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Dual signaling is differentially activated by different active states of the metabotropic glutamate receptor 1alpha.

ABSTRACT: The metabotropic glutamate receptor 1alpha (mGluR1alpha) is crucial for some forms of synaptic plasticity, by inducing various cell responses via coupling to various types of G proteins. Upon glutamate binding, an active conformation, closed-open/active, of the extracellular domain is stabilized, which induces dimeric rearrangement in the intracellular domains, resulting in the initiation of downstream signals. We have confirmed that mGluR1alpha functionally interacts with both Gq and Gs pathways; a combination of fluorescent indicators showed that glutamate increased intracellular Ca(2+) and cAMP concentration ([Ca(2+)](i) and [cAMP](i)). By contrast, Gd(3+), a different type of ligand whose recognition site on mGluR1alpha is distinct from the glutamate site, increased only [Ca(2+)](i) and the concentration-activation curve was bell-shaped. FRET analysis revealed that a low concentration of Gd(3+) induced dimeric rearrangement of the intracellular domains of mGluR1alpha as does glutamate, whereas a high concentration of Gd(3+) reversed the FRET efficiency, which was consistent with a bell-shaped relationship between concentration and Gq activation. These results suggest that Gd(3+) induces an active and a sort of "inactivated" conformation in mGluR1alpha. The Gd(3+)-induced active state is considered to correspond to the closed-closed/active conformation, revealed by previous x-ray crystallographic studies. In conclusion, the glutamate-induced closed-open/active state coupled both to Gs and Gq proteins whereas the Gd(3+)-induced closed-closed/active conformation state preferred Gq to Gs, suggesting that mGluR1alpha serves not only as a simple on/off switch but also as a multiple signaling path regulator.

SUBMITTER: Tateyama M

PROVIDER: S-EPMC1347971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Metabotropic glutamate receptors (mGluRs) are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects--enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%), the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%), the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86%) and 8/9 (89%) for ArgusLab and 10/14 (71%) and 7/9 (78%) for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by photochemical isomerization of the retinal ligand--despite the extensive differences in sequences between mGluRs and rhodopsin.

Project description:BackgroundHypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.Methodology/principal findingsWe investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Conclusions/significanceBoth HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

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Dual signaling is differentially activated by different active states of the metabotropic glutamate receptor 1alpha.

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