Vsevolod Gurevich - Profile on Academia.edu (original) (raw)
Papers by Vsevolod Gurevich
The Expression Of Phosphorylation–independent super–arrestins Protects Rod Outer Segments In Rhodopsin Kinase Knockout Mice
Investigative Ophthalmology & Visual Science, May 1, 2004
Current Biology, Apr 1, 2009
G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in eve... more G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in every cell in the body and are targeted by the majority of clinically used drugs . GPCR signaling, including rhodopsin-driven phototransduction, is terminated by receptor phosphorylation followed by arrestin binding . Genetic defects in receptor phosphorylation and excessive signaling by overactive GPCR mutants result in a wide variety of diseases, from retinal degeneration to cancer . Here, we tested whether arrestin1 mutants with enhanced ability to bind active unphosphorylated rhodopsin [7-10] can suppress uncontrolled signaling, bypassing receptor phosphorylation by rhodopsin kinase (RK) and replacing this two-step mechanism with a singlestep deactivation in rod photoreceptors. We show that in this precisely timed signaling system with single-photon sensitivity , an enhanced arrestin1 mutant partially compensates for defects in rhodopsin phosphorylation, promoting photoreceptor survival, improving functional performance, and facilitating photoresponse recovery. These proof-of-principle experiments demonstrate the feasibility of functional compensation in vivo for the first time, which is a promising approach for correcting genetic defects associated with gain-of-function mutations. Successful modification of protein-protein interactions by appropriate mutations paves the way to targeted redesign of signaling pathways to achieve desired functional outcomes. Enhanced Arrestin1 Mutant Protects Photoreceptors in the Absence of Rhodopsin Phosphorylation Wild-type (WT) arrestins bind active unphosphorylated G protein-coupled receptors (GPCRs) with low affinity . The
Enhanced Arrestin Mutant Protects Rod Photoreceptors and Facilitates Photoresponse Recovery in the Absence of Rhodopsin Phosphorylation
Investigative Ophthalmology & Visual Science, May 14, 2008
Journal of Biological Chemistry, Feb 1, 2013
Background: Arrestin-1 with enhanced binding to unphosphorylated active rhodopsin (Rh*) has thera... more Background: Arrestin-1 with enhanced binding to unphosphorylated active rhodopsin (Rh*) has therapeutic potential. Results: Manipulation of the rhodopsin binding surface of arrestin-1 greatly increases its binding to Rh*. Stable arrestin-1 with high binding to Rh* can be engineered with and without the ability to self-associate. Significance: The affinity of arrestin-1 for Rh* and its propensity to oligomerize can be independently changed by targeted mutagenesis. Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gainof-function receptor mutations.
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.
How Much Arrestin Is Good for Rod Photoreceptors
Investigative Ophthalmology & Visual Science, 2005
Solution Structure of the Rod Arrestin Tetramer Explains the Mode of Its Formation and Its Inability to Bind Rhodopsin
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.
Rod and Cone Arrestins Differentially Mobilize Signaling Proteins to Microtubules
Investigative Ophthalmology & Visual Science, May 10, 2007
Functional Capabilities of an N-Formyl Peptide Receptor−Gαi2 Fusion Protein: Assemblies with G Proteins and Arrestins
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.
The Expression Of Phosphorylation–independent super–arrestins Protects Rod Outer Segments In Rhodopsin Kinase Knockout Mice
Investigative Ophthalmology & Visual Science, May 1, 2004
Current Biology, Apr 1, 2009
G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in eve... more G protein-coupled receptors (GPCRs) are the largest family of signaling proteins expressed in every cell in the body and are targeted by the majority of clinically used drugs . GPCR signaling, including rhodopsin-driven phototransduction, is terminated by receptor phosphorylation followed by arrestin binding . Genetic defects in receptor phosphorylation and excessive signaling by overactive GPCR mutants result in a wide variety of diseases, from retinal degeneration to cancer . Here, we tested whether arrestin1 mutants with enhanced ability to bind active unphosphorylated rhodopsin [7-10] can suppress uncontrolled signaling, bypassing receptor phosphorylation by rhodopsin kinase (RK) and replacing this two-step mechanism with a singlestep deactivation in rod photoreceptors. We show that in this precisely timed signaling system with single-photon sensitivity , an enhanced arrestin1 mutant partially compensates for defects in rhodopsin phosphorylation, promoting photoreceptor survival, improving functional performance, and facilitating photoresponse recovery. These proof-of-principle experiments demonstrate the feasibility of functional compensation in vivo for the first time, which is a promising approach for correcting genetic defects associated with gain-of-function mutations. Successful modification of protein-protein interactions by appropriate mutations paves the way to targeted redesign of signaling pathways to achieve desired functional outcomes. Enhanced Arrestin1 Mutant Protects Photoreceptors in the Absence of Rhodopsin Phosphorylation Wild-type (WT) arrestins bind active unphosphorylated G protein-coupled receptors (GPCRs) with low affinity . The
Enhanced Arrestin Mutant Protects Rod Photoreceptors and Facilitates Photoresponse Recovery in the Absence of Rhodopsin Phosphorylation
Investigative Ophthalmology & Visual Science, May 14, 2008
Journal of Biological Chemistry, Feb 1, 2013
Background: Arrestin-1 with enhanced binding to unphosphorylated active rhodopsin (Rh*) has thera... more Background: Arrestin-1 with enhanced binding to unphosphorylated active rhodopsin (Rh*) has therapeutic potential. Results: Manipulation of the rhodopsin binding surface of arrestin-1 greatly increases its binding to Rh*. Stable arrestin-1 with high binding to Rh* can be engineered with and without the ability to self-associate. Significance: The affinity of arrestin-1 for Rh* and its propensity to oligomerize can be independently changed by targeted mutagenesis. Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gainof-function receptor mutations.
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.
How Much Arrestin Is Good for Rod Photoreceptors
Investigative Ophthalmology & Visual Science, 2005
Solution Structure of the Rod Arrestin Tetramer Explains the Mode of Its Formation and Its Inability to Bind Rhodopsin
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.
Rod and Cone Arrestins Differentially Mobilize Signaling Proteins to Microtubules
Investigative Ophthalmology & Visual Science, May 10, 2007
Functional Capabilities of an N-Formyl Peptide Receptor−Gαi2 Fusion Protein: Assemblies with G Proteins and Arrestins
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.