Vsevolod Gurevich | Vanderbilt University (original) (raw)

Papers by Vsevolod Gurevich

Journal of Biological Chemistry, Feb 1, 2013

Methods in molecular biology, 2019

Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor ... more Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cellbased functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.

ACS Chemical Biology, May 6, 2019

Figures S1-S18, Tables S1-S5, methods for expression and purification of ERK2 for crystallization... more Figures S1-S18, Tables S1-S5, methods for expression and purification of ERK2 for crystallization, mathematical anisotropy model for dose-response curves, and chemical synthesis of library compounds (PDF) Accession Codes The atomic coordinates of the 2507-8-ERK2 complex have been deposited in the Protein Data Bank as entry 6NBS.

PLOS Genetics, Oct 17, 2019

Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the k... more Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the key tissue for whole-body glucose disposal and utilization. New drugs aimed at improving insulin sensitivity of SKM would greatly expand available therapeutic options. βarrestin-1 and-2 (Barr1 and Barr2, respectively) are two intracellular proteins best known for their ability to mediate the desensitization and internalization of G protein-coupled receptors (GPCRs). Recent studies suggest that Barr1 and Barr2 regulate several important metabolic functions including insulin release and hepatic glucose production. Since SKM expresses many GPCRs, including the metabolically important β 2-adrenergic receptor, the goal of this study was to examine the potential roles of Barr1 and Barr2 in regulating SKM and whole-body glucose metabolism. Using SKM-specific knockout (KO) mouse lines, we showed that the loss of SKM Barr2, but not of SKM Barr1, resulted in mild improvements in glucose tolerance in diet-induced obese mice. SKM-specific Barr1-and Barr2-KO mice did not show any significant differences in exercise performance. However, lack of SKM Barr2 led to increased glycogen breakdown following a treadmill exercise challenge. Interestingly, mice that lacked both Barr1 and Barr2 in SKM showed no significant metabolic phenotypes. Thus, somewhat surprisingly, our data indicate that SKM β-arrestins play only rather subtle roles (SKM Barr2) in regulating whole-body glucose homeostasis and SKM insulin sensitivity.

Frontiers in Molecular Neuroscience

Accurate photon counting requires that rods generate highly amplified, reproducible single photon... more Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the bas...

Frontiers in Molecular Neuroscience

Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, ro... more Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, rods operate over many log units of light intensity. Several processes inside photoreceptor cells make this incredible light adaptation possible. Here, we added to our previously developed, fully space resolved biophysical model of rod phototransduction, some of the mechanisms that play significant roles in shaping the rod response under high illumination levels: the function of RGS9 in shutting off G protein transducin, and calcium dependences of the phosphorylation rates of activated rhodopsin, of the binding of cGMP to the light-regulated ion channel, and of two membrane guanylate cyclase activities. A well stirred version of this model captured the responses to bright, saturating flashes in WT and mutant mouse rods and was used to explain “Pepperberg plots,” that graph the time during which the response is saturated against the natural logarithm of flash strength for bright flashes. At...

PLOS ONE

Retinal rods function as accurate photon counters to provide for vision under very dim light. To ... more Retinal rods function as accurate photon counters to provide for vision under very dim light. To do so, rods must generate highly amplified, reproducible responses to single photons, yet outer segment architecture and randomness in the location of rhodopsin photoisomerization on the surface of an internal disk introduce variability to the rising phase of the photon response. Soon after a photoisomerization at a disk rim, depletion of cGMP near the plasma membrane closes ion channels and hyperpolarizes the rod. But with a photoisomerization in the center of a disk, local depletion of cGMP is distant from the channels in the plasma membrane. Thus, channel closure is delayed by the time required for the reduction of cGMP concentration to reach the plasma membrane. Moreover, the local fall in cGMP dissipates over a larger volume before affecting the channels, so response amplitude is reduced. This source of variability increases with disk radius. Using a fully space-resolved biophysical model of rod phototransduction, we quantified the variability attributable to randomness in the location of photoisomerization as a function of disk structure. In mouse rods that have small disks bearing a single incisure, this variability was negligible in the absence of the incisure. Variability was increased slightly by the incisure, but randomness in the shutoff of rhodopsin emerged as the main source of single photon response variability at all but the earliest times. Variability arising from randomness in the transverse location of photoisomerization increased in magnitude and persisted over a longer period in the photon response of large salamander rods. A symmetric arrangement of multiple incisures in the disks of salamander rods greatly reduced this variability during the rising phase, but the incisures had the opposite effect on variability arising from randomness in rhodopsin shutoff at later times.

Rod photoreceptors generate measurable responses to single-photon activation of individual molecu... more Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.

Investigative Ophthalmology & Visual Science, 2005

Investigative Ophthalmology & Visual Science, 2008

Journal of Neurochemistry, 2020

The finger loop in the central crest of the receptor‐binding site of arrestins engages the cavity... more The finger loop in the central crest of the receptor‐binding site of arrestins engages the cavity between the transmembrane helices of activated G‐protein‐coupled receptors. Therefore, it was hypothesized to serve as the sensor that detects the activation state of the receptor. We performed comprehensive mutagenesis of the finger loop in bovine visual arrestin‐1, generated mutant radiolabeled proteins by cell‐free translation, and determined the effects of mutations on the in vitro binding of arrestin‐1 to purified phosphorylated light‐activated rhodopsin. This interaction is driven by two factors, rhodopsin activation and rhodopsin‐attached phosphates. Therefore, the binding of arrestin‐1 to light‐activated unphosphorylated rhodopsin is low. To evaluate the role of the finger loop specifically in the recognition of the active receptor conformation, we tested the effects of these mutations in the context of truncated arrestin‐1 that demonstrates much higher binding to unphosphorylat...

Pharmacology & Therapeutics, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Journal of Biological Chemistry, 2020

The multi-faceted adaptor protein β-arrestin1 (β-arr1) promotes activation of focal adhesion kina... more The multi-faceted adaptor protein β-arrestin1 (β-arr1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal transducing adaptor molecule 1) as disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which b-arr1 together with STAM1 activates FAK we used site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy-based studies coupled with BRET-based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12 induced activation of FAK, but had no impact on ERK-1/2 activat...

Journal of Biological Chemistry, 1995

Trends in Biochemical Sciences, 2018

Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins ... more Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins regulate signaling from >800 G protein-coupled receptors (GPCRs) or non-receptor activators by simultaneously binding an activator and one out of hundreds of other signaling proteins. When arrestins are bound to GPCRs or other activators, the affinity for these signaling partners changes. Thus, it is proposed that an activator alters arrestin's ability to transduce a signal. The comparison of all available arrestin structures identifies several common conformational rearrangements associated with activation. In particular, it identifies elements that are directly involved in binding to GPCRs or other activators, elements that likely engage distinct downstream effectors, and elements that likely link the activator-binding sites with the effector-binding sites.

Applied Magnetic Resonance

In this article, the second author's name Eugenia V. Gurevich was incorrectly written as Eugenis ... more In this article, the second author's name Eugenia V. Gurevich was incorrectly written as Eugenis V. Gurevich. The original article has been corrected. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Investigative Ophthalmology & Visual Science, May 10, 2007

Biochemistry, May 30, 2003

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kin... more G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

Journal of Molecular Biology, Aug 1, 2005

Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G pro... more Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G protein-coupled receptors to terminate G proteinmediated 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 wildtype, 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.

The FASEB Journal, Mar 1, 2006

Journal of Biological Chemistry, Feb 1, 2013

Methods in molecular biology, 2019

Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor ... more Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cellbased functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.

ACS Chemical Biology, May 6, 2019

Figures S1-S18, Tables S1-S5, methods for expression and purification of ERK2 for crystallization... more Figures S1-S18, Tables S1-S5, methods for expression and purification of ERK2 for crystallization, mathematical anisotropy model for dose-response curves, and chemical synthesis of library compounds (PDF) Accession Codes The atomic coordinates of the 2507-8-ERK2 complex have been deposited in the Protein Data Bank as entry 6NBS.

PLOS Genetics, Oct 17, 2019

Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the k... more Type 2 diabetes (T2D) has become a major health problem worldwide. Skeletal muscle (SKM) is the key tissue for whole-body glucose disposal and utilization. New drugs aimed at improving insulin sensitivity of SKM would greatly expand available therapeutic options. βarrestin-1 and-2 (Barr1 and Barr2, respectively) are two intracellular proteins best known for their ability to mediate the desensitization and internalization of G protein-coupled receptors (GPCRs). Recent studies suggest that Barr1 and Barr2 regulate several important metabolic functions including insulin release and hepatic glucose production. Since SKM expresses many GPCRs, including the metabolically important β 2-adrenergic receptor, the goal of this study was to examine the potential roles of Barr1 and Barr2 in regulating SKM and whole-body glucose metabolism. Using SKM-specific knockout (KO) mouse lines, we showed that the loss of SKM Barr2, but not of SKM Barr1, resulted in mild improvements in glucose tolerance in diet-induced obese mice. SKM-specific Barr1-and Barr2-KO mice did not show any significant differences in exercise performance. However, lack of SKM Barr2 led to increased glycogen breakdown following a treadmill exercise challenge. Interestingly, mice that lacked both Barr1 and Barr2 in SKM showed no significant metabolic phenotypes. Thus, somewhat surprisingly, our data indicate that SKM β-arrestins play only rather subtle roles (SKM Barr2) in regulating whole-body glucose homeostasis and SKM insulin sensitivity.

Frontiers in Molecular Neuroscience

Accurate photon counting requires that rods generate highly amplified, reproducible single photon... more Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the bas...

Frontiers in Molecular Neuroscience

Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, ro... more Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, rods operate over many log units of light intensity. Several processes inside photoreceptor cells make this incredible light adaptation possible. Here, we added to our previously developed, fully space resolved biophysical model of rod phototransduction, some of the mechanisms that play significant roles in shaping the rod response under high illumination levels: the function of RGS9 in shutting off G protein transducin, and calcium dependences of the phosphorylation rates of activated rhodopsin, of the binding of cGMP to the light-regulated ion channel, and of two membrane guanylate cyclase activities. A well stirred version of this model captured the responses to bright, saturating flashes in WT and mutant mouse rods and was used to explain “Pepperberg plots,” that graph the time during which the response is saturated against the natural logarithm of flash strength for bright flashes. At...

PLOS ONE

Retinal rods function as accurate photon counters to provide for vision under very dim light. To ... more Retinal rods function as accurate photon counters to provide for vision under very dim light. To do so, rods must generate highly amplified, reproducible responses to single photons, yet outer segment architecture and randomness in the location of rhodopsin photoisomerization on the surface of an internal disk introduce variability to the rising phase of the photon response. Soon after a photoisomerization at a disk rim, depletion of cGMP near the plasma membrane closes ion channels and hyperpolarizes the rod. But with a photoisomerization in the center of a disk, local depletion of cGMP is distant from the channels in the plasma membrane. Thus, channel closure is delayed by the time required for the reduction of cGMP concentration to reach the plasma membrane. Moreover, the local fall in cGMP dissipates over a larger volume before affecting the channels, so response amplitude is reduced. This source of variability increases with disk radius. Using a fully space-resolved biophysical model of rod phototransduction, we quantified the variability attributable to randomness in the location of photoisomerization as a function of disk structure. In mouse rods that have small disks bearing a single incisure, this variability was negligible in the absence of the incisure. Variability was increased slightly by the incisure, but randomness in the shutoff of rhodopsin emerged as the main source of single photon response variability at all but the earliest times. Variability arising from randomness in the transverse location of photoisomerization increased in magnitude and persisted over a longer period in the photon response of large salamander rods. A symmetric arrangement of multiple incisures in the disks of salamander rods greatly reduced this variability during the rising phase, but the incisures had the opposite effect on variability arising from randomness in rhodopsin shutoff at later times.

Rod photoreceptors generate measurable responses to single-photon activation of individual molecu... more Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.

Investigative Ophthalmology & Visual Science, 2005

Investigative Ophthalmology & Visual Science, 2008

Journal of Neurochemistry, 2020

The finger loop in the central crest of the receptor‐binding site of arrestins engages the cavity... more The finger loop in the central crest of the receptor‐binding site of arrestins engages the cavity between the transmembrane helices of activated G‐protein‐coupled receptors. Therefore, it was hypothesized to serve as the sensor that detects the activation state of the receptor. We performed comprehensive mutagenesis of the finger loop in bovine visual arrestin‐1, generated mutant radiolabeled proteins by cell‐free translation, and determined the effects of mutations on the in vitro binding of arrestin‐1 to purified phosphorylated light‐activated rhodopsin. This interaction is driven by two factors, rhodopsin activation and rhodopsin‐attached phosphates. Therefore, the binding of arrestin‐1 to light‐activated unphosphorylated rhodopsin is low. To evaluate the role of the finger loop specifically in the recognition of the active receptor conformation, we tested the effects of these mutations in the context of truncated arrestin‐1 that demonstrates much higher binding to unphosphorylat...

Pharmacology & Therapeutics, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Journal of Biological Chemistry, 2020

The multi-faceted adaptor protein β-arrestin1 (β-arr1) promotes activation of focal adhesion kina... more The multi-faceted adaptor protein β-arrestin1 (β-arr1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal transducing adaptor molecule 1) as disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which b-arr1 together with STAM1 activates FAK we used site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy-based studies coupled with BRET-based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12 induced activation of FAK, but had no impact on ERK-1/2 activat...

Journal of Biological Chemistry, 1995

Trends in Biochemical Sciences, 2018

Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins ... more Arrestins are a small family of proteins with four isoforms in humans. Remarkably, two arrestins regulate signaling from >800 G protein-coupled receptors (GPCRs) or non-receptor activators by simultaneously binding an activator and one out of hundreds of other signaling proteins. When arrestins are bound to GPCRs or other activators, the affinity for these signaling partners changes. Thus, it is proposed that an activator alters arrestin's ability to transduce a signal. The comparison of all available arrestin structures identifies several common conformational rearrangements associated with activation. In particular, it identifies elements that are directly involved in binding to GPCRs or other activators, elements that likely engage distinct downstream effectors, and elements that likely link the activator-binding sites with the effector-binding sites.

Applied Magnetic Resonance

In this article, the second author's name Eugenia V. Gurevich was incorrectly written as Eugenis ... more In this article, the second author's name Eugenia V. Gurevich was incorrectly written as Eugenis V. Gurevich. The original article has been corrected. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Investigative Ophthalmology & Visual Science, May 10, 2007

Biochemistry, May 30, 2003

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kin... more G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

Journal of Molecular Biology, Aug 1, 2005

Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G pro... more Arrestins are regulatory proteins that bind specifically to ligand-activated phosphorylated G protein-coupled receptors to terminate G proteinmediated 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 wildtype, 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.

The FASEB Journal, Mar 1, 2006