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 decreasing order) are pregnenolone sulfate, progesterone, Delta5-pregnene-3beta,20alpha-diol, and 3beta-hydroxy-5alpha-pregnan-20-one. It was hypothesized that the pregnenolone-binding protein pertained to microtubule-associated proteins (MAPs). Indeed, partial purification of fetal brain cytosol by fast pressure liquid chromatography with sequential ion-exchange and gel-filtration columns yielded two fractions, one of very high molecular mass, >200 kDa, and the other of 40-60 kDa, enriched in [(3)H]pregnenolone-binding activity and in proteins immunolabeled with monoclonal anti-tubulin and anti-MAP2 antibodies. Because many proteins are associated with microtubules, binding assays were repeated with purified calf-brain tubulin, MAP2, and Tau protein. Only the MAP2 fraction showed saturable [(3)H]pregnenolone binding with an affinity very close to that of rat-brain microtubules, but with a much larger concentration of binding sites (16 pmol/mg MAP2), which was increased more than 8-fold after copolymerization of MAP2 with tubulin. Finally, steroid effects on microtubule-assembly kinetics were assayed. Pregnenolone induced a large, dose-related increase of both the rate and extent of MAP2-induced tubulin assembly, whereas progesterone, inactive per se, counteracted the stimulatory effect of pregnenolone. Electron microscopic analysis confirmed that pregnenolone-increased assembly of microtubules produced a completely normal structure. The stimulatory effect on MAP2-tubulin interaction was also observed in fetal rat-brain neuron cultures. Therefore, we propose a mechanism of neurosteroid action, the control of microtubule or, more generally, of neural cytoskeleton dynamics, with potential roles in brain development, plasticity, and aging.
Murakami
K
K
Institut National de la Santé et de la Recherche Médicale U488, Stéroides et Système Nerveux, 80 Rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France.
Fellous
A
A
Baulieu
E E
EE
Robel
P
P
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007700:Kinetics; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000648:ultrastructure*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D011284:Pregnenolone / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D008869:Microtubule-Associated Proteins; D011284:Pregnenolone