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.     

Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization

Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of beta-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with beta-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, beta-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for beta-arrestin, B2R remains associated with beta-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a beta-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/beta-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of beta-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.     

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.     

Cellular trafficking of G protein-coupled receptor/beta-arrestin endocytic complexes

beta-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta-arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta-arrestin2 in response to activation of several distinct GPCRs including the beta2-adrenergic receptor (beta2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta2AR activation, beta-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta-arrestins are confined to the periphery of cells and do not traffic along with activated beta2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta-arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta2AR carboxyl-terminal tail results in a beta-arrestin membrane localization pattern similar to that observed in response to beta2AR activation. In contrast, the corresponding chimeric beta2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/betaarrestin complexes and cellular distribution of beta-arrestins.     

Internalization determinants of the parathyroid hormone receptor differentially regulate beta-arrestin/receptor association.

beta-Arrestins have been implicated in regulating internalization of the parathyroid hormone receptor (PTHR), but the structural features in the receptor required for this effect are unknown. In the present study performed in HEK-293 cells, we demonstrated that different topological domains of PTHR are implicated in agonist-dependent receptor internalization; truncation of the cytoplasmic tail (PTHR-TR), selective mutations of the cytoplasmic tail to remove the sites of parathyroid hormone (PTH)-stimulated phosphorylation (PTHR-PD), and mutations in the third transmembrane helix (N289A) or in the third cytoplasmic loop (K382A) resulted in a 30-60% reduction in (125)I-PTH-related protein internalization. To better define the role of these internalization determinants, we have tested the ability of these mutant PTHRs to associate with beta-arrestins by using three different methodological approaches: 1) ability of overexpression of beta-arrestins to restore the internalization of (125)I-PTH-related protein for the mutant PTHRs; 2) visualization of PTH-mediated trafficking of beta-arrestin1 and -2 fused to the green fluorescent protein with receptors by confocal microscopy; 3) quantification of beta-arrestin1-green fluorescent protein translocation by Western blot. Our data reveal that the receptor' cytoplasmic tail contains determinants of beta-arrestin interaction that are distinct from the phosphorylation sites and are sufficient for transient association of beta-arrestin2, but stable association requires receptor phosphorylation. Determinants in the receptor's core (Asn-289 and Lys-382) appear to regulate internalization of the receptor/beta-arrestin complex toward early endocytic endosomes during the initial step of endocytosis.     

Agonist-specific regulation of delta-opioid receptor trafficking by G protein-coupled receptor kinase and beta-arrestin

Opioid receptors mediate multiple biological functions through their interaction with endogenous opioid peptides as well as opioid alkaloids including morphine and etorphine. Previously we have reported that the ability of distinct opioid agonists to differentially regulate mu-opioid receptor (mu OR) responsiveness is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the receptor (1). In the present study, we further examined the role of GRK and beta-arrestin in agonist-specific regulation of the delta-opioid receptor (delta OR). While both etorphine and morphine effectively activate the delta OR, only etorphine triggers robust delta OR phosphorylation followed by plasma membrane translocation of beta-arrestin and receptor internalization. In contrast, morphine is unable to either elicit delta OR phosphorylation or stimulate beta-arrestin translocation, correlating with its inability to cause delta OR internalization. Unlike for the mu OR, overexpression of GRK2 results in neither the enhancement of delta OR sequestration nor the rescue of delta OR-mediated beta-arrestin translocation. Therefore, our findings not only point to the existence of marked differences in the ability of different opioid agonists to promote delta OR phosphorylation by GRK and binding to beta-arrestin, but also demonstrate differences in the regulation of two opioid receptor subtypes. These observations may have important implications for our understanding of the distinct ability of various opioids in inducing opioid tolerance and addiction.     

Agonist-specific regulation of parathyroid hormone (PTH) receptor type 2 activity: structural and functional analysis of PTH- and tuberoinfundibular peptide (TIP) 39-stimulated desensitization and internalization

The human PTH receptor type 2 (PTH2R) is activated by PTH and tuberoinfundibular peptide of 39 residues (TIP39), resulting in cAMP and intracellular Ca signaling. We now report that, despite these similarities, PTH and TIP39 elicit distinct responses from PTH2R. First, TIP39 induced beta-arrestin and protein kinase Cbeta mobilization and receptor internalization, whereas PTH did not. However, PTH stimulated trafficking of these molecules for a chimeric PTH2R containing the N terminus and third extracellular loop of PTH receptor type 1 (PTH1R). Second, whereas PTH-stimulated cAMP activity was brief and rapidly resensitized, the response to TIP39 was sustained and partly desensitized for a prolonged period. PTH2R desensitization was mediated by beta-arrestin interaction with the C terminus (amino acids 426-457) of PTH2R, whereas beta-arrestin mobilization had a minor influence on PTH2R internalization in response to TIP39, as shown with C terminus deletion mutants and/or dominant negative forms of beta-arrestin and dynamin. These data contrast with PTH1R, at which these dominant negative mutants markedly inhibited receptor internalization. Collectively, these results further highlight how specific interactions within the ligand-receptor bimolecular complex mediate distinct postactivation responses of class II G protein- coupled receptors and provide novel insights into the physiological regulation of PTH2R activity.     

Distinct regulation of internalization and mitogen-activated protein kinase activation by two isoforms of the dopamine D2 receptor

Two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of a 29-amino acid specific to D2L within the putative third intracellular loop of the receptor. Here, we examined D2 receptor-mediated MAPK activation in association with receptor internalization. Overexpression of beta-arrestin 1 and 2 increased the D2S-mediated activation of MAPK, whereas it did not affect the activation of MAPK by D2L. Expression of a dominant negative beta-arrestin 2 (319-418) mutant and of a dominant negative dynamin I (K44A) mutant inhibited the activation of MAPK by D2S, but not the activation of MAPK by D2L. Treatment with inhibitors of internalization, i.e. concanavalin A and monodansylcadaverin, blocked D2S-mediated MAPK activation but not D2L-mediated activation. By confocal microscopy, we observed beta-arrestin 1 and 2, translocated to the plasma membrane and colocalized with D2L and D2S receptors upon stimulation with dopamine, and this was followed by the translocation of receptors into endocytic vesicles. Moreover, the expression of the beta-arrestin 2 (319-418) mutant blocked the internalization of both D2L and D2S. In addition, although K44A dynamin mutant expression did not alter D2L internalization, it completely blocked the internalization of D2S. The stimulation of D2L induces activation of MAPK via transactivation of the platelet-derived growth factor receptor, whereas D2S does not. Taken together, these data suggest that D2L activates MAPK signaling by mobilizing the growth factor receptor, platelet-derived growth factor receptor, whereas D2S appears to activate MAPK signaling by mobilizing clathrin-mediated endocytosis in a beta-arrestin/dynamin-dependent manner.     

beta-Arrestin-dependent constitutive internalization of the human chemokine decoy receptor D6

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor beta-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with beta-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from beta-arrestin activate any G-protein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.     

Mu-opioid receptor desensitization: role of receptor phosphorylation,internalization, and representation

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized 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.     

Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration,activation, and regulation

IL-8 (or CXCL8) activates the receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB) to induce chemotaxis in leukocytes, but only CXCR1 mediates cytotoxic and cross-regulatory signals. This may be due to the rapid internalization of CXCR2. To investigate the roles of the intracellular domains in receptor regulation, wild-type, chimeric, phosphorylation-deficient, and cytoplasmic tail (C-tail) deletion mutants of both receptors were expressed in RBL-2H3 cells and studied for cellular activation, receptor phosphorylation, desensitization, and internalization. All but one chimeric receptor bound IL-8 and mediated signal transduction, chemotaxis, and exocytosis. Upon IL-8 activation, the chimeric receptors underwent receptor phosphorylation and desensitization. One was resistant to internalization, yet it mediated normal levels of beta-arrestin 2 (beta arr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with beta arr-2 and the adaptor protein-2 beta. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca(2+) mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis.     

Comparative studies of molecular mechanisms of dopamine D2 and D3 receptors for the activation of extracellular signal-regulated kinase

Dopamine D(2) and D(3) receptors (D(2)R/D(3)R), which have similar structural architecture as well as functional similarities, are expressed in the same brain dopaminergic neurons. It is intriguing that two receptor proteins with virtually the same functional roles are expressed in the same neuron. Recently we have shown that D(2)R and D(3)R possess different regulatory processes including intracellular trafficking properties, which implies that they might employ different signaling mechanisms for regulation of the same cellular processes. Here we studied the signaling pathways of ERK activation mediated by D(2)R and D(3)R in HEK-293 cells and corroborated them with concomitant studies in COS-7 cells and C6 cells. Our results show that Src, phosphatidylinositol 3-kinase, and atypical protein kinase C were commonly involved in D(2)R-/D(3)R-mediated ERK activation. However, beta-arrestin and sequestration of D(2)R/D(3)R were found not to be involved. ERK activations mediated by D(3)R, but not D(2)R, were blocked by betaARK-CT, AG1478 epidermal growth factor receptor (EGFR) inhibitor, and by dominant negative mutants of Ras and Raf, suggesting the involvement of the Gbetagamma(i) pathway. The alpha-subunit of G(o) (Galpha(o)) was able to couple with D(3)R to mediate ERK activation. We conclude that D(3)R mainly utilizes the betagamma pathway of G(i) protein, which involves the transactivation of EGFR in HEK-293 cells. In contrast, the alpha-subunit of the G(i) protein plays a main role in D(2)R-mediated ERK activation. Our study suggests one example of intricate cellular regulations in the brain, that is, dopaminergic neurons could regulate ERK activity more flexibly through alternative usage of either the D(2)R or D(3)R pathway depending on the cellular situation.     

Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration

The vasoactive intestinal polypeptide type-1 (VPAC(1)) receptor is a class II G protein-coupled receptor, distinct from the adrenergic receptor superfamily. The mechanisms involved in the regulation of the VPAC(1) receptor are largely unknown. We examined agonist-dependent VPAC(1) receptor signaling, phosphorylation, desensitization, and sequestration in human embryonic kidney 293 cells. Agonist stimulation of cells overexpressing this receptor led to a dose-dependent increase in cAMP that peaked within 5-10 min and was completely desensitized after 20 min. Cells cotransfected with the VPAC(1) receptor (VPAC(1)R) and G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6 exhibited enhanced desensitization that was not evident with GRK 4. Immunoprecipitation of the epitope-tagged VPAC(1) receptor revealed dose-dependent phosphorylation that was increased with cotransfection of any GRK. Agonist-stimulated internalization of the VPAC(1)R peaked in 10 min, and neither overexpressed beta-arrestin nor its dominant-negative mutant altered internalization. However, a dynamin-dominant negative mutant did inhibit VPAC(1) receptor internalization. Interestingly, VPAC(1)R specificity in desensitization was not evident by study of the overexpressed receptor; however, we determined that human embryonic kidney 293 cells express an endogenous VPAC(1)R that did demonstrate dose-dependent GRK specificity. Therefore, VPAC(1) receptor regulation involves agonist-stimulated, GRK-mediated phosphorylation, beta-arrestin translocation, and dynamin-dependent receptor internalization. Moreover, study of endogenously expressed receptors may provide information not evident in overexpressed systems.     

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.     

Dual role of the beta2-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta2-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta2-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta2-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.     

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.     

Differential beta-arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, beta-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII-bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of beta-arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.     

Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors

Leukotriene B4 (LTB4) activates the G-protein-coupled receptor leukotriene B4 receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB4 with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced G-protein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of beta-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of beta-arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged beta-arrestin confirmed phosphorylation-independent beta-arrestin translocation and internalization of BLT1. In beta-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of beta-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and beta-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate beta-arrestin association and internalization of BLT1.     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.