Cone arrestin (CAR) is a novel member of the arrestin superfamily expressed in retinal cone photoreceptors and the pineal gland. To understand the regulatory mechanisms controlling its cone- and pineal-specific expression, and to facilitate further functional studies using gene knockout approaches, we characterized the genomic organization and the 5'-flanking region of the mouse CAR (mCAR) gene. The mCAR gene is comprised of 17 exons and 16 introns, encoding five alternatively spliced transcripts. A 215-bp proximal promoter fragment containing a TATA box, an Sp1 site and four cone-rod homeobox-binding sites is sufficient to direct expression in cultured retinoblastoma cells and in cone photoreceptors and the pineal gland in transgenic Xenopus laevis. (+info)
The solution structure and activation of visual arrestin studied by small-angle X-ray scattering.
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Visual arrestin is converted from a 'basal' state to an 'activated' state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 micro m. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257-269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*. (+info)
Arrestin and its splice variant Arr1-370A (p44). Mechanism and biological role of their interaction with rhodopsin.
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Deactivation of G-protein-coupled receptors relies on a timely blockade by arrestin. However, under dim light conditions, virtually all arrestin is in the rod inner segment, and the splice variant p(44) (Arr(1-370A)) is the stop protein responsible for receptor deactivation. Using size exclusion chromatography and biophysical assays for membrane-bound protein-protein interaction, membrane binding, and G-protein activation, we have investigated the interactions of Arr(1-370A) and proteolytically truncated Arr(3-367) with rhodopsin. We find that these short arrestins do not only interact with the phosphorylated active receptor but also with inactive phosphorylated rhodopsin or opsin in membranes or solution. Because of the latter interaction they are not soluble (like arrestin) but membrane-bound in the dark. Upon photoexcitation, Arr(3-367) and Arr(1-370A) interact with prephosphorylated rhodopsin faster than arrestin and start to quench G(t) activation on a subsecond time scale. The data indicate that in the course of rhodopsin deactivation, Arr(1-370A) is handed over from inactive to active phosphorylated rhodopsin. This mechanism could provide a new aspect of receptor shutoff in the single photon operating range of the rod cell. (+info)
Spontaneous induction of immunoregulation by an endogenous retinal antigen.
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PURPOSE: Ocular immune deviation studies have relied on intraocular injections of antigen to induce altered immune responses. Contributions of the injection process itself have complicated study of the mechanisms and interpretations of biological significance. In the current study, transgenic mice were used to determine the presence of immune deviation or other evidence of immunoregulation, elicited by endogenous Escherichia coli beta-galactosidase (beta-gal) expressed through a retinal arrestin promoter. METHODS: Mice that express beta-gal in the retina and various control mice were immunized with beta-gal and tested for immune responsiveness by the ear-swelling test for delayed-type hypersensitivity (DTH), in vitro proliferation assays, and cytokine assays. Spleen cells from transgenic mice were also transferred to normal recipients to test for transfer of immune deviation. RESULTS: Endogenous retinal beta-gal expression depressed the DTH response and proliferation assays after beta-gal immunization. The ability to depress DTH was transferred by naive spleen cells from transgenic mice to nontransgenic mice. Use of an immunization protocol that included high-dose Mycobacterium tuberculosis (M tb) H37Ra adjuvant and concurrent administration of pertussis toxin reversed the inhibition of DTH. CONCLUSIONS: An endogenous neo self-retinal antigen alters subsequent immune responses without intraocular injection, suggesting that normal retinal proteins in quiet eyes induce immunoregulatory responses. Based on cytokine assays, there were similarities to the immune deviation induced by intraocular inoculation in the IL-4 response, but the IL-10, IFN-gamma, and TGF-beta1 results were not similar, indicating that the mechanisms differ. The ability of supplemented adjuvant to overcome endogenous tolerance may be related to the requirement for supplemented adjuvants in the induction of experimental autoimmune uveoretinitis. (+info)
The third intracellular loop of alpha 2-adrenergic receptors determines subtype specificity of arrestin interaction.
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Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the alpha(2b)- and alpha(2c)-adrenergic receptors (ARs) while having no effect on the alpha(2a)AR, here we used alpha(2)ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the alpha(2a)AR, alpha(2b)AR, or alpha(2c)AR. These studies revealed that arrestin-3 directly binds to the alpha(2b)AR and alpha(2c)AR but not the alpha(2a)AR, whereas arrestin-2 only binds to the alpha(2b)AR. Truncation mutagenesis of the alpha(2b)AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the alpha(2b)AR third intracellular loop. Mutation of these residues in the holo-alpha(2b)AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant alpha(2b)AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the alpha(2b)AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization. (+info)
Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.
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Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms. (+info)
Ascidian arrestin (Ci-arr), the origin of the visual and nonvisual arrestins of vertebrate.
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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. (+info)
Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains. (+info)