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

Abstract

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 Gβ and Gγ subunits and the effects of different Gβγ 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 Gβ and Gγ subunits.

Methods: Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi₃ in combination with Gβ₁ or Gβ₂ 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 Gβ and Gγ subunits. Heterotrimers containing Gβ₂ showed slightly lower membrane localization compared to those containing Gβ₁, but certain combinations, such as Gαi₃β₂γ₈ and Gαi₃β₂γ₁₀, 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 Gβ₁ 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 Gβ and Gγ subunits in G-protein signaling pathways. Video Abstract.

Keywords: Docking simulations; Dopamine D2 receptor; FRET; GPCR; Gαi3 subunit; Gβγ dimer; Heterotrimeric G proteins; Intracellular cAMP levels; Live-cell imaging; Subcellular localization.