Palmitoylation regulates GDP/GTP exchange of G protein by affecting the GTP-binding activity of Goα

The effect of palmitoylation on the GTP-binding activity and conformation of Goα protein in hydrophobic and hydrophilic environments was studied. The binding assay was performed with an isotope labeled analog of GTP, GTP-γ-35S, and its fluorescent analog, BODIPY FL-GTPγS was used to detect conformational change in the GTP-binding domain of Goα. Investigation of the GTP-γ-35S binding activity of Goα shows that in a hydrophobic environment, mimicked by the presence of detergent, the apparent dissociation constant for palmitoylated Goα (KD = 25.5 × 10−9 ± 1.7 × 10−9 M) increased threefold compared with that of non-palmitoylated Goα (KD = 9.9 × 10−9 ± 0.8 × 10−9 M), while in an aqueous environment without detergent there is no significant difference between palmitoylated (KD = 50.1 × 10−9 ± 5.2 × 10−9 M) and non-palmitoylated (KD = 65.5 × 10−9 ± 7.6 × 10−9 M) Go(. This indicates that in a membrane environment palmitoylation may weaken the GTPγS binding ability of Go(. Fluorescent quenching studies using BODIPY FL-GTPγS as a probe showed that the conformation of the GTP-binding domain of Go( tends to become more compact after palmitoylation. These results imply that palmitoylation may regulate the GTP/GDP exchange of Goα by influencing the GTP-binding activity of Goα and facilitating the on-off switch function of the G protein in G protein-coupled signal transduction.