Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 65
Filter
Add more filters










Publication year range
1.
Prog Retin Eye Res ; 93: 101116, 2023 03.
Article in English | MEDLINE | ID: mdl-36273969

ABSTRACT

The light sensor of vertebrate scotopic (low-light) vision, rhodopsin, is a G-protein-coupled receptor comprising a polypeptide chain with bound chromophore, 11-cis-retinal, that exhibits remarkable physicochemical properties. This photopigment is extremely stable in the dark, yet its chromophore isomerises upon photon absorption with 70% efficiency, enabling the activation of its G-protein, transducin, with high efficiency. Rhodopsin's photochemical and biochemical activities occur over very different time-scales: the energy of retinaldehyde's excited state is stored in <1 ps in retinal-protein interactions, but it takes milliseconds for the catalytically active state to form, and many tens of minutes for the resting state to be restored. In this review, we describe the properties of rhodopsin and its role in rod phototransduction. We first introduce rhodopsin's gross structural features, its evolution, and the basic mechanisms of its activation. We then discuss light absorption and spectral sensitivity, photoreceptor electrical responses that result from the activity of individual rhodopsin molecules, and recovery of rhodopsin and the visual system from intense bleaching exposures. We then provide a detailed examination of rhodopsin's molecular structure and function, first in its dark state, and then in the active Meta states that govern its interactions with transducin, rhodopsin kinase and arrestin. While it is clear that rhodopsin's molecular properties are exquisitely honed for phototransduction, from starlight to dawn/dusk intensity levels, our understanding of how its molecular interactions determine the properties of scotopic vision remains incomplete. We describe potential future directions of research, and outline several major problems that remain to be solved.


Subject(s)
Rhodopsin , Transducin , Photoreceptor Cells/metabolism , Retina/metabolism , Rhodopsin/metabolism , Transducin/metabolism , Vision, Ocular , Animals
2.
J Phys Chem Lett ; 10(24): 7672-7677, 2019 Dec 19.
Article in English | MEDLINE | ID: mdl-31763851

ABSTRACT

Current submillisecond time-resolved broad-band infrared spectroscopy, one of the most frequently used techniques for studying structure-function relationships in life sciences, is typically limited to fast-cycling reactions that can be repeated thousands of times with high frequency. Notably, a majority of chemical and biological processes do not comply with this requirement. For example, the activation of vertebrate rhodopsin, a prototype of many protein receptors in biological organisms that mediate basic functions of life, including vision, smell, and taste, is irreversible. Here we present a dispersive single-shot Féry spectrometer setup that extends such spectroscopy to irreversible and slow-cycling systems by exploiting the unique properties of brilliant synchrotron infrared light combined with an advanced focal plane detector array embedded in a dispersive optical concept. We demonstrate our single-shot method on microbial actinorhodopsin with a slow photocycle and on vertebrate rhodopsin with irreversible activation.


Subject(s)
Rhodopsin/chemistry , Single Molecule Imaging/instrumentation , Single Molecule Imaging/methods , Spectrophotometry, Infrared/instrumentation , Spectrophotometry, Infrared/methods , Kinetics , Light , Photochemical Processes , Protein Conformation
4.
Open Biol ; 8(8)2018 08.
Article in English | MEDLINE | ID: mdl-30068566

ABSTRACT

Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein α-subunits (Gα*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction-diffusion simulations. Gα* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of Gα* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 Gα* · PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that Gα* · PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of Gα* which binds with lower affinity, forming Gα* · PDE6 · Gα*. Reaction-diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of Gα* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated Gα* fails to activate the effector enzyme.


Subject(s)
Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Transducin/metabolism , Animals , Binding Sites , Cattle , Cell Membrane/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 6/chemistry , Enzyme Activation , Hydrolysis , Protein Binding , Transducin/chemistry
5.
J Biol Chem ; 293(12): 4403-4410, 2018 03 23.
Article in English | MEDLINE | ID: mdl-29363577

ABSTRACT

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (Gt) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the Gt α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, Gt α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling.


Subject(s)
Arrestin/chemistry , Arrestin/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Conformation , Rhodopsin/chemistry , Rhodopsin/metabolism , Crystallography, X-Ray , Humans , Phosphorylation , Protein Binding , Signal Transduction
6.
Proc Natl Acad Sci U S A ; 114(16): E3268-E3275, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28373559

ABSTRACT

Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.


Subject(s)
Light , Nanostructures/chemistry , Protein Conformation/radiation effects , Rhodopsin/chemistry , Transducin/chemistry , Animals , Cattle , Protein Structure, Secondary , Rhodopsin/metabolism , Rhodopsin/radiation effects , Spin Labels , Transducin/metabolism , Transducin/radiation effects
7.
PLoS One ; 10(11): e0143399, 2015.
Article in English | MEDLINE | ID: mdl-26606751

ABSTRACT

GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.


Subject(s)
Models, Molecular , Protein Conformation , Receptors, G-Protein-Coupled/chemistry , Guanosine Diphosphate/metabolism , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction
8.
Mol Pharmacol ; 88(3): 572-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26184590

ABSTRACT

Signaling by G-protein-coupled receptors is often considered a uniform process, whereby a homogeneously activated proportion of randomly distributed receptors are activated under equilibrium conditions and produce homogeneous, steady-state intracellular signals. While this may be the case in some biologic systems, the example of rhodopsin with its strictly local single-quantum mode of function shows that homogeneity in space and time cannot be a general property of G-protein-coupled systems. Recent work has now revealed many other systems where such simplicity does not prevail. Instead, a plethora of mechanisms allows much more complex patterns of receptor activation and signaling: different mechanisms of protein-protein interaction; temporal changes under nonequilibrium conditions; localized receptor activation; and localized second messenger generation and degradation-all of which shape receptor-generated signals and permit the creation of multiple signal types. Here, we review the evidence for such pleiotropic receptor signaling in space and time.


Subject(s)
Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Humans , Kinetics , Molecular Sequence Data , Protein Binding , Protein Transport , Receptors, G-Protein-Coupled/chemistry
9.
J Biol Chem ; 290(33): 20117-27, 2015 Aug 14.
Article in English | MEDLINE | ID: mdl-26105054

ABSTRACT

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.


Subject(s)
Rhodopsin/metabolism , Amino Acid Sequence , Animals , Cattle , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Rhodopsin/chemistry , Sequence Homology, Amino Acid , Spectroscopy, Fourier Transform Infrared
10.
J Biol Chem ; 290(20): 12919-28, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25847250

ABSTRACT

In the G protein-coupled receptor rhodopsin, light-induced cis/trans isomerization of the retinal ligand triggers a series of distinct receptor states culminating in the active Metarhodopsin II (Meta II) state, which binds and activates the G protein transducin (Gt). Long before Meta II decays into the aporeceptor opsin and free all-trans-retinal, its signaling is quenched by receptor phosphorylation and binding of the protein arrestin-1, which blocks further access of Gt to Meta II. Although recent crystal structures of arrestin indicate how it might look in a precomplex with the phosphorylated receptor, the transition into the high affinity complex is not understood. Here we applied Fourier transform infrared spectroscopy to monitor the interaction of arrestin-1 and phosphorylated rhodopsin in native disc membranes. By isolating the unique infrared signature of arrestin binding, we directly observed the structural alterations in both reaction partners. In the high affinity complex, rhodopsin adopts a structure similar to Gt-bound Meta II. In arrestin, a modest loss of ß-sheet structure indicates an increase in flexibility but is inconsistent with a large scale structural change. During Meta II decay, the arrestin-rhodopsin stoichiometry shifts from 1:1 to 1:2. Arrestin stabilizes half of the receptor population in a specific Meta II protein conformation, whereas the other half decays to inactive opsin. Altogether these results illustrate the distinct binding modes used by arrestin to interact with different functional forms of the receptor.


Subject(s)
Arrestin/chemistry , Cell Membrane/chemistry , Multiprotein Complexes/chemistry , Rhodopsin/chemistry , Animals , Arrestin/genetics , Arrestin/metabolism , Cattle , Cell Membrane/genetics , Cell Membrane/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Binding , Protein Stability , Rhodopsin/genetics , Rhodopsin/metabolism , Spectroscopy, Fourier Transform Infrared
11.
Methods Enzymol ; 556: 563-608, 2015.
Article in English | MEDLINE | ID: mdl-25857800

ABSTRACT

In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.


Subject(s)
Arrestin/metabolism , GTP-Binding Proteins/metabolism , Protein Interaction Mapping/methods , Rhodopsin/metabolism , Animals , Arrestin/chemistry , Cattle , Crystallography, X-Ray/methods , GTP-Binding Proteins/chemistry , Models, Molecular , Protein Binding , Protein Conformation , Rhodopsin/chemistry , Spectrometry, Fluorescence/methods , Spectrophotometry, Ultraviolet/methods , Spectroscopy, Fourier Transform Infrared/methods
13.
Biophys J ; 107(5): 1042-1053, 2014 Sep 02.
Article in English | MEDLINE | ID: mdl-25185540

ABSTRACT

Dim-light vision is mediated by retinal rod cells. Rhodopsin (R), a G-protein-coupled receptor, switches to its active form (R(∗)) in response to absorbing a single photon and activates multiple copies of the G-protein transducin (G) that trigger further downstream reactions of the phototransduction cascade. The classical assumption is that R and G are uniformly distributed and freely diffusing on disk membranes. Recent experimental findings have challenged this view by showing specific R architectures, including RG precomplexes, nonuniform R density, specific R arrangements, and immobile fractions of R. Here, we derive a physical model that describes the first steps of the photoactivation cascade in spatiotemporal detail and single-molecule resolution. The model was implemented in the ReaDDy software for particle-based reaction-diffusion simulations. Detailed kinetic in vitro experiments are used to parametrize the reaction rates and diffusion constants of R and G. Particle diffusion and G activation are then studied under different conditions of R-R interaction. It is found that the classical free-diffusion model is consistent with the available kinetic data. The existence of precomplexes between inactive R and G is only consistent with the data if these precomplexes are weak, with much larger dissociation rates than suggested elsewhere. Microarchitectures of R, such as dimer racks, would effectively immobilize R but have little impact on the diffusivity of G and on the overall amplification of the cascade at the level of the G protein.


Subject(s)
Retinal Rod Photoreceptor Cells/chemistry , Rhodopsin/chemistry , Transducin/chemistry , Computer Simulation , Diffusion , Kinetics , Models, Molecular , Photochemical Processes , Software , Video Recording
14.
Nat Commun ; 5: 4801, 2014 Sep 10.
Article in English | MEDLINE | ID: mdl-25205354

ABSTRACT

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαßγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the 'finger loop' region (ArrFL1-4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.


Subject(s)
Arrestins/metabolism , Binding, Competitive , Receptors, G-Protein-Coupled/metabolism , Amino Acid Motifs , Animals , Cattle , Crystallography, X-Ray , Models, Molecular , Protein Structure, Tertiary , Rhodopsin/metabolism , Signal Transduction , Spectroscopy, Fourier Transform Infrared , X-Ray Absorption Spectroscopy
15.
J Am Chem Soc ; 136(32): 11244-7, 2014 Aug 13.
Article in English | MEDLINE | ID: mdl-25046433

ABSTRACT

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (Gαßγ, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active ß2-adrenoceptor (ß2AR*) in complex with C-terminal peptides derived from the key interaction site of Gα (GαCT) as surrogate of Gαßγ. We find that GiαCT and GsαCT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed ß2AR*. The slim GiαCT stabilizes a ß2AR* conformation, not accessible to the bulkier GsαCT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of ß2AR* toward Gi or Gs.


Subject(s)
Receptors, Adrenergic, beta-2/chemistry , Receptors, G-Protein-Coupled/chemistry , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Cattle , Cell Membrane/metabolism , Computer Simulation , Cytoplasm/metabolism , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rhodopsin/chemistry , Signal Transduction
16.
Handb Exp Pharmacol ; 219: 101-16, 2014.
Article in English | MEDLINE | ID: mdl-24292826

ABSTRACT

The retinal rod cell is an exquisitely sensitive single-photon detector that primarily functions in dim light (e.g., moonlight). However, rod cells must routinely survive light intensities more than a billion times greater (e.g., bright daylight). One serious challenge to rod cell survival in daylight is the massive amount of all-trans-retinal that is released by Meta II, the light-activated form of the photoreceptor rhodopsin. All-trans-retinal is toxic, and its condensation products have been implicated in disease. Our recent work has developed the concept that rod arrestin (arrestin-1), which terminates Meta II signaling, has an additional role in protecting rod cells from the consequences of bright light by limiting free all-trans-retinal. In this chapter we will elaborate upon the molecular mechanisms by which arrestin-1 serves as both a single-photon response quencher as well as an instrument of rod cell survival in bright light. This discussion will take place within the framework of three distinct functional modules of vision: signal transduction, the retinoid cycle, and protein translocation.


Subject(s)
Arrestin/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Signal Transduction/physiology , Animals , Humans , Light , Phosphorylation , Protein Transport/physiology , Retinoids/physiology , Rhodopsin/metabolism
17.
Angew Chem Int Ed Engl ; 52(42): 11021-4, 2013 Oct 11.
Article in English | MEDLINE | ID: mdl-24038729

ABSTRACT

Receptor-ligand interaction: Olfactory receptors (ORs) are G-protein-coupled receptors (GPCRs), which detect signaling molecules such as hormones and odorants. The structure of opsin, the GPCR employed in vision, with a detergent molecule bound deep in its orthosteric ligand-binding pocket provides a template for OR homology modeling, thus enabling investigation of the structural basis of the mechanism of odorant-receptor recognition.


Subject(s)
Olfactory Receptor Neurons/chemistry , Opsins/chemistry , Receptors, G-Protein-Coupled/chemistry , Humans , Models, Molecular , Olfactory Receptor Neurons/metabolism , Opsins/metabolism , Receptors, G-Protein-Coupled/metabolism
18.
J Am Chem Soc ; 135(33): 12305-12, 2013 Aug 21.
Article in English | MEDLINE | ID: mdl-23883288

ABSTRACT

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric G protein transducin (Gt) to transmit the light signal into retinal rod cells. The rhodopsin activity is virtually zero in the dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity and speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy and supporting all-atom molecular dynamics (MD) simulations to study the conformational diversity of rhodopsin in membrane environment and extend the static picture provided by the available crystal structures. The FTIR results show how the equilibria of inactive and active protein states of the receptor (so-called metarhodopsin states) are regulated by the highly conserved E(D)RY and Yx7K(R) motives. The MD data identify an intrinsically unstructured cytoplasmic loop region connecting transmembrane helices 5 and 6 (CL3) and show how each protein state is split into conformational substates. The C-termini of the Gtγ- and Gtα-subunits (GαCT and GγCT), prepared as synthetic peptides, are likely to bind sequentially and at different sites of the active receptor. The peptides have different effects on the receptor conformation. While GγCT stabilizes the active states but preserves CL3 flexibility, GαCT selectively stabilizes a single conformational substate with largely helical CL3, as it is found in crystal structures. Based on these results we propose a mechanism for the fast and precise signal transfer from rhodopsin to Gt, which assumes a stepwise and mutual reduction of their conformational space. The mechanism relies on conserved amino acids and may therefore underlie GPCR/G protein coupling in general.


Subject(s)
Rhodopsin/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Binding Sites , Cattle , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Conformation , Rhodopsin/agonists , Rhodopsin/chemistry , Rhodopsin/genetics , Spectroscopy, Fourier Transform Infrared , Transducin/chemistry
19.
Nature ; 497(7447): 142-6, 2013 May 02.
Article in English | MEDLINE | ID: mdl-23604253

ABSTRACT

Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins and initiate G-protein-independent signalling. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs. Structures of the inactive state of arrestin are available, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a ∼21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop, loop 139 (refs 8, 10, 11) and the sequence Asp 296-Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.


Subject(s)
Arrestins/chemistry , Arrestins/metabolism , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Alternative Splicing , Animals , Arrestins/genetics , Cattle , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Molecular Weight , Protein Conformation , Protein Isoforms/genetics , Rotation , Sequence Deletion , Static Electricity , beta-Arrestins
20.
Nat Commun ; 3: 995, 2012.
Article in English | MEDLINE | ID: mdl-22871814

ABSTRACT

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod arrestin induces uptake of the agonist all-trans-retinal [corrected] in only half the population of phosphorylated opsin in the native membrane. Agonist uptake blocks subsequent entry of the inverse agonist 11-cis-retinal (that is, regeneration of rhodopsin), but regeneration is not blocked in the other half of aporeceptors. Environmentally sensitive fluorophores attached to arrestin reported that conformational changes in loop(V-VI) (N-domain) are coupled to the entry of agonist, while loop(XVIII-XIX) (C-domain) engages the aporeceptor even before agonist is added. The data are most consistent with a model in which each domain of arrestin engages its own aporeceptor, and the different binding preferences of the domains lead to asymmetric ligand binding by the aporeceptors. Such a mechanism would protect the rod cell in bright light by concurrently sequestering toxic all-trans-retinal [corrected] and allowing regeneration with 11-cis-retinal.


Subject(s)
Arrestin/metabolism , Animals , Arrestin/chemistry , Cattle , Models, Molecular , Models, Theoretical , Opsins/metabolism , Protein Binding , Protein Structure, Secondary , Retinaldehyde/metabolism , Spectrometry, Fluorescence
SELECTION OF CITATIONS
SEARCH DETAIL
...