Palmitoylation regulates GDP/GTP exchange of G protein by affecting the GTP-binding activity of Goalpha.
The effect of palmitoylation on the GTP-binding activity and conformation of Goalpha protein in hydrophobic and hydrophilic environments was studied. The binding assay was performed with an isotope labeled analog of GTP, GTP-gamma-35S, and its fluorescent analog, BODIPY FL-GTPgammaS was used to detect conformational change in the GTP-binding domain of Goalpha. Investigation of the GTP-gamma-35S binding activity of Goalpha shows that in a hydrophobic environment, mimicked by the presence of detergent, the apparent dissociation constant for palmitoylated Goalpha (K(D)=25.5x10(-9)+/-1.7x10(-9)M) increased threefold compared with that of non-palmitoylated Goalpha (K(D)=9.9x10(-9)+/-0.8x10(-9)M), while in an aqueous environment without detergent there is no significant difference between palmitoylated (K(D)=50.1 x 10(-9)+/-5.2x10(-9)M) and non-palmitoylated (K(D)=65.5x10(-9)+/-7.6x10(-9)M) Go(. This indicates that in a membrane environment palmitoylation may weaken the GTPgammaS binding ability of Go(. Fluorescent quenching studies using BODIPY FL-GTPgammaS 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 Goalpha by influencing the GTP-binding activity of Goalpha and facilitating the on-off switch function of the G protein in G protein-coupled signal transduction.
Cao
Yu
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China.
Huang
Youguo
Y
D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D019308:Palmitic Acid / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013462:Sulfur Radioisotopes / Q000378:metabolism; D014867:Water / Q000737:chemistry
D005456:Fluorescent Dyes; D013462:Sulfur Radioisotopes; D014867:Water; D006153:Guanosine Diphosphate; D019308:Palmitic Acid; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go