DYNAMIN GTPASE IS STIMULATED BY CROSS-LINKING THROUGH THE C-TERMINAL PROLINE-RICH DOMAIN

Dynamin is a 100 kDa GTPase required for endocytic-coated vesicle form ation. Recombinant human neuronal dynamin (dynamin-1) was used for mon oclonal antibody (mAb) production. Two mAbs, designated hudy-2 (for hu man dynamin) and hudy-4, were chosen for further study based on their differential ability to recognize dynamin-1 and its non-neuronal isofo rm, dynamin-2. Both bind to the proline-rich C-terminal domain (PRD) o f dynamin and inhibit the ability of microtubules and grb2 to stimulat e GTPase activity. Hudy-4 binds to an epitope within the last 20 amino acids of dynamin-1 and has no effect on its intrinsic GTPase activity . Hudy-2 binds to an epitope within amino acids 822-838 that is common to dynamin-1 and dynamin-2. Hudy-2 stimulates dynamin's intrinsic GTP ase activity in a manner proportional to the valency of the immunoglob ulin (Ig) G. Crosslinking IgGs with secondary antibodies caused a 2-fo ld increase in GTPase activity, while F(ab)s were inactive. Importantl y, our findings suggest that the stimulation of dynamin GTPase activit y by multivalent proteins which bind in vitro to the PRD may not be a valid criterion on its own for assessing the in vivo functional signif icance of these interactions.