Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP
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Transducin
PDE10A
Phosphodiesterase 3
Transducin
Visual phototransduction
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Transducin
Visual phototransduction
Arrestin
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Obtaining a reliable 3D model for the complex formed by photoactivated rhodopsin (R*) and its G-protein, transducin (Gtalphabetagamma), would significantly benefit the entire field of structural biology of G-protein-coupled receptors (GPCRs). In this study, we have performed extensive configurational sampling for the isolated C-terminal fragment of the alpha-subunit of transducin, Gtalpha 340-350, within cavities of photoactivated rhodopsin formed by different energetically feasible conformations of the intracellular loops. Our results suggested a new 3D model of the rhodopsin-transducin complex that fully satisfied all available experimental data on site-directed mutagenesis of rhodopsin and Gtalphabetagamma as well as data from disulfide-linking experiments. Importantly, the experimental data were not used as a priori constraints in model building. We performed a thorough comparison of existing computational models of the rhodopsin-transducin complex with each other and with current experimental data. It was found that different models suggest interactions with different molecules in the rhodopsin oligomer, that providing valuable guidance in design of specific novel experimental studies of the R*-Gtalphabetagamma complex. Finally, we demonstrated that the isolated Gtalpha 340-350 fragment does not necessarily bind rhodopsin in the same binding mode as the same segment in intact Gtalpha.
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Arrestin
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Transducin
Arrestin
Visual phototransduction
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A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transduction lowers the cGMP binding affinity for noncatalytic sites on PDE and accelerates the dissociation of cGMP from these sites. These sites can exist in three states: high affinity (Kd = 60 nM) for the nonactivated PDE, intermediate affinity (Kd approximately 180 nM) when the enzyme is activated in a complex with transducin, and low affinity (Kd > 1 microM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by transducin causes a 10-fold increase in the rate of cGMP dissociation from one of the two noncatalytic sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP dissociation rate from both sites > 50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of light-adapted photoreceptors.
Transducin
PDE10A
Phosphodiesterase 3
Transduction (biophysics)
Visual phototransduction
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Transducin
Visual phototransduction
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Abstract Rhodopsin, upon activation by light, transduces the photon signal by activation of the G‐protein, transducin. The well‐studied rhodopsin/transducin system serves as a model for the understanding of signal transduction by the large class of G‐protein‐coupled receptors. The interactive form of rhodopsin, R*, is conformationally similar or identical to rhodopsin's photolysis intermediate Metarhodopsin II (MII). Formation of MII requires deprotonation of rhodopsin's protonated Schiff base which appears to facilitate some opening of the rhodopsin structure. This allows a change in conformation at rhodopsin's cytoplasmic surface that provides binding sites for transducin. Rhodopsin's 2nd, 3rd and putative 4th cytoplasmic loops bind transducin at sites including transducin's 5 kDa carboxyl‐terminal region. Site‐specific mutagenesis of rhodopsin is being used to distinguish sites on rhodopsin's surface that are important in binding transducin from those that function in activating transducin. These observations are consistent with and extend studies on the action of other G‐protein‐coupled receptors and their interactions with their respective G proteins.
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Transducin
Visual phototransduction
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The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtα and Gtβγ subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtγ(60−71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II [Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367−373]. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin−Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtγ C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtγ for alanine. The mutants were co-expressed with six-histidine-tagged Gtβ subunits in Sf9 insect cells. The Gtβ-6-His-γ mutant proteins were purified and assayed in the presence of Gtα for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*−Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtγ(60−71) region has the most significant effect on rhodopsin−Gt interactions, which places important constraints on the model of the rhodopsin−Gt complex.
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Alanine
C-terminus
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