The polybasic region in Gαi proteins: Relevant or not? Insights from Gαi₃ research

Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several signals, including modification by fatty acid moieties, interaction with the Gβγ complex, and, as observed in some Gα proteins, the presence of basic amino acid residues in the N-terminal region. In this work, we focused on investigating the influence of the polybasic region on the localization and function of a representative member of the Gαi family, Gαi₃. Through the use of confocal microscopy and fluorescence lifetime microscopy, we showed that, in the case of this protein, neutralizing the positive charge does not significantly affect its abundance in the cell membrane. However, it does affect its spatial arrangement concerning the dopamine D₂ receptor and influences inhibitory effect of Gαi₃ on intracellular cAMP production triggered by D₂ receptor stimulation. Moreover, in this work, we have shown, for the first time, that nonlipidated Gαi₃ binds to negatively charged lipids through electrostatic interactions, and membrane fluidity plays a significant role in this interaction.