs not only contribute to regulating the patterns of receptor desensitization, but also modulate receptor intracellular trafficking and resensitization patterns.     

Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and beta-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with beta-arrestin 1 (betaarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas beta-arrestin 2 (betaarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by betaarr1 compared with betaarr2. In contrast, both beta-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated betaarr1 in an agonist-dependent manner, whereas betaarr2 association was virtually undetectable. Remarkably, betaarr1 also interacted with phosphorylation defective PAR1 mutant, whereas betaarr2 did not. Moreover, constitutively active beta-arrestin mutants, betaarr1 R169E and betaarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, beta-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine(2A) receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, beta-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.     

Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis*

beta-Arrestins bind agonist-activated G protein-coupled receptors (GPCRs) and mediate their desensitization and internalization. Although beta-arrestins dissociate from some receptors at the plasma membrane, such as the beta2 adrenergic receptor, they remain associated with other GPCRs and internalize with them into endocytic vesicles. Formation of stable receptor-beta-arrestin complexes that persist inside the cell impedes receptor resensitization, and the aberrant formation of these complexes may play a role in GPCR-based diseases (Barak, L. S., Oakley, R. H., Laporte, S. A., and Caron, M. G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 93-98). Here, we investigate the molecular determinants responsible for sustained receptor/beta-arrestin interactions. We show in real time and in live human embryonic kidney (HEK-293) cells that a beta-arrestin-2-green fluorescent protein conjugate internalizes into endocytic vesicles with agonist-activated neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A receptor, and substance P receptor. Using receptor mutagenesis, we demonstrate that the ability of beta-arrestin to remain associated with these receptors is mediated by specific clusters of serine and threonine residues located in the receptor carboxyl-terminal tail. These clusters are remarkably conserved in their position within the carboxyl-terminal domain and serve as primary sites of agonist-dependent receptor phosphorylation. In addition, we identify a beta-arrestin mutant with enhanced affinity for the agonist-activated beta2-adrenergic receptor that traffics into endocytic vesicles with receptors that lack serine/threonine clusters and normally dissociate from wild-type beta-arrestin at the plasma membrane. By identifying receptor and beta-arrestin residues critical for the formation of stable receptor-beta-arrestin complexes, these studies provide novel targets for regulating GPCR responsiveness and treating diseases resulting from abnormal GPCR/beta-arrestin interactions.     

Low affinity of beta1-adrenergic receptor for beta-arrestins explains the resistance to agonist-induced internalization

It has been reported that beta-arrestin is essential for the internalization of many G protein-coupled receptors. Since beta1-adrenergic receptor (beta1AR) shows the resistance to agonist-induced internalization, we examine the interaction of beta-arrestin with beta1AR with three different approaches: translocation of beta-arrestin to the plasma membrane, direct binding of in vitro translated beta-arrestin to intracellular domains of beta1- and beta2ARs, inhibition of beta1- and beta2AR-stimulated adenylyl cyclase activities by beta-arrestin. The enhanced green fluorescent protein (EGFP)-tagged beta-arrestin 2 (beta-arrestin 2-GFP) translocates to and stays at the plasma membrane by beta2AR stimulation. Beta-arrestin 2-GFP also translocates to the plasma membrane upon beta1AR stimulation. However, it returns to the cytoplasm 10 - 30 min after agonist stimulation. The amount of beta-arrestin bound to the third intracellular loop and the carboxyl tail of beta1AR is lower than that of beta2AR. The fusion protein of beta-arrestin 1 with glutathione-S-transferase inhibits the beta1- and beta2AR-stimulated adenylyl cyclase activities. However, inhibition of the beta1AR-stimulated activity requires a higher amount of the fusion protein than that of the beta2AR-stimulated activity. These results suggest that affinity of beta1AR for beta-arrestins is lower than that of beta2AR, and explains the resistance to agonist-induced internalization. This conclusion is further supported by the finding that beta-arrestin can induce internalization of beta1AR when beta-arrestin 1 fused to the carboxyl tail of beta1AR.     

Heterologous inhibition of G protein-coupled receptor endocytosis mediated by receptor-specific trafficking of beta-arrestins

We have observed an unexpected type of nonreciprocal "cross-regulation" of the agonist-induced endocytosis of G protein-coupled receptors by clathrin-coated pits. Isoproterenol-dependent internalization of beta2-adrenergic receptors in stably transfected HEK293 cells was specifically blocked (>65% inhibition) by vasopressin-induced activation of V2 vasopressin receptors co-expressed at similar levels. In contrast, activation of beta2 receptors caused no detectable effect on V2 receptor internalization in the same cells. Several pieces of evidence suggest that this nonreciprocal inhibition of endocytosis is mediated by receptor-specific intracellular trafficking of beta-arrestins. First, previous studies showed that the activation of V2 but not beta2 receptors caused pronounced recruitment of beta-arrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257). Second, overexpression of arrestin 2 or 3 (beta-arrestin 1 or 2) abolished the V2 receptor-mediated inhibition of beta2 receptor internalization. Third, mutations of the V2 receptor that block endomembrane recruitment of beta-arrestins eliminated the V2 receptor-dependent blockade of beta2 receptor internalization. These results identify a novel type of heterologous regulation of G protein-coupled receptors, define a new functional role of receptor-specific intracellular trafficking of beta-arrestins, and suggest an experimental method to rapidly modulate the functional activity of beta-arrestins in intact cells.     

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.     

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.     

Distinct roles for Src tyrosine kinase in beta2-adrenergic receptor signaling to MAPK and in receptor internalization

G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the G(s)-coupled beta2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in beta2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the beta2-adrenergic receptor could activate the MAPK pathway. However, the internalization of beta2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in beta-arrestin 2 the internalization of the beta2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the beta2-adrenergic receptor was normal. Our data demonstrate that although Src and beta-arrestin 2 play essential roles in beta2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the beta2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for beta2-adrenergic receptor signaling to the MAPK pathway in MEF cells.     

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization

Beta2-ARs (beta2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta2-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the beta-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta2-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu215-His220, which showed little or no PDE4D5 binding, but was still recruited to the beta2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta2-AR regulation.     

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.     

Phosphorylation of the delta-opioid receptor regulates its beta-arrestins selectivity and subsequent receptor internalization and adenylyl cyclase desensitization

In the current study, we investigated the role of receptor phosphorylation and beta-arrestins in delta-opioid receptor (DOR) signaling and trafficking by using a DOR mutant in which all Ser/Thr residues in the C terminus were mutated to Ala (DTS). We demonstrated that the DOR agonist D-[Pen(2),Pen(5)]enkephalin could induce receptor internalization and adenylyl cyclase (AC) desensitization of DTS, but with comparatively slower kinetics than those observed with wild type DOR. Blockade of the internalization of DTS by the dominant-negative mutant dynamin, dynamin K44E, did not affect AC desensitization. However, depletion of beta-arrestins almost totally blocked both internalization and AC desensitization of DTS. A BRET assay suggested that DOR phosphorylation promotes receptor selectivity for beta-arrestin 2 over beta-arrestin 1. Furthermore, in mouse embryonic fibroblast (MEF) cells lacking either beta-arrestin 1 (beta arr1(-/-)) or beta-arrestin 2 (beta arr2(-/-)), agonist-induced DTS desensitization and internalization were similar to that observed in wild type MEFs. In contrast, although DOR internalization decreased in both beta arr1(-/-) MEFs and beta arr2(-/-) MEFs, DPDPE-induced DOR desensitization was significantly reduced in beta arr2(-/-) MEFs, but not in beta arr1(-/-) MEFs. Additionally, the BRET assay suggested that depletion of phosphorylation did not influence the stability of the receptor-beta-arrestin complex. Consistent with this observation, DTS did not recycle after internalization, which is like wild type DOR. Taken together, these results indicate that receptor phosphorylation confers DOR selectivity for beta-arrestin 2 without affecting the stability of the receptor-beta-arrestin complex and the fate of the internalized receptor.     

Exposure to TARC alters beta2-adrenergic receptor signaling in human peripheral blood T lymphocytes

The beta(2)-adrenergic receptor (beta(2)-AR) negatively regulates T cell activity through the activation of the G(s)/adenylyl cyclase/cAMP pathway. beta(2)-AR desensitization, which can be induced by its phosphorylation, may have important consequences for the regulation of T cell function in asthma. In the present study we demonstrate that the C-C chemokine thymus and activation-regulated chemokine (TARC) impairs the ability of beta(2)-agonist fenoterol to activate the cAMP downstream effector cAMP-responsive element binding protein (CREB) in freshly isolated human T cells. The TARC-induced activation of Src kinases resulted in membrane translocation of both G protein-coupled receptor kinase (GRK) 2 and beta-arrestin. Moreover, TARC was able to induce Src-dependent serine phosphorylation of the beta(2)-AR as well as its association with GRK2 and beta-arrestin. Finally, in contrast to CREB, phosphorylation of Src and extracellular signal-regulated kinase was enhanced by fenoterol upon TARC pretreatment. In summary, we show for the first time that TARC exposure impairs beta(2)-AR function in T cells. Our data suggest that this is mediated by Src-dependent activation of GRK2, resulting in receptor phosphorylation, binding to beta-arrestin, and a switch from cAMP-dependent signaling to activation of the MAPK pathway. We propose that aberrant T cell control in the presence of endogenous beta-agonists promotes T cell-mediated inflammation in asthma.