Use of estradiol-BSA conjugates to characterize membrane estrogen binding sites in brain cells: Biochemical and physiological studies

Rapid nongenomic actions of estrogen probably involve one or several membrane estrogen receptors/binding proteins, which are different from classical estrogen receptor (ER). Specific membrane binding sites for estrogen (mEBS) in female rat brain were demonstrated by a novel and sensitive radioligand binding assay using 17β-estradiol covalently linked with ($\sp{125}$I) -labeled bovine serum albumin (17β-E-6- ($\sp{125}$I) BSA conjugate), as a ligand. These membrane binding sites (dissociation constants of 3-34 nM and total binding sites of 3-40 pmol/mg protein) in crude synaptosomal fractions (P2) from hypothalamus, olfactory bulb, and cerebellum show distinct binding specificity from classical ER. To establish if the mEBS identified by 17β-E-6- ($\sp{125}$I) BSA represent putative membrane estrogen binding proteins, a ligand blotting technique was used. Three major binding proteins with molecular weights of about 23, 28, and 32 kDa, and three additional proteins (18, 40, and 130 kDa), were specifically labeled. The 23 and 40 kDa proteins were concentrated in mitochondrial fractions (mP2) whereas 18, 28, and 32 kDa proteins were enriched in microsomal fractions (P3). One of these membrane estrogen binding proteins (23 kDa) was purified from digitonin-solubilized P2 fractions by affinity columns coupled with 17β-E-6-BSA and was identified as the oligomycin-sensitivity conferring protein (OSCP), a subunit of the F$\sb0$F$\sb1$ ATP-synthase/ATPase required for the coupling of a proton gradient across the F$\sb0$ sector of the mitochondrial membrane to ATP synthesis in the F$\sb1$ sector. OSCP, therefore, represents an estrogen target site in addition to ER in the cells and may play an important role in estrogen-induced changes in cell energy metabolism. The unlabeled ligand, 17β-E-6-BSA (1-100 nM), was biologically active in rapidly facilitating the release of dopamine from corpus striatum, an important estrogen target site that lacks classical ER. This rapid action of estrogen is consistent with the presence of mEBS in the plasma membranes of these cells and future work is ongoing to purify this putative mEBS and/or clone its cDNA.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Neuroscience

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Zheng, Jianbiao

Contributors dc:contributor

• Ramirez, Victor D.

• Biology, Molecular
• Biology, Neuroscience
• Biology, Animal Physiology

Statement dc:rights

• Copyright 1996 Zheng, Jianbiao

Language dc:language

eng

Identifier

9780591199864
AAI9712504
(UMI)AAI9712504

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/21798