RESUMO
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.
Assuntos
Arrestina/química , Membrana Celular/química , Complexos Multiproteicos/química , Rodopsina/química , Animais , Arrestina/genética , Arrestina/metabolismo , Bovinos , Membrana Celular/genética , Membrana Celular/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estabilidade Proteica , Rodopsina/genética , Rodopsina/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In the retinal binding pocket of rhodopsin, a Schiff base links the retinal ligand covalently to the Lys296 side chain. Light transforms the inverse agonist 11-cis-retinal into the agonist all-trans-retinal, leading to the active Meta II state. Crystal structures of Meta II and the active conformation of the opsin apoprotein revealed two openings of the 7-transmembrane (TM) bundle towards the hydrophobic core of the membrane, one between TM1/TM7 and one between TM5/TM6, respectively. Computational analysis revealed a putative ligand channel connecting the openings and traversing the binding pocket. Identified constrictions within the channel motivated this study of 35 rhodopsin mutants in which single amino acids lining the channel were replaced. 11-cis-retinal uptake and all-trans-retinal release were measured using UV/visible and fluorescence spectroscopy. Most mutations slow or accelerate both uptake and release, often with opposite effects. Mutations closer to the Lys296 active site show larger effects. The nucleophile hydroxylamine accelerates retinal release 80 times but the action profile of the mutants remains very similar. The data show that the mutations do not probe local channel permeability but rather affect global protein dynamics, with the focal point in the ligand pocket. We propose a model for retinal/receptor interaction in which the active receptor conformation sets the open state of the channel for 11-cis-retinal and all-trans-retinal, with positioning of the ligand at the active site as the kinetic bottleneck. Although other G protein-coupled receptors lack the covalent link to the protein, the access of ligands to their binding pocket may follow similar schemes.
Assuntos
Mutação , Rodopsina/metabolismo , Ligantes , Modelos Moleculares , Rodopsina/química , Rodopsina/genética , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/química , Rodopsina/química , Transducina/química , Simulação por Computador , Difusão , Cinética , Modelos Moleculares , Processos Fotoquímicos , Software , Gravação em VídeoRESUMO
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.
Assuntos
Arrestina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Luz , Fosforilação , Transporte Proteico/fisiologia , Retinoides/fisiologia , Rodopsina/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are ubiquitous signal transducers in cell membranes, as well as important drug targets. Interaction with extracellular agonists turns the seven transmembrane helix (7TM) scaffold of a GPCR into a catalyst for GDP and GTP exchange in heterotrimeric Galphabetagamma proteins. Activation of the model GPCR, rhodopsin, is triggered by photoisomerization of its retinal ligand. From the augmentation of biochemical and biophysical studies by recent high-resolution 3D structures, its activation intermediates can now be interpreted as the stepwise engagement of protein domains. Rearrangement of TM5-TM6 opens a crevice at the cytoplasmic side of the receptor into which the C terminus of the Galpha subunit can bind. The Galpha C-terminal helix is used as a transmission rod to the nucleotide binding site. The mechanism relies on dynamic interactions between conserved residues and could therefore be common to other GPCRs.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Rodopsina/química , Animais , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Rodopsina/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Rodopsina/agonistas , Rodopsina/química , Rodopsina/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Transducina/químicaRESUMO
In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.
Assuntos
Arrestina/química , Arrestina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/metabolismo , Visão Ocular/fisiologia , Animais , Arrestina/genética , Bovinos , Modelos Químicos , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the alpha-subunit of intracellular G proteins (Galphabetagamma). Kinetic analysis of transducin (G(t)alphabetagamma) activation shows that an intermediary R*xG(t)alphabetagamma.GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*xG protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a G(t)alpha C-terminal peptide (GalphaCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal alpha-helix of G(t)alpha (alpha5) and shows how alpha5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them--termed stable or S-interaction--matches the position of the GalphaCT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of GalphaCT and conserved E(D)RY and NPxxY(x)(5,6)F regions of the GPCR. The alternative fit--termed intermediary or I-interaction--is distinguished by a tilt (42 degrees ) and rotation (90 degrees ) of alpha5 relative to the S-interaction and shows different alpha5 contacts with the NPxxY(x)(5,6)F region and the second cytoplasmic loop of R*. From the 2 alpha5 interactions, we derive a "helix switch" mechanism for the transition of R*xG(t)alphabetagamma.GDP to the nucleotide-free R*xG protein complex that illustrates how alpha5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.
Assuntos
Modelos Químicos , Modelos Moleculares , Rodopsina/química , Transducina/química , Algoritmos , Sítios de Ligação , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Cinética , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Rodopsina/metabolismo , Transducina/metabolismoRESUMO
Besides Lys-296 in the binding pocket of opsin, all-trans-retinal forms adducts with peripheral lysine residues and phospholipids, thereby mimicking the spectral and chemical properties of metarhodopsin species. These pseudophotoproducts composed of nonspecific retinylidene Schiff bases have long plagued the investigation of rhodopsin deactivation and identification of decay products. We discovered that, while hydroxylamine can enter the retinal binding pocket of light-activated rhodopsin, the modified hydroxylamine compounds o-methylhydroxylamine (mHA), o-ethylhydroxylamine (eHA), o-tert-butylhydroxylamine (t-bHA), and o-(carboxymethyl)hydroxylamine (cmHA) are excluded. However, the alkylated hydroxylamines react quickly and efficiently with exposed retinylidene Schiff bases to form their respective retinal oximes. We further investigated how t-bHA affects light-activated rhodopsin and its interaction with binding partners. We found that both metarhodopsin II (Meta II) and Meta III are resistant to t-bHA, and neither arrestin nor transducin binding is affected by t-bHA. This discovery suggests that the hypothetical solvent channel that opens in light-activated rhodopsin is extremely stringent with regard to size and/or polarity. We believe that alkylated hydroxylamines will prove to be extremely useful reagents for the investigation of rhodopsin activation and decay mechanisms. Furthermore, the use of alkylated hydroxylamines should not be limited to in vitro studies and could help elucidate visual signal transduction mechanisms in the living cells of the retina.
Assuntos
Hidroxilaminas/química , Retinaldeído/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Bases de Schiff , Alquilação , Animais , Arrestina/metabolismo , Bovinos , Células Cultivadas , Hidroxilaminas/metabolismo , Cinética , Luz , Ligação Proteica , Retina/citologia , Retina/metabolismo , Rodopsina/efeitos da radiação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Transducina/metabolismoRESUMO
Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I â Meta IIa â Meta IIb â Meta IIbH(+). The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) is mandatory for Tyr223(5.58) to engage in the interaction with the key player Arg135(3.50) predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation.
Assuntos
Sequência Conservada , Rodopsina/agonistas , Rodopsina/química , Transducina/química , Tirosina/química , Sequência de Aminoácidos , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Retinaldeído/química , Rodopsina/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Tirosina/genéticaRESUMO
Bet3, a transport protein particle component involved in vesicular trafficking, contains a hydrophobic tunnel occupied by a fatty acid linked to cysteine 68. We reported that Bet3 has a unique self-palmitoylating activity. Here we show that mutation of arginine 67 reduced self-palmitoylation of Bet3, but the effect was compensated by increasing the pH. Thus, arginine helps to deprotonate cysteine such that it could function as a nucleophile in the acylation reaction which is supported by the structural analysis of non-acylated Bet3. Using fluorescence spectroscopy we show that long-chain acyl-CoAs bind with micromolar affinity to Bet3, whereas shorter-chain acyl-CoAs do not interact. Mutants with a deleted acylation site or a blocked tunnel bind to Pal-CoA, only the latter with slightly reduced affinity. Bet3 contains three binding sites for Pal-CoA, but their number was reduced to two in the mutant with an obstructed tunnel, indicating that Bet3 contains binding sites on its surface.
Assuntos
Proteínas de Transporte/metabolismo , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Acilação , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Proteínas de Transporte/genética , Cisteína/genética , Cisteína/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , LipoilaçãoRESUMO
G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Galphabetagamma). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Galphabetagamma. To investigate whether a G protein-coupled receptor monomer can activate Galphabetagamma, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G(t)) to determine the stoichiometry of rhodopsin/G(t) binding and the rate of catalyzed nucleotide exchange in G(t). Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G(t) was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G(t) interaction yielded a maximum of approximately 50 G(t) molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of G(t) with an activated rhodopsin monomer is sufficient for fully functional G(t) activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated.
Assuntos
Rodopsina/metabolismo , Transducina/metabolismo , Membrana Celular , Difusão , Dimerização , Proteínas de Ligação ao GTP/metabolismo , Nucleotídeos de Guanina/metabolismo , Cinética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , SoluçõesRESUMO
Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.
Assuntos
Arrestina/química , Rodopsina/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fotoquímica , Conformação ProteicaRESUMO
Multilayered samples consisting of Al, Co and Ni nanolayers were produced by MBE and characterized nondestructively by means of SRXRF, mu-XRF, WDXRF, RBS, XRR, and destructively with SIMS. The main aims were to identify the elements, to determine their purity and their sequence, and also to examine the roughness, density, homogeneity and thickness of each layer. Most of these important properties could be determined by XRF methods, e.g., on commercial devices. For the thickness, it was found that all of the results obtained via XRR, RBS, SIMS and various XRF methods (SRXRF, mu-XRF, WDXRF) agreed with each other within the limits of uncertainty, and a constant deviation from the presets used in the MBE production method was observed. Some serious preliminary discrepancies in the results from the XRF methods were examined, but all deviations could be explained by introducing various corrections into the evaluation methods and/or redetermining some fundamental parameters.
Assuntos
Alumínio/análise , Cobalto/análise , Cobre/análise , Nanotecnologia , Níquel/análise , Nanoestruturas/análise , Tamanho da Partícula , Sensibilidade e Especificidade , Espectrometria por Raios X , Propriedades de SuperfícieRESUMO
We examine the implications of a recent report providing evidence that two transducins must bind to the rod phosphodiesterase to elicit significant hydrolytic activity. To predict the rod photoreceptor's electrical response, we use numerical simulation of the two-dimensional diffusional contact of interacting molecules at the surface of the disc membrane, and then we use the simulated PDE activity as the driving function for the downstream reaction cascade. The results account for a number of aspects of rod phototransduction that have previously been puzzling. For example, they explain the existence of a greater initial delay in rods than in cones. Furthermore, our analysis suggests that the 'continuous' noise recorded in rods in darkness is likely to arise from spontaneous activation of individual molecules of PDE at a rate of a few tens per second per rod, probably as a consequence of spontaneous activation of transducins at a rate of thousands per second per rod. Hence, the dimeric activation of PDE in rods provides immunity against spontaneous transducin activation, thereby reducing the continuous noise. Our analysis also provides a coherent quantitative explanation of the amplification underlying the single photon response. Overall, numerical analysis of the dimeric activation of PDE places rod phototransduction in a new light.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Transdução de Sinal Luminoso , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Simulação por Computador , Ativação Enzimática , Humanos , Mamíferos , Transducina/metabolismoRESUMO
Isoprenylated proteins are associated with membranes and their inter-compartmental distribution is regulated by solubilization factors, which incorporate lipid moieties in hydrophobic cavities and thereby facilitate free diffusion during trafficking. Here we report the crystal structure of a solubilization factor, the prenyl-binding protein (PrBP/δ), at 1.81 Šresolution in its ligand-free apo-form. Apo-PrBP/δ harbors a preshaped, deep hydrophobic cavity, capacitating apo-PrBP/δ to readily bind its prenylated cargo. To investigate the molecular mechanism of cargo solubilization we analyzed the PrBP/δ-induced membrane dissociation of rod photoreceptor phosphodiesterase (PDE6). The results suggest that PrBP/δ exclusively interacts with the soluble fraction of PDE6. Depletion of soluble species in turn leads to dissociation of membrane-bound PDE6, as both are in equilibrium. This "solubilization by depletion" mechanism of PrBP/δ differs from the extraction of prenylated proteins by the similar folded solubilization factor RhoGDI, which interacts with membrane bound cargo via an N-terminal structural element lacking in PrBP/δ.
Assuntos
Proteínas de Transporte/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Neopreno/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Proteínas de Transporte/química , Bovinos , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Neopreno/química , Ligação Proteica , Domínios Proteicos , Prenilação de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/química , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismoRESUMO
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.
Assuntos
GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/metabolismo , Animais , Sítios de Ligação , Bovinos , Membrana Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/química , Ativação Enzimática , Hidrólise , Ligação Proteica , Transducina/químicaRESUMO
Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca(2+)-binding protein centrin 1 in a specific domain of this cilium. Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca(2+)-activated centrin 1 binds with high affinity and specificity to transducin. The assembly of centrin-G-protein complex is mediated by the betagamma-complex. The Ca(2+)-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.