Role of the C-terminal basic amino acids and the lipid anchor of the Gγ2 protein in membrane interactions and cell localization

Heterotrimeric G proteins are peripheral membrane proteins that frequently localize to the plasma membrane where their presence in molar excess over G protein coupled receptors permits signal amplification. Their distribution is regulated by protein-lipid interactions, which has a clear influence on their activity. Gβγ dimer drives the interaction between G protein heterotrimers with cell membranes. We focused our study on the role of the C-terminal region of the Gγ2 protein in G protein interactions with cell membranes. The Gγ2 subunit is modified at cysteine (Cys) 68 by the addition of an isoprenyl lipid, which is followed by the proteolytic removal of the last three residues that leaves an isoprenylated and carboxyl methylated Cys-68 as the terminal amino acid. The role of Cys isoprenylation of the CAAX box has been defined for other proteins, yet the importance of proteolysis and carboxyl methylation of isoprenylated proteins is less clear. Here, we showed that not only geranylgeranylation but also proteolysis and carboxyl methylation are essential for the correct localization of Gγ2 in the plasma membrane. Moreover, we showed the importance of electrostatic interactions between the inner leaflet of the plasma membrane and the positively charged C-terminal domain of the Gγ2 subunit (amino acids Arg-62, Lys-64 and Lys-65) as a second signal to reach the plasma membrane. Indeed, single or multiple point mutations at Gγ2 C-terminal amino acids have a significant effect on Gγ2 protein-plasma membrane interactions and its localization to charged Ld (liquid disordered) membrane microdomains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.