title: Evaluation of a novel photoactive and biotinylated dehydroepiandrosterone analog.
abstract: 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.
authors.affiliation: Research Service, Department of Veterans Affairs Medical Center, Iowa City, IA, USA.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Liu
authors.forename: Brianne
authors.initials: B
authors.lastname: O'Leary
authors.forename: Mary
authors.initials: M
authors.lastname: Iruthayanathan
authors.forename: Laurie
authors.initials: L
authors.lastname: Love-Homan
authors.forename: Nury
authors.initials: N
authors.lastname: Perez-Hernandez
authors.forename: Horacio F
authors.initials: HF
authors.lastname: Olivo
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Dillon
mesh_terms: 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
chemical_list: D001577:Benzophenones; D019920:Photoaffinity Labels; D017319:Photosensitizing Agents; D003687:Dehydroepiandrosterone; D001710:Biotin; C047723:benzophenone; D000738:Androsterone