Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly

Fetal or adult rat-brain cytosol and fetal rat-brain microtubules contain a high-affinity, low-capacity pregnenolone-binding protein. The equilibrium dissociation constant is in the 30-50 nM range. The best competitors (in de creasing order) are pregnenolone sulfate, progesterone, Delta 5-pregnene-3 beta,20 alpha-diol, and 3 beta-hydroxy-5 alpha-pregnan-20-one. It was hypot hesized that the pregnenolone-binding protein pertained to microtubule-asso ciated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and ge l-filtration columns yielded two fractions, one of very high molecular mass , >200 kDa, and the other of 40-60 kDa, enriched in [H-3]pregnenolone-bindi ng activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubule s, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [H-3]pregnenolone bin ding with an affinity very close to that of rat-brain microtubules, but wit h a much larger concentration of binding sites (16 pmol/mg MBP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin, Fi nally, steroid effects on microtubule-assembly kinetics were assayed. Pregn enolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, co unteracted the stimulatory effect of pregnenolone. Electron microscopic ana lysis confirmed that pregnenolone-increased assembly of microtubules produc ed a completely normal structure. The stimulatory effect on MAP2-tubulin in teraction was also observed in fetal rat-brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule o r, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging.