SYNERGISTIC ACTIVATION OF DYNAMIN GTPASE BY GRB2 AND PHOSPHOINOSITIDES

Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microt ubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich do main (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoin ositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by -sn-glycerol-3-be nzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinos itol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bis phosphate did not activate GTPase at all. We then addressed the questi on of whether both domains of dynamin, PRD and PH, can be engaged simu ltaneously, and determined the effects of dual occupancy on dynamin GT Pase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphos phate together increased the dynamin GTPase activity up to 4-fold high er than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to d ynamin simultaneously resulting in superactivation of dynamin GTPase a ctivity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.