G protein-associated, specific membrane binding sites mediate the neuroprotective effect of dehydroepiandrosterone.
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.
Charalampopoulos
Ioannis
I
Department of Pharmacology, School of Medicine, University of Crete, Heraklion, Greece.
Alexaki
Vassilia-Ismini
VI
Lazaridis
Iakovos
I
Dermitzaki
Erene
E
Avlonitis
Nicolaos
N
Tsatsanis
Christos
C
Calogeropoulou
Theodora
T
Margioris
Andrew N
AN
Castanas
Elias
E
Gravanis
Achille
A
D000313:Adrenal Medulla / Q000166:cytology; D000728:Androgens / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003687:Dehydroepiandrosterone / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004967:Estrogens / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D018696:Neuroprotective Agents / Q000378:metabolism / Q000494:pharmacology*; D016716:PC12 Cells / Q000187:drug effects; D037342:Pertussis Toxin / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects* / Q000378:metabolism; D011963:Receptors, GABA-A / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D011987:Receptors, Steroid / Q000187:drug effects* / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D051020:bcl-X Protein / Q000502:physiology
D000728:Androgens; D016895:Culture Media, Serum-Free; D004967:Estrogens; D005938:Glucocorticoids; D018696:Neuroprotective Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D043562:Receptors, G-Protein-Coupled; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate; D011987:Receptors, Steroid; D051020:bcl-X Protein; C095972:dehydroepiandrosterone receptor; C509625:dehydroepiandrosterone-bovine serum albumin; D012710:Serum Albumin, Bovine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D003687:Dehydroepiandrosterone; D037342:Pertussis Toxin