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At least 19 recordsLinked to original sources
PubMed2003

Arrestin migrates in photoreceptors in response to light: a study of arrestin localization using an arrestin-GFP fusion protein in transgenic frogs.

Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45-75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25-2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results.

Adaptation, Ocular↗
PubMed1999

Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutively active arrestin.

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the synthetic phosphopeptide comprising the fully phosphorylated carboxyl-terminal 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhydryls was enhanced while that of another was greatly reduced. Since this synthetic peptide was shown to activate arrestin with respect to its binding to unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhydryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rapidly with DTNB, but not as rapidly as with SDS-denatured arrestin. The arrestin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in ROS disk membranes. The arrestin mutant R175Q also inhibited the light-activated PDE activity with an IC50 of 1.3 microM under the experimental conditions that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that binds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain several different conformations, all of which are able to attain the activated functional state of arrestin.

Animals↗
PubMed2003

Differential regulation of beta-arrestin 1 and beta-arrestin 2 gene expression in rat brain by morphine.

Beta-arrestins are a family of regulatory and scaffold proteins functioning in signal transduction of G protein-coupled receptors including opioid receptors. Upon agonist stimulation, beta-arrestins bind to opioid receptors phosphorylated by G protein-coupled receptor kinases and promote receptor internalization and desensitization. Studies indicated that beta-arrestins are required in the development of morphine tolerance in mice. In the current study, we investigated the potential regulatory effects of morphine administration on beta-arrestin 1 and beta-arrestin 2 mRNA levels in different brain regions in rat using in situ hybridization method. Our results showed that the acute morphine administration (10 mg/kg) resulted in approximately 30% reduction in both beta-arrestin 1 and beta-arrestin 2 mRNA levels in hippocampus while the chronic morphine treatment (10 mg/kg, b.i.d., for 9 days) caused no significant change in level of either beta-arrestin mRNA. In locus coeruleus, both acute and chronic morphine treatments resulted in significant decreases (over 50%) in beta-arrestin 1 mRNA level but failed to induce any change in the level of beta-arrestin 2 gene expression. The acute morphine administration had no significant effect on beta-arrestin 1 or beta-arrestin 2 mRNA level in periaqueductal gray and cerebral cortex. However, after chronic morphine treatment, beta-arrestin 2 mRNA level decreased by 40% in periaqueductal gray and increased by 25% in cerebral cortex, in strong contrast to the unchanged beta-arrestin 1 mRNA level in these two brain regions. Furthermore, spontaneous or naloxone-precipitated withdrawal of morphine that did not affect the level of beta-arrestin 1 mRNA resulted in an aberrant increase (100% over control) in beta-arrestin 2 mRNA level in hippocampus. Our results thus demonstrated for the first time that opiate administration regulates level of beta-arrestin mRNAs in brain and the expression of beta-arrestin 1 and beta-arrestin 2 subtypes is differentially regulated in locus coeruleus, periaqueductal gray, and cerebral cortex by morphine. These data suggest that beta-arrestin 1 and beta-arrestin 2 may play different roles in the development of opioid tolerance and dependence.

Animals↗
PubMed2002

Ascidian arrestin (Ci-arr), the origin of the visual and nonvisual arrestins of vertebrate.

Arrestin is one of the key proteins for the termination of G protein signaling. Activated G protein-coupled receptors (GPCRs) are specifically phosphorylated by G protein-coupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Vertebrates possess two types of arrestin; visual arrestin expressed exclusively in photoreceptor cells in retinae and pineal organs, and beta-arrestin, which is expressed ubiquitously. Unlike visual arrestin, beta-arrestin contains the clathrin-binding domain at the C-terminus, responsible for the agonist-induced internalization of GPCRs. Here, we isolated a novel arrestin gene (Ci-arr) from the primitive chordate, the ascidian Ciona intestinalis larvae. The deduced amino acid sequence suggests that Ci-Arr be closely related to vertebrate arrestins. Interestingly, this arrestin has the feature of both visual and beta-arrestin. Whereas the expression of Ci-arr was restricted to the photoreceptors in the larvae similarly to visual arrestin, the gene product, containing the clathrin-binding domain, promoted the GPCR internalization in HEK293tsA201 cells similarly to beta-arrestin. The phylogenetic tree shows that Ci-Arr is branched from a common root of visual and beta-arrestins. Southern analysis suggests that the Ciona genome contains only one gene for the arrestin family. These results suggest that the visual and beta-arrestin genes were generated by the duplication of the prototypical arrestin gene like Ci-arr in the early evolution of vertebrates.

Adrenergic beta-Agonists↗
PubMed2003

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.

Animals↗
PubMed1994

Visual arrestin binding to rhodopsin. Intramolecular interaction between the basic N terminus and acidic C terminus of arrestin may regulate binding selectivity.

Visual arrestin plays an important role in quenching phototransduction via its ability to preferentially bind to phosphorylated light-activated rhodopsin (P-Rh*). Recently we proposed a mechanism for the binding of visual arrestin to P-Rh* that helps to explain the nature of the conformational changes in arrestin observed upon binding. This mechanism involves a multisite interaction between arrestin and P-Rh* and implies an interaction between the C-terminal and N-terminal domains of arrestin. To obtain further insight into the mechanism of arrestin-rhodopsin interaction we have characterized the ability of polyanions to inhibit the interaction of wild type and mutant arrestins to different functional forms of rhodopsin. These studies reveal that: 1) heparin is most potent at inhibiting arrestin binding to dark phosphorylated rhodopsin > light-activated rhodopsin > P-Rh*; 2) C-terminal deletions in arrestin increase arrestin sensitivity to heparin inhibition while an N-terminal deletion (residues 2-16) decreases heparin inhibition; 3) the sensitivity of chimeric arrestins to heparin inhibition is determined by the origin of the N terminus of the chimera; and 4) heparin also inhibits arrestin binding to truncated 329G-Rh*, suggesting it does not mimic the phosphorylated C terminus of rhodopsin. Taken together, these data suggest that heparin mimics the regulatory acidic C terminus of arrestin. Since the basic N-terminal region of arrestin appears to serve as a site of heparin binding it is a likely candidate to be involved in the intramolecular interaction with the C-terminal region. The interaction of the N- and C-terminal domains of arrestin may control the conformational rearrangements in arrestin that occur upon binding to P-Rh*.

Animals↗
PubMed1995

Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors.

Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

Animals↗
PubMed1997

Modulation of the arrestin-clathrin interaction in cells. Characterization of beta-arrestin dominant-negative mutants.

We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote beta2-adrenergic receptor (beta2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) to function as a dominant-negative with respect to beta2AR internalization and compared its properties with those of beta-arrestin and beta-arrestin-V53D, a previously reported dominant-negative mutant. In vitro studies demonstrated that beta-arrestin-V53D bound better to clathrin than beta-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas beta-arrestin (319-418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the beta2AR in HEK293 cells, beta-arrestin (319-418) effectively inhibited agonist-promoted receptor internalization, whereas beta-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of beta2AR internalization by beta-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both beta-arrestin (319-418) and beta-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of beta-arrestin (319-418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking.

Animals↗
PubMed1992

Receptor-specific desensitization with purified proteins. Kinase dependence and receptor specificity of beta-arrestin and arrestin in the beta 2-adrenergic receptor and rhodopsin systems.

Homologous desensitization of beta-adrenergic receptors, as well as adaptation of rhodopsin, are thought to be triggered by specific phosphorylation of the receptor proteins. However, phosphorylation alone seems insufficient to inhibit receptor function, and it has been proposed that the inhibition is mediated, following receptor phosphorylation, by the additional proteins beta-arrestin in the case of beta-adrenergic receptors and arrestin in the case of rhodopsin. In order to test this hypothesis with isolated proteins, beta-arrestin and arrestin were produced by transient overexpression of their cDNAs in COS7 cells and purified to apparent homogeneity. Their functional effects were assessed in reconstituted receptor/G protein systems using either beta 2-adrenergic receptors with Gs or rhodopsin with Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated by their specific kinases beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry of about 1:1. More than 100-fold higher concentrations of arrestin were required to inhibit beta 2-receptor function. Conversely, arrestin caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated rhodopsin when present in concentrations about equal to those of rhodopsin, whereas beta-arrestin at 100-fold higher concentrations had little inhibitory effect. The potency of beta-arrestin in inhibiting beta 2-receptor function was increased over 10-fold following phosphorylation of the receptors by beta ARK, but was not affected by receptor phosphorylation using protein kinase A. This suggests that beta-arrestin plays a role in beta ARK-mediated homologous, but not in protein kinase A-mediated heterologous desensitization of beta-adrenergic receptors. It is concluded that even though arrestin and beta-arrestin are similar proteins, they display marked specificity for their respective receptors and that phosphorylation of the receptors by the receptor-specific kinases serves to permit the inhibitory effects of the "arresting" proteins by allowing them to bind to the receptors and thereby inhibit their signaling properties. Furthermore, it is shown that this mechanism of receptor inhibition can be reproduced with isolated purified proteins.

Adrenergic beta-Antagonists↗
PubMed2000

Interactions of metarhodopsin II. Arrestin peptides compete with arrestin and transducin.

Arrestin blocks the interaction of rhodopsin with the G protein transducin (G(t)). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G(t) and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10-30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11-30 and 51-70 (IC(50) < 100 microm) and residues 231-260 (IC(50) < 200 microm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G(t) or the farnesylated G(t)gamma C-terminal peptide. Only arrestin-(11-30) inhibited MII.G(t) less (IC(50) = 300 microm) than phosphorylated MII.arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and G(t). The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure.

Amino Acid Sequence↗
PubMed2002

beta-Arrestin/AP-2 interaction in G protein-coupled receptor internalization: identification of a beta-arrestin binging site in beta 2-adaptin.

beta-Arrestins, proteins involved in the turn-off of G protein-coupled receptor (GPCR) activation, bind to the beta(2)-adaptin subunit of the clathrin adaptor AP-2. The interaction of beta(2)-adaptin with beta-arrestin involves critical arginine residues in the C-terminal domain of beta-arrestin and plays an important role in initiating clathrin-mediated endocytosis of the beta(2)-adrenergic receptor (beta(2)AR) (Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S., and Caron, M. G. (2000) J. Biol. Chem. 275, 23120--23126). However, the beta-arrestin-binding site in beta(2)-adaptin has not been identified, and little is known about the role of beta-arrestin/AP-2 interaction in the endocytosis of other GPCRs. Using in vitro binding assays, we have identified two glutamate residues (Glu-849 and Glu-902) in beta(2)-adaptin that are important in beta-arrestin binding. These residues are located in the platform subdomain of the C terminus of beta(2)-adaptin, where accessory/adapter endocytic proteins for other classes of receptors interact, distinct from the main site where clathrin interacts. The functional significance of the beta-arrestin/AP-2/clathrin complex in the endocytosis of GPCRs such as the beta(2)AR and vasopressin type II receptor was evaluated using mutant constructs of the beta(2)-adaptin C terminus containing either the clathrin and the beta-arrestin binding domains or the beta-arrestin-binding domain alone. When expressed in human embryonic kidney 293 cells, both constructs acted as dominant negatives inhibiting the agonist-induced internalization of the beta(2)AR and the vasopressin type II receptor. In addition, although the beta(2)-adaptin construct containing both the clathrin and beta-arrestin binding domains was able to block the endocytosis of transferrin receptors, a beta(2)-adaptin construct capable of associating with beta-arrestin but lacking its high affinity clathrin interaction did not interfere with transferrin receptor endocytosis. These results suggest that the interaction of beta-arrestin with beta(2)-adaptin represents a selective endocytic trigger for several members of the GPCR family.

Adaptor Protein Complex beta Subunits↗
PubMed2000

An additional phosphate-binding element in arrestin molecule. Implications for the mechanism of arrestin activation.

Arrestins quench the signaling of a wide variety of G protein-coupled receptors by virtue of high-affinity binding to phosphorylated activated receptors. The high selectivity of arrestins for this particular functional form of receptor ensures their timely binding and dissociation. In a continuing effort to elucidate the molecular mechanisms responsible for arrestin's selectivity, we used the visual arrestin model to probe the functions of its N-terminal beta-strand I comprising the highly conserved hydrophobic element Val-Ile-Phe (residues 11-13) and the adjacent positively charged Lys(14) and Lys(15). Charge elimination and reversal in positions 14 and 15 dramatically reduce arrestin binding to phosphorylated light-activated rhodopsin (P-Rh*). The same mutations in the context of various constitutively active arrestin mutants (which bind to P-Rh*, dark phosphorylated rhodopsin (P-Rh), and unphosphorylated light-activated rhodopsin (Rh*)) have minimum impact on P-Rh* and Rh* binding and virtually eliminate P-Rh binding. These results suggest that the two lysines "guide" receptor-attached phosphates toward the phosphorylation-sensitive trigger Arg(175) and participate in phosphate binding in the active state of arrestin. The elimination of the hydrophobic side chains of residues 11-13 (triple mutation V11A, I12A, and F13A) moderately enhances arrestin binding to P-Rh and Rh*. The effects of triple mutation V11A, I12A, and F13A in the context of phosphorylation-independent mutants suggest that residues 11-13 play a dual role. They stabilize arrestin's basal conformation via interaction with hydrophobic elements in arrestin's C-tail and alpha-helix I as well as its active state by interactions with alternative partners. In the context of the recently solved crystal structure of arrestin's basal state, these findings allow us to propose a model of initial phosphate-driven structural rearrangements in arrestin that ultimately result in its transition into the active receptor-binding state.

Amino Acid Sequence↗
PubMed2000

beta-arrestin differentially regulates the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this implicates multiple interaction sites between beta-arrestin and CXCR4.

The chemokine receptor CXCR4 has recently been shown to be a co-receptor involved in the entry of human immunodeficiency virus type 1 into target cells. This study shows that coexpression of beta-arrestin with CXCR4 in human embryonic kidney 293 cells attenuated chemokine-stimulated G protein activation and inhibition of cAMP production. Truncation of the C-terminal 34 amino acids of CXCR4 (CXCR4-T) abolished the effects of beta-arrestin on CXCR4/G protein signaling, indicating the functional interaction of the receptor C terminus with beta-arrestin. On the other hand, receptor internalization and the subsequent activation of extracellular signal-regulated kinases were significantly promoted by coexpression of beta-arrestin with CXCR4, whereas the C-terminal truncation of CXCR4 did not affect this regulation of beta-arrestin, suggesting that beta-arrestin can functionally interact with CXCR4 with or without the C terminus. Moreover, beta(2)V54D, the dominant inhibitory mutant of beta-arrestin 2, exerted no effects on CXCR4/G protein signaling, but strongly influenced receptor internalization and extracellular signal-regulated kinase activation. Further cross-linking experiments demonstrated that beta-arrestin as well as beta(2)V54D could physically contact both CXCR4 and CXCR4-T. Glutathione S-transferase pull-down assay showed that beta-arrestin was able to bind efficiently in vitro to both the third intracellular loop and the 34-amino acid C terminus of CXCR4. Taken together, our data clearly establish that beta-arrestin can effectively regulate different functions of CXCR4 and that this is mediated through its distinct interactions with the C terminus and other regions including the third loop of CXCR4.

Arrestins↗
PubMed1994

Chromosome mapping of the human arrestin (SAG), beta-arrestin 2 (ARRB2), and beta-adrenergic receptor kinase 2 (ADRBK2) genes.

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: beta-adrenergic receptor kinase (beta ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, beta-arrestin. Both beta ARK and beta-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, beta ARK1, beta ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/beta-arrestin gene family includes arrestin (also known as S-antigen), beta-arrestin 1, and beta-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), beta-arrestin 2 (ARRB2), and beta ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for beta-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for beta ARK2 (ADRBK2) to chromosome 22q11.

Antigens↗
PubMed1996

Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr.

Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

Amino Acid Sequence↗
PubMed2003

The interaction of a constitutively active arrestin with the arrestin-insensitive 5-HT(2A) receptor induces agonist-independent internalization.

5-HT(2A) serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT(2A) receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT(2A) receptor phosphorylation. Because the arrestin-insensitivity of 5-HT(2A) receptors is cell-type selective, we used a "constitutively active" arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this "constitutively active" arrestin mutant (Arr2-R169E) can force 5-HT(2A) receptors to be regulated by arrestins. Cotransfection of 5-HT(2A) receptors with Arr2-R169E induced agonist-independent 5-HT(2A) receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT(2A) receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT(2A) receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT(2A) receptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor.

Amino Acid Substitution↗
PubMed2004

Stable interaction between beta-arrestin 2 and angiotensin type 1A receptor is required for beta-arrestin 2-mediated activation of extracellular signal-regulated kinases 1 and 2.

Binding of beta-arrestins to seven-membrane-spanning receptors (7MSRs) not only leads to receptor desensitization and endocytosis but also elicits additional signaling processes. We recently proposed that stimulation of the angiotensin type 1A (AT(1A)) receptor results in independent beta-arrestin 2- and G protein-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation. Here we utilize two AT(1A) mutant receptors to study these independent pathways, one truncated at residue 324, thus removing all potential carboxyl-terminal phosphorylation sites, and the other bearing four mutations in the serine/threonine-rich clusters in the carboxyl terminus. As assessed by confocal microscopy, the two mutant receptors interacted with beta-arrestin 2-green fluorescent protein with much lower affinity than did the wild-type receptor. In addition, the mutant receptors more robustly stimulated G protein-mediated inositol phosphate production. Approximately one-half of the wild-type AT(1A) receptor-stimulated ERK1/2 activation was via a beta-arrestin 2-dependent pathway (suppressed by beta-arrestin 2 small interfering RNA), whereas the rest was mediated by a G protein-dependent pathway (suppressed by protein kinase C inhibitor). ERK1/2 activation by the mutant receptors was insensitive to beta-arrestin 2 small interfering RNA but was reduced more than 80% by a protein kinase C inhibitor. The biochemical consequences of ERK activation by the G protein and beta-arrestin 2-dependent pathways were also distinct. G-protein-mediated ERK activation enhanced the transcription of early growth response 1, whereas beta-arrestin 2-dependent ERK activation did not. In addition, stimulation of the truncated AT(1A) mutant receptor caused significantly greater early growth response 1 transcription than did the wild-type receptor. These findings demonstrate how the ability of receptors to interact with beta-arrestins determines both the mechanism of ERK activation as well as the physiological consequences of this activation.

Amino Acid Sequence↗
PubMed2003

Mapping the arrestin-receptor interface. Structural elements responsible for receptor specificity of arrestin proteins.

Arrestins selectively bind to phosphorylated activated forms of their cognate G protein-coupled receptors. Arrestin binding prevents further G protein activation and often redirects signaling to other pathways. The comparison of the high-resolution crystal structures of arrestin2, visual arrestin, and rhodopsin as well as earlier mutagenesis and peptide inhibition data collectively suggest that the elements on the concave sides of both arrestin domains most likely participate in receptor binding directly, thereby dictating its receptor preference. Using comparative binding of visual arrestin/arrestin2 chimeras to the preferred target of visual arrestin, light-activated phosphorylated rhodopsin (PRh*), and to the arrestin2 target, phosphorylated activated m2 muscarinic receptor (P-m2 mAChR*), we identified the elements that determine the receptor specificity of arrestins. We found that residues 49-90 (beta-strands V and VI and adjacent loops in the N-domain) and 237-268 (beta-strands XV and XVI in the C-domain) in visual arrestin and homologous regions in arrestin2 are largely responsible for their receptor preference. Only 35 amino acids (22 of which are nonconservative substitutions) in the two elements are different. Simultaneous exchange of both elements between visual arrestin and arrestin2 fully reverses their receptor specificity, demonstrating that these two elements in the two domains of arrestin are necessary and sufficient to determine their preferred receptor targets.

Animals↗