TY - JOUR
T1 - Evaluation of a novel photoactive and biotinylated dehydroepiandrosterone analog
AU - Liu, Dongmin
AU - O'Leary, Brianne
AU - Iruthayanathan, Mary
AU - Love-Homan, Laurie
AU - Perez-Hernandez, Nury
AU - Olivo, Horacio F.
AU - Dillon, Joseph S.
N1 - Funding Information:
This material is based upon work supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development Program . The contents do not represent the views of the Department of Veterans Affairs or the United States Government. The studies were also supported by grants from the American Heart Association and the NIH ( AG018928 ). We thank Dr. Rebeca Lopez-Marure for critical reading of the manuscript.
PY - 2010/10
Y1 - 2010/10
N2 - 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 [3H]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 [3H]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 55kDa and 80kDa, 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.
AB - 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 [3H]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 [3H]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 55kDa and 80kDa, 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.
KW - Benzophenone
KW - Biotin
KW - DHEA
KW - Nitric oxide
KW - Non-genomic
UR - http://www.scopus.com/inward/record.url?scp=77956338888&partnerID=8YFLogxK
U2 - 10.1016/j.mce.2010.07.002
DO - 10.1016/j.mce.2010.07.002
M3 - Artículo
SN - 0303-7207
VL - 328
SP - 56
EP - 62
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
IS - 1-2
ER -