Structure of the visual signaling complex between transducin and phosphodiesterase 6.
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ABSTRACT:
SUBMITTER: Gao Y
PROVIDER: S-EPMC8208777 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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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.
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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 .
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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.
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