Beyond the G protein α subunit: investigating the functional impact of other components of the Gαi3 heterotrimers.

BACKGROUND: Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gß and Gγ subunits and the effects of different Gßγ dimer compositions on GPCR signaling are poorly understood. In this study, we aimed to investigate the subcellular localization and functional response of Gαi3-based heterotrimers with different combinations of Gß and Gγ subunits. METHODS: Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi3 in combination with Gß1 or Gß2 heterotrimers, along with representative Gγ subunits. Furthermore, fluorescence lifetime imaging microscopy (FLIM-FRET) was used to investigate the nanoscale distribution of Gαi3-based heterotrimers in the plasma membrane, specifically with the dopamine D2 receptor (D2R). In addition, the functional response of the system was assessed by monitoring intracellular cAMP levels and conducting bioinformatics analysis to further characterize the heterotrimer complexes. RESULTS: Our results show that Gαi3 heterotrimers mainly localize to the plasma membrane, although the degree of colocalization is influenced by the accompanying Gß and Gγ subunits. Heterotrimers containing Gß2 showed slightly lower membrane localization compared to those containing Gß1, but certain combinations, such as Gαi3ß2γ8 and Gαi3ß2γ10, deviated from this trend. Examination of the spatial arrangement of Gαi3 in relation to D2R and of changes in intracellular cAMP level showed that the strongest functional response is observed for those trimers for which the distance between the receptor and the Gα subunit is smallest, i.e. complexes containing Gß1 and Gγ8 or Gγ10 subunit. Deprivation of Gαi3 lipid modifications resulted in a significant decrease in the amount of protein present in the cell membrane, but did not always affect intracellular cAMP levels. CONCLUSION: Our studies show that the composition of G protein heterotrimers has a significant impact on the strength and specificity of GPCR-mediated signaling. Different heterotrimers may exhibit different conformations, which further affects the interactions of heterotrimers and GPCRs, as well as their interactions with membrane lipids. This study contributes to the understanding of the complex signaling mechanisms underlying GPCR-G-protein interactions and highlights the importance of the diversity of Gß and Gγ subunits in G-protein signaling pathways. Video Abstract.

On the mechanism of GIRK2 channel gating by phosphatidylinositol bisphosphate, sodium, and the Gßγ dimer.

G protein-gated inwardly rectifying K+ (GIRK) channels belong to the inward-rectifier K+ (Kir) family, are abundantly expressed in the heart and the brain, and require that phosphatidylinositol bisphosphate is present so that intracellular channel-gating regulators such as Gßγ and Na+ ions can maintain the channel-open state. However, despite high-resolution structures (GIRK2) and a large number of functional studies, we do not have a coherent picture of how Gßγ and Na+ ions control gating of GIRK2 channels. Here, we utilized computational modeling and all-atom microsecond-scale molecular dynamics simulations to determine which gates are controlled by Na+ and Gßγ and how each regulator uses the channel domain movements to control gate transitions. We found that Na+ ions control the cytosolic gate of the channel through an anti-clockwise rotation, whereas Gßγ stabilizes the transmembrane gate in the open state through a rocking movement of the cytosolic domain. Both effects alter the way in which the channel interacts with phosphatidylinositol bisphosphate and thereby stabilizes the open states of the respective gates. These studies of GIRK channel dynamics present for the first time a comprehensive structural model that is consistent with the great body of literature on GIRK channel function.