N-terminus of MAP2C as a neurosteroid-binding site

The kinetics of neurosteroid binding to recombinant human microtubule-associated protein 2C (rhMAP2C) and neurosteroid regulation of MAP2C-stimulated tubulin assembly were studied. In a quartz crystal microbalance assay, progesterone-BSA at 1-10 nM showed concentration-dependent binding to rhMAP2C, and this binding was competitively inhibited by pregnenolone or progesterone. However, no progesterone-BSA binding to N-terminal 71 amino acid residues rhMAP2C was found. In an rhMAP2C-stimulated tubulin assembly assay, pregnenolone enhanced the assembly of an rhMAP2C-progesterone-BSA complex in a progesterone-reversible manner, progesterone alone had no effect. Although N-terminal 71 amino acid residues rhMAP2C retains an activity to stimulate this assembly, this effect was not affected by pregnenolone or progesterone. These findings suggest that neurosteroids specifically bind to the N-terminus of rhMAP2 and regulate tubulin assembly.