FASEB J. 2006 Mar;20(3):577-9. Epub 2006 Jan 11.
G protein-associated, specific membrane binding sites mediate the
neuroprotective effect of dehydroepiandrosterone.
Charalampopoulos I, Alexaki VI, Lazaridis I, Dermitzaki E, Avlonitis N,
Tsatsanis C, Calogeropoulou T, Margioris AN, Castanas E, Gravanis A.
Department of Pharmacology, School of Medicine, University of Crete,
The neurosteroid dehydroepiandrosterone (DHEA) at 1 nM protects
NMDA-/GABAA-receptor negative neural crest-derived PC12 cells from
apoptosis. We now report that membrane-impermeable DHEA-BSA conjugate
replaces unconjugated DHEA in protecting serum-deprived PC12 cells from
apoptosis (IC50=1.5 nM). Protection involves phosphorylation of the
prosurvival factor Src and induction of the anti-apoptotic protein Bcl-2
and is sensitive to pertussis toxin. Binding assays of [3H]DHEA on isolated
PC12 cell membranes revealed saturation within 30 min and binding of DHEA
with a Kd of 0.9 nM. A similar binding activity was detectable in isolated
membranes from rat hippocampus and from normal human adrenal chromaffin
cells. The presence of DHEA-specific membrane binding sites was confirmed
by flow cytometry and confocal laser microscopy of DHEA-BSA-FITC stained
cells. In contrast to estrogens and progestins, glucocorticoids and
androgens displaced DHEA from its membrane binding sites but with a 10-fold
lower affinity than DHEA (IC50=9.3 and 13.6 nM, respectively). These agents
acted as pure antagonists, blocking the antiapoptotic effect of DHEA as
well as the induction of Bcl-2 proteins and Src kinase activation. In
conclusion, our findings suggest that neural crest-derived cells possess
specific DHEA membrane binding sites coupled to G proteins. Binding to
these sites confers neuroprotection.