Structure of the visual signaling complex between transducin and phosphodiesterase 6.
Ontology highlight
ABSTRACT:
SUBMITTER: Gao Y
PROVIDER: S-EPMC8208777 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is composed of the GTP-bound transducin α subunit (GαT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis, leading to hyperpolarization of the photoreceptor cell. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GαT. The structure reveals two GαT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEγ subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GαT·GTP subunits and PDE6 that supports an alternating-site catalytic mechanism.
| S-EPMC7597677 | biostudies-literature
Project description:Phosphodiesterase 10A (PDE10A) is a dual substrate PDE that can hydrolyze both cGMP and cAMP. In brain, PDE10A is almost exclusively expressed in the striatum. In several studies, PDE10A has been implicated in regulation of striatal output using either specific inhibitors or PDE10A knock-out mice and has been suggested as a promising target for novel antipsychotic drugs. In striatal medium spiny neurons, PDE10A is localized at the plasma membrane and in dendritic spines close to postsynaptic densities. In the present study, we identify PDE10A as the major cAMP PDE in mouse striatum and monitor PKA-dependent PDE10A phosphorylation. With recombinantly expressed PDE10A we demonstrate that phosphorylation does not alter PDE10A activity. In striatum, PDE10A was found to be associated with the A kinase anchoring protein AKAP150 suggesting the existence of a multiprotein signaling complex localizing PDE10A to a specific functional context at synaptic membranes. Furthermore, the cAMP effector PKA, the NMDA receptor subunits NR2A and -B, as well as PSD95, were tethered to the complex. In agreement, PDE10A was almost exclusively found in multiprotein complexes as indicated by migration in high molecular weight fractions in size exclusion chromatography. Finally, affinity of PDE10A to the signaling complexes formed around AKAP150 was reduced by PDE10A phosphorylation. The data indicate that phosphorylation of PDE10 has an impact on the interaction with other signaling proteins and adds an additional line of complexity to the role of PDE10 in regulation of synaptic transmission.
| S-EPMC4424333 | biostudies-literature
Project description:Uncoordinated 119 protein (UNC119) is a partner of transducin-? subunit (G?t ) that is essential for transducin trafficking in rod photoreceptors. The interaction is known to involve binding of the acylated N terminus of G?t to the hydrophobic pocket of UNC119. To gain insights into the mechanism of transducin trafficking, we isolated a highly pure protein complex between myristoylated chimeric G?t (G?t *) and UNC119??????, and examined the solution structure by small angle X-ray scattering and chemical crosslinking. The solution structure of the G?t -UNC119?????? complex was derived with rigid body/ab initio modeling against the small angle X-ray scattering data by use of known atomic structures of G?t and UNC119, and a distance constraint based on the protein crosslinking with p-phenyldimaleimide. The model of the G?t -UNC119?????? complex indicates rotation and bending of the N-terminal ?-helix of G?t from its position in the structure of the heterotrimeric G-protein transducin (Gt ). This allows a considerably more compact complex conformation, which also suggests a novel interface involving the switch II/?3-?5 surface of G?t . Supporting a novel interface, UNC119 was found to bind full-length G?t * more strongly than the G?t N-terminal peptide. Furthermore, UNC119 competed with the effector molecule phosphodiesterase-6 ?-subunit, which is known to bind to the same surface of G?t . The solution structure of the G?t -UNC119 complex suggests that the ability of UNC119 to dissociate Gt subunits and release G?t from the membrane is attributable to disruption and sterical occlusion of the G????-binding sites on G?t .
| S-EPMC4310827 | biostudies-literature
Project description:Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho(?), which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1mM n-dodecyl-?-d-maltoside (DDM) and Rho monomers in 5mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho(?)·Gt and sConA-Rho(?)·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho(?)·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho(?)·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho(?)·Gt complex in which the photoactivated Rho(?) dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho(?) dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal.
| S-EPMC3633645 | biostudies-literature
Project description:Activation of cGMP phosphodiesterase (PDE) by activated transducin α subunit (Tα*) is a necessary step to generate a light response in vertebrate photoreceptors. PDE in rods is a heterotetramer composed of two catalytic subunits, PDEα and PDEβ, and two inhibitory PDEγ subunits, each binding to PDEα or PDEβ. Activation of PDE is achieved by relief of the inhibitory constraint of PDEγ on the catalytic subunit. In this activation mechanism, it is widely believed that Tα* binds to PDEγ still bound to the catalytic subunit, and removes or displaces PDEγ from the catalytic subunit. However, recent structural analysis showed that the binding of Tα* to PDEγ still bound to PDEα or PDEβ seems to be difficult because the binding site of PDEγ to PDEα or PDEβ overlaps with the binding site to Tα*. In the present study, we propose a novel activation mechanism of PDE, the trapping mechanism, in which Tα* activates PDE by trapping PDEγ released reversibly and spontaneously from the catalytic subunit. This mechanism well explains PDE activation by Tα* in solution. Our further analysis with this mechanism suggests that more effective PDE activation in disk membranes is highly dependent on the membrane environment.
| S-EPMC6510727 | biostudies-literature
Project description:The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho(?) form. Rho(?) binds the heterotrimeric G protein, transducin (G(t)) inducing GDP to GTP exchange and G(t) dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho(?)·G(t) complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho(?) to G(t) in the complex and its mass determined by scanning transmission electron microscopy was 221±12kDa. A 21.6Å structure was calculated from projections of negatively stained Rho(?)·G(t) complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G(t) heterotrimer, corroborating the heteropentameric structure of the Rho(?)·G(t) complex.
| S-EPMC3210324 | biostudies-literature
Project description:Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein alpha subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein alpha subunit and the structure of the GPCR-G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR-G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein-GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR-G-protein complexes is a prerequisite for structural studies designed to address these possibilities.
| S-EPMC2875673 | biostudies-literature
Project description:Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its ? subunit (G?T). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the G?T helical domain (?HD) contacting the G?? subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by G?? during receptor-catalyzed nucleotide exchange.
| S-EPMC6707884 | biostudies-literature
Project description:The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.
| S-EPMC1149622 | biostudies-other
Project description:Conventional functional connectivity analysis using functional magnetic resonance imaging (fMRI) measures the correlation of temporally synchronized brain activities between brain regions. Lag structure analysis relaxes the synchronicity constraint of fMRI signals, and thus, this approach might be better at explaining functional connectivity. However, the sources of the lag structure in fMRI are primarily unknown. Here, we applied lag structure analysis to the human visual cortex to identify the possible sources of lag structure. A total of 1,250 fMRI data from two independent databases were considered. We explored the temporal lag patterns between the central and peripheral visual fields in early visual cortex and those in two visual pathways of dorsal and ventral streams. We also compared the lag patterns with effective connectivity obtained with dynamic causal modeling. We found that the lag structure in early visual cortex flows from the central to peripheral visual fields and the order of the lag structure flow was consistent with the order of signal flows in visual pathways. The effective connectivity computed by dynamic causal modeling exhibited similar patterns with the lag structure results. This study suggests that signal flows in visual streams are possible sources of the lag structure in human visual cortex.
| S-EPMC6414616 | biostudies-literature