Biomedical subjects

Sergey A Vishnivetskiy

Publications and source records attributed to Sergey A Vishnivetskiy.

10 recordsLinked to original sources

PubMed2006

Arrestin binding to calmodulin: a direct interaction between two ubiquitous signaling proteins.

Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.

Arrestins↗

PubMed2006

Visual and both non-visual arrestins in their "inactive" conformation bind JNK3 and Mdm2 and relocalize them from the nucleus to the cytoplasm.

Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a "nuclear exclusion assay" to study their interactions with two proteins involved in "life-and-death" decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants "frozen" in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely "preloaded" with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration.

Animals↗

PubMed2006

Soluble mimics of the cytoplasmic face of the human V1-vascular vasopressin receptor bind arrestin2 and calmodulin.

Signal transduction by G protein-coupled receptors (GPCRs) is mediated by interactions between intracellular proteins and exposed motifs on the cytoplasmic face of these receptors. Arrestins bind to GPCRs and modulate receptor function either by interfering with heterotrimeric G protein signaling or by serving as signaling adaptors themselves. Calmodulin interacts with GPCRs triggering a calcium response. We have studied the interaction of arrestin2 and calmodulin with intracellular elements of the human V1-vascular vasopressin receptor (hV1R). For this purpose, we designed, expressed, and purified soluble fusion proteins with the maltose-binding protein (MBP) from Escherichia coli that mimic the intracellular surface of the hV1R. These MBP fusion proteins bind arrestin2 and calmodulin with affinities in the micromolar range. A different series of soluble receptor analogs, named vasopressin receptor 1 elements on a soluble scaffold (V1ROSS) proteins, consist of the third intracellular loop and/or the C-terminal segment of the hV1R receptor juxtaposed on the surface of the MBP. V1ROSS proteins bind calmodulin and a truncated, phosphorylation-independent form of arrestin2 more tightly than the corresponding linear fusion proteins. Thus, embedding receptor loops within the three-dimensional structure of the MBP yields a better representation of the active conformation of these receptor loops than linear receptor peptides fused onto the C terminus of the MBP. V1ROSS proteins provide a valuable tool to study receptor interactions because they are more amenable to structural analysis than the native membrane receptor. These findings set the stage for the detailed structural analysis of these protein-protein interactions that are important for understanding the mechanism of signaling.

Amino Acid Sequence↗

PubMed2006

Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin.

Arrestins regulate signaling and trafficking of G protein-coupled receptors by virtue of their preferential binding to the phosphorylated active form of the receptor. To identify sites in arrestin involved in receptor interaction, a nitroxide-containing side chain was introduced at each of 28 different positions in visual arrestin, and the dynamics of the side chain was used to monitor arrestin interaction with phosphorylated forms of its cognate receptor, rhodopsin. At physiological concentrations, visual arrestin associates with both inactive dark phosphorylated rhodopsin (P-Rh) and light-activated phosphorylated rhodopsin (P-Rh*). Residues distributed over the concave surfaces of the two arrestin domains are involved in weak interactions with both states of phosphorhodopsin, and the flexible C-terminal sequence (C-tail) of arrestin becomes dynamically disordered in both complexes. A large-scale movement of the C-tail is demonstrated by direct distance measurements using a doubly labeled arrestin with one nitroxide in the C-tail and the other in the N-domain. Despite some overlap, the molecular "footprint" of arrestin bound to P-Rh and P-Rh* is different, showing the structure of the complexes to be unique. Strong immobilizing interactions with residues in a highly flexible loop between beta-strands V and VI are only observed in complex with the activated state. This result identifies this loop as a key recognition site in the arrestin-P-Rh* complex and supports the view that flexible sequences are key elements in protein-protein interactions.

Arrestin↗

PubMed2006

Visual arrestin binding to microtubules involves a distinct conformational change.

Recently we found that visual arrestin binds microtubules and that this interaction plays an important role in arrestin localization in photoreceptor cells. Here we use site-directed mutagenesis and spin labeling to explore the molecular mechanism of this novel regulatory interaction. The microtubule binding site maps to the concave sides of the two arrestin domains, overlapping with the rhodopsin binding site, which makes arrestin interactions with rhodopsin and microtubules mutually exclusive. Arrestin interaction with microtubules is enhanced by several "activating mutations" and involves multiple positive charges and hydrophobic elements. The comparable affinity of visual arrestin for microtubules and unpolymerized tubulin (K(D) > 40 mum and >65 mum, respectively) suggests that the arrestin binding site is largely localized on the individual alphabeta-dimer. The changes in the spin-spin interaction of a double-labeled arrestin indicate that the conformation of microtubule-bound arrestin differs from that of free arrestin in solution. In sharp contrast to rhodopsin, where tight binding requires an extended interdomain hinge, arrestin binding to microtubules is enhanced by deletions in this region, suggesting that in the process of microtubule binding the domains may move in the opposite direction. Thus, microtubule and rhodopsin binding induce different conformational changes in arrestin, suggesting that arrestin assumes three distinct conformations in the cell, likely with different functional properties.

Arrestin↗

PubMed2005

Crystal structure of cone arrestin at 2.3A: evolution of receptor specificity.

Arrestins play a fundamental role in the regulation and signal transduction of G protein-coupled receptors. Here we describe the crystal structure of cone arrestin at 2.3A resolution. The overall structure of cone visual arrestin is similar to the crystal structures of rod visual and the non-visual arrestin-2, consisting of two domains, each containing ten beta-sheets. However, at the tertiary structure level, there are two major differences, in particular on the concave surfaces of the two domains implicated in receptor binding and in the loop between beta-strands I and II. Functional analysis shows that cone arrestin, in sharp contrast to its rod counterpart, bound cone pigments and non-visual receptors. Conversely, non-visual arrestin-2 bound cone pigments, suggesting that it may also regulate phototransduction and/or photopigment trafficking in cone photoreceptors. These findings indicate that cone arrestin displays structural and functional features intermediate between the specialized rod arrestin and the non-visual arrestins, which have broad receptor specificity. A unique functional feature of cone arrestin was the low affinity for its cognate receptor, resulting in an unusually rapid dissociation of the complex. Transient arrestin binding to the photopigment in cones may be responsible for the extremely rapid regeneration and reuse of the photopigment that is essential for cone function at high levels of illumination.

Alanine↗

PubMed2005

Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions.

In rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.

Adenosine Triphosphate↗

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↗

PubMed2002

Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

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.

Animals↗

PubMed2002

Conservation of the phosphate-sensitive elements in the arrestin family of proteins.

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the beta(2)-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the beta(2)-adrenergic and delta-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated delta-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in non-visual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.

Animals↗