Evaluation of a novel photoactive and biotinylated dehydroepiandrosterone analog.
To characterize the cell surface receptor for dehydroepiandrosterone (DHEA), we synthesized a DHEA analog containing biotin and benzophenone groups (DHEA-BP-Bt). DHEA-BP-Bt was equipotent with DHEA in competing with [(3)H]DHEA for binding to solubilized plasma membranes of bovine aortic endothelial cells (BAEC). Additionally, DHEA-BP-Bt pre-conjugated to avidin and immobilized on agarose, also inhibited plasma membrane binding of [(3)H]DHEA. Furthermore, DHEA-BP-Bt activated endothelial nitric oxide synthase, similar to DHEA. Confocal micrographs showed that, upon photoirradiation, DHEA-BP-Bt bound to sites on the cell surface of BAEC in a DHEA inhibitable manner. Finally, DHEA-BP-Bt bound specifically to proteins of approximately 55 kDa and 80 kDa, either when live cells were UV irradiated with the analog and plasma membrane proteins separated by SDS-PAGE or in a ligand blot analysis. These data confirm the successful synthesis of a photoactive, biotinylated DHEA analog which is capable of cross-linking to and identifying plasma membrane DHEA binding sites and which will allow us to further purify this receptor.
Liu
Dongmin
D
Research Service, Department of Veterans Affairs Medical Center, Iowa City, IA, USA.
O'Leary
Brianne
B
Iruthayanathan
Mary
M
Love-Homan
Laurie
L
Perez-Hernandez
Nury
N
Olivo
Horacio F
HF
Dillon
Joseph S
JS
D000738:Androsterone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019921:Biotinylation; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003687:Dehydroepiandrosterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D018613:Microscopy, Confocal; D008954:Models, Biological; D019920:Photoaffinity Labels / Q000378:metabolism / Q000494:pharmacology*; D017319:Photosensitizing Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
D001577:Benzophenones; D019920:Photoaffinity Labels; D017319:Photosensitizing Agents; D003687:Dehydroepiandrosterone; D001710:Biotin; C047723:benzophenone; D000738:Androsterone