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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↗

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↗

PubMed1993

Beta-arrestin and arrestin are recognized by autoantibodies in sera from multiple sclerosis patients.

Multiple sclerosis (MS), one of the most common chronic neurologic diseases, is characterized by the presence of multiple plaques of demyelination throughout the central nervous system. Although the etiology of the disease has not been established, it is believed to involve autoimmune mechanisms. We have examined sera from patients with MS for the presence of antibodies to antigens from brain and retina. Immunoblot analysis of soluble fraction of proteins from bovine brain revealed a prominent band at 45 kDa stained with sera of 8-14 patients with MS. In two patients with MS, serum antibody titers during relapse were higher compared with those when the patients were in remission. These antibodies were undetectable in cerebrospinal fluid of our MS patients and additionally were absent in sera of patients with other neurological diseases and normal control subjects. Furthermore, immunoblot analysis of the soluble fraction from bovine retinal rod outer segments revealed a prominent protein band at 48 kDa stained with MS sera. This antigen was purified to homogeneity from bovine retinal outer segments and identified as arrestin. Additionally, sera from MS patients reacted with purified beta-arrestin 1, a 45-kDa protein homologous to arrestin that is found in various tissues. Using limited proteolysis of arrestin and a competitive ELISA test with a synthetic peptide, we identified the recognition site(s) for antibodies in sera of MS patients at a dominant immunogenic site on arrestin located at the C-terminal region of the molecule. We suggest that the presence of circulating antibodies reactive with beta-arrestin or arrestin may be related to the course of MS progression.

Amino Acid Sequence↗

PubMed1995

The arrestin superfamily: cone arrestins are a fourth family.

Arrestins constitute a superfamily of regulatory proteins that down-regulate phosphorylated G-protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone-like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.

Amino Acid Sequence↗

PubMed1995

Visual arrestin binding to rhodopsin. Diverse functional roles of positively charged residues within the phosphorylation-recognition region of arrestin.

Arrestin plays a critical role in quenching phototransduction via its ability to specifically interact with the phosphorylated light-activated form of the visual receptor rhodopsin. In an effort to identify the residues involved in interaction with the phosphorylated C terminus of rhodopsin, we introduced point mutations into a basic region in visual arrestin previously implicated in phosphorylation-recognition (residues 163-189). A total of nine point mutations were made, each substituting a neutral hydrophilic residue for a positively charged Lys, Arg, or His. The functional consequences of these mutations were then analyzed by comparing the binding of full-length and truncated wild-type and mutant arrestin to various functional forms of rhodopsin. These studies demonstrate that Arg-171, Arg-175, and Lys-176 in bovine arrestin play a primary role in phosphate interaction, while Lys-166 and Lys-167 likely play a minor role in phosphate binding. In contrast, Lys-163 and His-179 appear to play a regulatory role, while Arg-182 and Arg-189 are not directly involved in arrestin binding to rhodopsin. Arg-175 also appears to function as a phosphorylation-sensitive trigger since charge neutralization by mutagenesis enables arrestin-R175N to bind to light-activated rhodopsin as well as wild-type arrestin binds to phosphorylated light-activated rhodopsin. The implications of these findings for the sequential multisite binding of arrestin to rhodopsin are discussed.

Acetates↗

PubMed1997

Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state.

Arrestin plays an important role in quenching phototransduction via its ability to interact specifically with the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Previous studies have demonstrated that Arg175 in bovine arrestin is directly involved in the phosphorylation-dependent binding of arrestin to rhodopsin and seems to function as a phosphorylation-sensitive trigger. In this study, we further probed the molecular mechanism of phosphorylation recognition by substituting 19 different amino acids for Arg175. We also assessed the effects of mutagenesis of several other highly conserved residues within the phosphorylation-recognition region (Val170, Leu172, Leu173, Ile174, Val177, and Gln178). The binding of all of these mutants to P-Rh*, light-activated rhodopsin, and truncated rhodopsin, which lacks the carboxyl-terminal phosphorylation sites, was then characterized. Overall, our results suggest that arrestin interaction with the phosphorylated carboxyl-terminal domain of rhodopsin activates two relatively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recognition region for the rhodopsin carboxyl-terminal domain. Together, these two mechanisms ensure the exquisite selectivity of arrestin toward P-Rh*. Mutagenesis of residues that play a major role in binding site mobilization and phosphorylation-recognition enabled us to create "constitutively active" (phosphorylation-independent) arrestin mutants that have high affinity for both P-Rh* and light-activated rhodopsin. The introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylation-recognition is proposed.

Amino Acid Sequence↗

PubMed1993

X-arrestin: a new retinal arrestin mapping to the X chromosome.

We have been using a differential cDNA cloning approach to isolate human retina-specific and retina-enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina-specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine beta-arrestin, and bovine and human retinal arrestin (S-antigen), respectively. Chromosomal mapping localized this new arrestin gene to the proximal long arm of the X chromosome, hence it was named X-arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.

Amino Acid Sequence↗

PubMed1993

Binding of wild type and chimeric arrestins to the m2 muscarinic cholinergic receptor.

Arrestins play an important role in regulating the activity of the G protein-coupled receptors rhodopsin and the beta 2-adrenergic receptor. Recently, we described the expression and functional characterization of visual arrestin using an in vitro translation system. Here we report the expression of beta-arrestin and development of a direct binding assay to study the interaction of arrestins with a muscarinic cholinergic receptor. In vitro translated beta-arrestin was found to specifically bind to purified reconstituted human m2 muscarinic cholinergic receptor (hm2 mAChR) in an agonist- and phosphorylation-dependent manner. Visual arrestin also bound to the hm2 mAChR, albeit to a lesser extent and with lower affinity. In an attempt to dissect the major domains responsible for determining the receptor binding specificity of arrestin and beta-arrestin, we generated several chimeric arrestins. One contained the first 340 residues of beta-arrestin followed by residues 346-404 of arrestin (BRV4), another consisted of the first 207 residues of beta-arrestin and residues 214-404 of visual arrestin (BV3), and a third had residues 1-43 of beta-arrestin replaced by residues 1-47 of arrestin (VIN1). All of these arrestins were able to specifically bind to the activated and phosphorylated form of both the hm2 mAChR and rhodopsin, with a clear preference for the muscarinic receptor. The Kd values for beta-arrestin, BRV4, BV3, VIN1, and visual arrestin binding to the hm2 mAChR were 0.48 +/- 0.06, 0.51 +/- 0.19, 1.38 +/- 0.26, 1.13 +/- 0.26, and 7.2 +/- 1.2 nM, respectively. These data demonstrate that: 1) beta-arrestin binds to the hm2 mAChR in an activation- and phosphorylation-dependent fashion, 2) visual arrestin has 15-fold lower affinity for the hm2 mAChR as compared to beta-arrestin, and 3) the N-terminal half of beta-arrestin plays a key role in determining receptor binding specificity. The use of in vitro translated arrestins to directly assess receptor binding may serve as a viable approach for elucidating the specificity and molecular mechanisms involved in receptor-arrestin interaction.

Animals↗

PubMed1994

Cone arrestin identified by targeting expression of a functional family.

High acuity, color vision in humans is initiated in cones by a receptor/G-protein-linked phototransduction cascade. G-protein-linked receptors are rapidly deactivated by receptor phosphorylation and the binding of a member of the "arrestin" family of proteins. Divergence in amino acid sequence at the carboxyl terminus of S-antigen (rod photoreceptor arrestin) and beta-arrestin 1 and beta-arrestin 2 (beta-adrenergic receptors) suggests that receptor specificity may be coded within this region. An anchor primer strategy was utilized to screen for carboxyl-terminal variability with a rat pineal library, identifying three known arrestins plus three unknowns (C-arrestin, D-arrestin, and E-arrestin). cDNA was prepared by reverse transcription of mRNA from 12 rat tissues, and members of the arrestin family were amplified by polymerase chain reaction using the anchor primer and customized 3'-primers for the individual arrestins. The amplified arrestins were then digested by selected restriction endonucleases, producing a pattern of characteristic cleavage products for each arrestin isoform. The procedural combination of epitope domain anchor and tissue screening demonstrated that C-arrestin is enriched in the retina. C-arrestin was isolated from a lambda MAX1 human retinal cDNA library and sequenced, revealing significant identity to known arrestins and divergence within the 3'-region. The mRNA for C-arrestin was visualized by in situ hybridization, localizing in the retina with cone photoreceptors and in the pineal to a subpopulation of pinealocytes. A gene for human C-arrestin was mapped to the X chromosome, making C-arrestin a candidate for several inherited X-linked retinopathies. The localization of C-arrestin to cone photoreceptors suggests that it, like others in the arrestin family, may bind to phosphorylated receptors and participate in deactivation of the phototransduction cascade.

Amino Acid Sequence↗

PubMed1998

Characterization of dominant negative arrestins that inhibit beta2-adrenergic receptor internalization by distinct mechanisms.

Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1-320 or 201-409 resulted in the inhibition of beta2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1-320), but not arrestin-3(201-409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284-409), and arrestin-3(290-409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284-409) and arrestin-3(290-409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371-379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain.

Adrenergic beta-2 Receptor Antagonists↗

PubMed1993

Molecular analysis of human beta-arrestin-1: cloning, tissue distribution, and regulation of expression. Identification of two isoforms generated by alternative splicing.

The cDNA for human beta-arrestin-1 was cloned by polymerase chain reaction (PCR) and identified based on its remarkably high amino acid identity (98.6%) with the bovine sequence. Two alternatively spliced isoforms of human beta-arrestin-1, differing only in the presence or absence of 24 base pairs/8 amino acids within the sequence, were identified and called beta-arrestin-1A and beta-arrestin-1B, respectively. Both isoforms were found in all tissues tested. Southern blot analysis revealed the existence of a single gene for beta-arrestin-1, suggesting that the two isoforms are generated by alternative mRNA splicing. The possible presence of similar isoforms was investigated for the other members of the arrestin/beta-arrestin gene family by PCR. Two isoforms of arrestin were revealed in bovine peripheral blood leukocytes. The expression of beta-arrestin-1 was studied in several human tissues and cell types. High levels of beta-arrestin-1 mRNA and immunoreactivity were found in peripheral blood leukocytes. The possible regulation of the expression of beta-arrestin-1 was also investigated. Our work documents for the first time that the expression of beta-arrestin-1 is modulated by intracellular cAMP. Using two cell types, human endothelial cells and smooth muscle cells, we found that 6-8-h treatments with the cAMP-inducing agents cholera toxin, forskolin, iloprost, and isoproterenol raised beta-arrestin-1 mRNA by 2-4-fold. Forskolin preferentially increased beta-arrestin-1A expression in smooth muscle cells, as assessed by PCR. beta-Arrestin-1 immunoreactivity was 2-3-fold higher in smooth muscle cells exposed to forskolin for 8 h, compared with untreated controls. We conclude that (i) the finding of alternatively spliced isoforms of beta-arrestin-1 and arrestin documents a novel mechanism to generate diversity within the arrestin/beta-arrestin gene family; (ii) the abundant expression of beta-arrestin-1 in peripheral blood leukocytes further supports our previous suggestion of a major role for the beta ARK/beta-arrestin system in regulating receptor-mediated immune functions; (iii) the increased expression of beta-arrestin-1 by cAMP suggests a new mechanism for the regulation of receptor-mediated responses.

Alternative Splicing↗

PubMed1992

Cell-free expression of visual arrestin. Truncation mutagenesis identifies multiple domains involved in rhodopsin interaction.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. To further characterize rhodopsin/arrestin interactions we have utilized a rabbit reticulocyte lysate translation system to synthesize bovine visual arrestin. The translated arrestin (404 amino acids) was demonstrated to be fully functional in terms of its ability to specifically recognize and bind to phosphorylated light-activated rhodopsin (P-Rh*). Competitive binding studies revealed that the in vitro synthesized arrestin and purified bovine visual arrestin had comparable affinities for P-Rh*. In an effort to assess the functional role of different regions of the arrestin molecule, two truncated arrestin mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes. In vitro translation of the transcribed truncated mRNAs resulted in the production of arrestins truncated from the carboxyl terminus. The ability of each of the mutant arrestins to bind to dark (Rh), light-activated (Rh*), dark phosphorylated (P-Rh), and light-activated phosphorylated rhodopsin were then compared. Arrestin lacking 39 carboxyl-terminal residues binds specifically not only to P-Rh* but also to Rh* and P-Rh. This suggests that the carboxyl-terminal domain of arrestin plays an important regulatory role in ensuring strict arrestin binding selectivity to P-Rh*. Arrestin that has only the first 191 amino-terminal residues predominately discriminates the phosphorylation state of the rhodopsin; however, it also retains some binding specificity for the activation state. These results suggest that the amino-terminal half of arrestin contains key rhodopsin recognition sites responsible for interaction with both the phosphorylated and light-activated forms of rhodopsin.

Animals↗

PubMed1997

Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization.

Two of the common mechanisms regulating G protein-coupled receptor (GPCR) signal transduction are phosphorylation and sequestration (internalization). Agonist-mediated receptor phosphorylation by the beta-adrenergic receptor kinase (betaARK) facilitates subsequent interaction with an arrestin protein, resulting in receptor desensitization. Studies of the beta2-adrenergic receptor (beta2AR) receptor in human embryonic kidney (HEK) 293 cells indicate that betaARK and arrestin proteins (beta-arrestins) also regulate sequestration. Consistent with this notion, we show in HEK 293 cells that reduction in or removal of the ability of the beta2AR to be phosphorylated by betaARK or to interact normally with beta-arrestin substantially reduces agonist-mediated sequestration. To evaluate betaARK and beta-arrestin regulation of beta2AR sequestration, we examined the relationship between betaARK and/or beta-arrestin expression and beta2AR sequestration in a variety of cultured cells, including HEK 293, COS 7, CHO, A431, and CHW. COS cells had both the lowest levels of endogenous beta-arrestin expression and beta2AR sequestration, whereas HEK 293 had the highest. Overexpression of beta-arrestin, but not betaARK, in COS cells increased the extent of wild-type beta2AR sequestration to levels observed in HEK 293 cells. However, a betaARK phosphorylation-impaired beta2AR mutant (Y326A) required the simultaneous overexpression of both betaARK and beta-arrestin for this to occur. Among all cell lines, sequestration correlated best with the product of betaARK and beta-arrestin expression. Moreover, an agonist-mediated translocation of wild-type beta2AR and endogenous beta-arrestin 2 to endocytic vesicles prepared from CHO fibroblasts was observed. These data suggest not only that the complement of cellular betaARK and arrestin proteins synergistically regulate beta2AR sequestration but also that beta-arrestins directly regulate beta2AR trafficking as well as desensitization.

Animals↗

PubMed1997

Interaction of arrestins with intracellular domains of muscarinic and alpha2-adrenergic receptors.

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208-Arg387), M3-MR (Gly308-Leu497), or alpha2A/D-adrenergic receptor (AR) (Lys224-Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3 fusion protein, but not to the control GST peptide or i3 peptides derived from the alpha2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified beta-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]beta-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vitro translated [3H]beta-arrestin, [3H]visual arrestin, and [3H]beta-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of beta-arrestin with the M3-MR peptide required both the amino (Gly308-Leu368) and carboxyl portions (Lys425-Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.

Animals↗