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Folding of G? Subunits: Implications for Disease States.

ABSTRACT: G-proteins play a central role in signal transduction by fluctuating between "on" and "off" phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated Gi?1 or Gs? subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of Gi?1 and Gs?. For all proteins studied, we found that the active conformations were more stable than the inactive conformations, and upon refolding from higher temperatures, activated wild-type subunits recovered significantly more native structure. We also observed that the wild-type subunits partially regained the ability to bind nucleotide. The increased compactness observed upon activation was consistent with the calculated decrease in solvent accessible surface area for wild-type Gi?1. We found that as the temperature increased, G? subunits, which are known to be rich in ?-helices, converted to proteins with increased content of ?-sheets and random coil. For active conformations from wild-type and tryptophan mutants of Gi?1, melting temperatures indicated that denaturation starts around hydrophobic tryptophan microenvironments and then radiates toward tyrosine residues at the surface, followed by alteration of the secondary structure. For Gs?, however, disruption of secondary structure preceded unfolding around tyrosine residues. In the active conformations, a ?-cation interaction between essential arginine and tryptophan residues, which was characterized by a fluorescence-measured red shift and modeled by molecular dynamics, was also shown to be a contributor to the stability of G? subunits. The folding properties of G? subunits reported here are discussed in the context of diseases associated to G-proteins.

SUBMITTER: Najor M

PROVIDER: S-EPMC6210069 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Folding of Gα Subunits: Implications for Disease States.

Najor Matthew M Leverson Brian D BD Goossens Jesse L JL Kothawala Saad S Olsen Kenneth W KW Mota de Freitas Duarte D

ACS omega 20181001 10

G-proteins play a central role in signal transduction by fluctuating between "on" and "off" phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated Giα1 or Gsα subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of Giα1 and G< ...[more]

PMID: 30411001

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Project description:GABAergic parvalbumin-expressing (PV+) interneurons provide powerful inhibitory modulation of grid cells in layer II of the medial entorhinal cortex (MEC LII). However, the molecular machinery through which PV+ cells regulate grid cell activity is poorly defined. PV+ interneurons impart inhibitory modulation primarily via GABA-A receptors (GABAARs). GABAARs are pentameric ion channels assembled from a repertoire of 19 subunits. Multiple subunit combinations result in a variety of receptor subtypes mediating functionally diverse postsynaptic inhibitory currents. Whilst the broad expression patterns of GABAAR subunits within the EC have been reported, those expressed by individual MEC LII cell types, in particular grid cells candidates, stellate and pyramidal cells, are less well described. Stellate and pyramidal cells are distinguished by their selective expression of reelin (RE+) and calbindin (CB+) respectively. Thus, the overall aim of this study was to provide a high resolution analysis of the major (α and γ) GABAAR subunits expressed in proximity to somato-dendritic PV+ boutons, on RE+ and CB+ cells, using immunohistochemistry, confocal microscopy and quantitative RT-PCR (qPCR). Clusters immunoreactive for the α1 and γ2 subunits decorated the somatic membranes of both RE+ and CB+ cells and were predominantly located in apposition to clusters immunoreactive for PV and vesicular GABA transporter (VGAT), suggesting expression in GABAergic synapses innervated by PV interneurons. Although intense α2 subunit-immunopositive clusters were evident in hippocampal fields located in close proximity to the EC, no specific signal was detected in MEC LII RE+ and CB+ profiles. Immunoreactivity for the α3 subunit was detected in all RE+ somata. In contrast, only a sub-population of CB+ cells was α3 immunopositive. These included CB-α3 cells which were both PV+ and PV-. Furthermore, α3 subunit mRNA and immunofluorescence decreased significantly between P 15 and P 25, a period implicated in the functional maturation of grid cells. Finally, α5 subunit immunoreactivity was detectable only on CB+ cells, not on RE+ cells. The present data demonstrates that physiologically distinct GABAAR subtypes are selectively expressed by CB+ and RE+ cells. This suggests that PV+ interneurons could utilize distinct postsynaptic signaling mechanisms to regulate the excitability of these different, candidate grid cell sub-populations.

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Folding of G? Subunits: Implications for Disease States.

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Folding of G<sub>α</sub> Subunits: Implications for Disease States.

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