The Importance of N-terminal polycysteine and polybasic sequences for G14αand G16α palmitoylation, plasma membrane localization, and signaling function

Abstract

Plasma membrane targeting of G protein α(Gα) subunits is essential for competent receptor-to-G protein signaling. Many Gα are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that Gq family members (Gqα, G11α, G14α, and G 16α) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G14α and G16α. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G14α and G16α. We find that G 14α and G16α are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G 16α-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G14α and G16α expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G16α, more so than G14α, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G14α and G16α are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Gα palmitoylation but is important for localization and functional activity of heterotrimeric G proteins. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.