Biomedical subjects

Vsevolod V Gurevich

Publications and source records attributed to Vsevolod V Gurevich.

At least 19 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↗

PubMed2006

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors.

The 4 mammalian arrestins serve as almost universal regulators of the largest known family of signaling proteins, G-protein-coupled receptors (GPCRs). Arrestins terminate receptor interactions with G proteins, redirect the signaling to a variety of alternative pathways, and orchestrate receptor internalization and subsequent intracellular trafficking. The elucidation of the structural basis and fine molecular mechanisms of the arrestin-receptor interaction paved the way to the targeted manipulation of this interaction from both sides to produce very stable or extremely transient complexes that helped to understand the regulation of many biologically important processes initiated by active GPCRs. The elucidation of the structural basis of arrestin interactions with numerous non-receptor-binding partners is long overdue. It will allow the construction of fully functional arrestins in which the ability to interact with individual partners is specifically disrupted or enhanced by targeted mutagenesis. These "custom-designed" arrestin mutants will be valuable tools in defining the role of various interactions in the intricate interplay of multiple signaling pathways in the living cell. The identification of arrestin-binding sites for various signaling molecules will also set the stage for designing molecular tools for therapeutic intervention that may prove useful in numerous disorders associated with congenital or acquired disregulation of GPCR signaling.

Animals↗

PubMed2006

Arrestins: ubiquitous regulators of cellular signaling pathways.

In vertebrates, the arrestins are a family of four proteins that regulate the signaling and trafficking of hundreds of different G-protein-coupled receptors (GPCRs). Arrestin homologs are also found in insects, protochordates and nematodes. Fungi and protists have related proteins but do not have true arrestins. Structural information is available only for free (unbound) vertebrate arrestins, and shows that the conserved overall fold is elongated and composed of two domains, with the core of each domain consisting of a seven-stranded beta-sandwich. Two main intramolecular interactions keep the two domains in the correct relative orientation, but both of these interactions are destabilized in the process of receptor binding, suggesting that the conformation of bound arrestin is quite different. As well as binding to hundreds of GPCR subtypes, arrestins interact with other classes of membrane receptors and more than 20 surprisingly diverse types of soluble signaling protein. Arrestins thus serve as ubiquitous signaling regulators in the cytoplasm and nucleus.

Animals↗

PubMed2005

The differential engagement of arrestin surface charges by the various functional forms of the receptor.

G-protein-coupled receptor signaling is terminated by arrestin proteins that preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, we performed an extensive analysis of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicated that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. We identified several additional phosphate-binding elements in the N-domain and demonstrated for the first time that the active receptor preferentially engages the arrestin C-domain. We also found that the interdomain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high affinity receptor binding state.

Animals↗

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

Conformational differences between arrestin2 and pre-activated mutants as revealed by hydrogen exchange mass spectrometry.

Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G protein-coupled receptors to terminate G protein-mediated signaling, cause the internalization of the receptor-arrestin complex, and initiate additional intracellular signaling cascades. Multiple lines of evidence suggest that arrestin normally exists in an inactive basal state and undergoes conformational activation in the process of receptor binding. "Pre-activated" phosphorylation-independent arrestin mutants display increased binding to ligand-activated but unphosphorylated receptors. The mutations are believed to expose key receptor-binding regions, allowing the mutants to mimic, to some extent, the transition of arrestin to its active state. In the present study, amide hydrogen exchange (HX) and mass spectrometry (MS) were used to examine the inactive conformation of wild-type arrestin2 and compare its solution conformation with two pre-activated mutants (R169E and 3A (I385A, V386A, F387A)). The results suggest an unexpected level of structural organization within arrestin elements containing clathrin and adaptin2-binding sites that were previously believed to be completely disordered. Increased deuterium incorporation was observed in both mutant forms compared with wild-type, indicating a change in the conformation of the mutants. Three regions demonstrated significant differences in deuterium incorporation: the first 33 residues of the N terminus and residues 243-255 (both previously implicated in receptor interaction), and residues 271-299. The results suggest that subtle differences in conformation are responsible for the significant difference in biological activity displayed by pre-activated arrestin mutants and that similar changes occur in the process of arrestin binding to the receptor.

Amino Acid Sequence↗

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↗

PubMed2005

Calmodulin kinase II inhibition protects against structural heart disease.

Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

Adrenergic beta-Antagonists↗

PubMed2005

Dopamine D1 receptor interaction with arrestin3 in neostriatal neurons.

Dopamine D1 receptor interactions with arrestins have been characterized using heterologously expressed D1 receptor and arrestins. The purpose of this study was to investigate the interaction of the endogenous D1 receptor with endogenous arrestin2 and 3 in neostriatal neurons. Endogenous arrestin2 and 3 in striatal homogenates bound to the C-terminus of the D1 receptor in a glutathione-S-transferase (GST) pulldown assay, with arrestin3 binding more strongly. The D1 C-terminus and, to a lesser extent, the third cytoplasmic loop also bound purified arrestin2 and 3. In neostriatal neurons, 2, 5, and 20 min agonist treatment increased the colocalization of the D1 receptor and arrestin3 immunoreactivity without altering the colocalization of the D1 receptor and arrestin2. Further, agonist treatment for 5 and 20 min caused translocation of arrestin3, but not arrestin2, to the membrane. The binding of arrestin3, but not arrestin2, to the D1 receptor was increased as assessed by coimmunoprecipitation after agonist treatment for 5 and 20 min. Agonist treatment of neurons induced D1 receptor internalization (35-45%) that was maximal within 2-5 min, a time-course similar to that of the increase in colocalization of the D1 receptor with arrestin3. These data indicate that the D1 receptor preferentially interacts with arrestin3 in neostriatal neurons.

Animals↗

PubMed2005

Inhibition of chemoattractant N-formyl peptide receptor trafficking by active arrestins.

Recent studies have highlighted the emergence of a class of G protein-coupled receptors that are internalized in an arrestin-independent manner. In addition to demonstrating that the N-formyl peptide receptor belongs in this family, we have recently shown that recycling of the receptor requires the presence of arrestins. To further elucidate mechanisms of arrestin-dependent regulation of G protein-coupled receptor processing, we examined the effects of altering the receptor-arrestin complex on ternary complex formation and cellular trafficking of the N-formyl peptide receptor by studying two active arrestin-2 mutants (truncated arrestin-2 [1-382], and arrestin-2 I386A, V387A, F388A). Complexes between the N-formyl peptide receptor and active arrestins exhibited higher affinity in vitro than the complex between the N-formyl peptide receptor and wild-type arrestin and furthermore were observed in vivo by colocalization studies using confocal microscopy. To assess the effects of these altered interactions on receptor trafficking, we demonstrated that active, but not wild-type, arrestin expression retards N-formyl peptide receptor internalization. Furthermore, expression of arrestin-2 I386A/V387A/F388A but not arrestin-2 [1-382] inhibited recycling of the N-formyl peptide receptor, reflecting an expanded role for arrestins in G protein-coupled receptor processing and trafficking. Whereas the extent of N-formyl peptide receptor phosphorylation had no effect on the inhibition of internalization, N-formyl peptide receptor recycling was restored when the receptor was only partially phosphorylated. These results indicate not only that a functional interaction between receptor and arrestin is required for recycling of certain G protein-coupled receptors, such as the N-formyl peptide receptor, but that the pattern of receptor phosphorylation further regulates this process.

Amino Acid Sequence↗

PubMed2004

Preferential Interaction between the dopamine D2 receptor and Arrestin2 in neostriatal neurons.

Dopamine D2 receptor interactions with arrestins and arrestin-dependent internalization have been characterized using heterologously expressed D2 receptor and arrestins. The purpose of this study was to investigate D2 receptor interaction with endogenous arrestins. Arrestin2 and arrestin3 in striatal homogenates bound to the third cytoplasmic loop of the D2 receptor, and purified arrestin2 and arrestin3 bound to the second and third loops and C terminus of the D2 receptor, in a glutathione S-transferase pull-down assay. In NS20Y neuroblastoma cells expressing an enhanced green-fluorescent protein-tagged D2 receptor (D2-EGFP), 2-h D2 agonist stimulation enhanced the colocalization of D2-EGFP with endogenous arrestin2 and arrestin3. These results suggest that the D2 receptor has the intrinsic ability to bind both nonvisual arrestins. Agonist treatment of D2-EGFP NS20Y cells induced D2 receptor internalization (36-46%) that was maximal within 20 min, but that was prevented by small interfering RNA-induced depletion of arrestin2 and arrestin3. In neostriatal neurons, 2-h agonist treatment selectively increased the colocalization of the endogenous D2 receptor with arrestin2, whereas receptor colocalization with arrestin3 was reduced. Agonist stimulation caused translocation of arrestin2, but not arrestin3, to the membrane in neurons and selectively enhanced the coimmunoprecipitation of the D2 receptor and arrestin2. All three measures of receptor/arrestin interaction (colocalization, translocation, and coprecipitation) demonstrated selective agonist-induced interaction between the D2 receptor and arrestin2 in neurons.

Animals↗

PubMed2004

Direct binding of visual arrestin to microtubules determines the differential subcellular localization of its splice variants in rod photoreceptors.

Proper function of visual arrestin is indispensable for rapid signal shut-off in rod photoreceptors. Dramatic light-dependent changes in its subcellular localization are believed to play an important role in light adaptation of photoreceptor cells. Here we show that visual arrestin binds microtubules. The truncated splice variant of visual arrestin, p44, demonstrates dramatically higher affinity for microtubules than the full-length protein (p48). Enhanced microtubule binding of p44 underlies its earlier reported preferential localization to detergent-resistant membranes, where it is anchored via membrane-associated microtubules in a rhodopsin-independent fashion. Experiments with purified proteins demonstrate that arrestin interaction with microtubules is direct and does not require any additional protein partners. Most importantly, arrestin interactions with microtubules and light-activated phosphorylated rhodopsin are mutually exclusive, suggesting that microtubule interaction may play a role in keeping p44 arrestin away from rhodopsin in dark-adapted photoreceptors.

Alternative Splicing↗

PubMed2004

The molecular acrobatics of arrestin activation.

Arrestin proteins play a key role in desensitizing G-protein-coupled receptors and re-directing their signaling to alternative pathways. The precise timing of arrestin binding to the receptor and its subsequent dissociation is ensured by its exquisite selectivity for the activated phosphorylated form of the receptor. The interaction between arrestin and the receptor involves the engagement of arrestin sensor sites that discriminate between active and inactive and phosphorylated and unphosphorylated forms of the receptor. This initial interaction is followed by a global conformational rearrangement of the arrestin molecule in the process of its transition into the high-affinity receptor-binding state that brings additional binding sites into action. In this article, we discuss the molecular mechanisms that underlie the sequential multi-site binding that ensures arrestin selectivity for the active phosphoreceptor and high fidelity of signal regulation by arrestin proteins.

Arrestins↗

PubMed2004

Arrestin2 expression selectively increases during neural differentiation.

Arrestins and G protein-coupled receptor kinases (GRKs) are key players in homologous desensitization of G protein-coupled receptors. Two non-visual arrestins, arrestin2 and 3, and five GRKs (GRK2, 3, 4, 5 and 6) are involved in desensitization of many receptors. Here, we demonstrate a steady increase in arrestin2 expression during prenatal development. The density of arrestin2 mRNA is higher in differentiated areas as compared with proliferative zones, whereas arrestin3 mRNA shows the opposite distribution. At embryonic day 14, concentrations of arrestin proteins are similar (32-34 nM). Later in development, arrestin2 expression rises, leading to a fourfold excess of arrestin2 over arrestin3 at birth (48 vs. 11 ng/mg protein or 102 vs. 25 nM). Among GRKs, only GRK5 increased with embryonic age from 124 nm at E14 to 359 nM at birth. Similarly, in vitro differentiation of cultured precursor cells, neurospheres, leads to a significant up-regulation of arrestin2 resulting in > 20-fold excess of arrestin2 (160 vs. 7 nM). GRK5 is the only subtype increased with neurosphere differentiation, although the change is only about twofold. The data demonstrate selective increases in the expression of arrestin2 associated with neural development and suggest specific yet unappreciated roles for arrestin2 in neural differentiation.

Animals↗