Demonstration of membrane estrogen binding proteins in rat brain by ligand blotting using a 17β-estradiol-[ 125I]bovine serum albumin conjugate

Abstract

This paper describes a ligand blotting procedure to visualize membrane estrogen receptors/binding proteins immobilized on nitrocellulose membranes. Using 17β-estradiol covalently linked with [ 125I]bovine serum albumin (BSA) at the C-6 position (17β-E-6-[ 125I]BSA) as a ligand, three major binding proteins with molecular masses of approximately 23, 28, and 32 kDa were identified from crude synaptosomal fractions (P2) of female rat brains. The binding of 17β-E-6-[ 125I]BSA to these proteins is selective for 17β-estradiol because BSA had no effect, and 17α-E-6-BSA was at least two orders of magnitude less potent than 17β-E-6-BSA in displacing the binding. In addition, [ 125I]BSA and 17α-E-6-[ 125I]BSA at similar concentrations did not bind to these proteins. Competition and saturation assays indicate that the binding affinity of 17β-E-6-[ 125I]BSA for these proteins was in the range of 1–10 nM. These proteins are not contaminants from cytosolic or serum estrogen binding proteins since no corresponding protein bands were found in cytosolic fractions. Three additional protein bands with molecular masses of approximately 18, 40, and 130 kDa were also detected, although inconsistently. The 23 and 40 kDa proteins seem to be concentrated in mitochondrial fractions (mP2), whereas the 28 and 32 kDa proteins are enriched in microsomal fractions (P3). Application of digitonin-solubilized P2 fractions to 17β-estradiol-coupled affinity columns resulted in significant purification of the 23 kDa protein as shown by ligand blotting. This protein was later identified as oligomycin-sensitivity conferring protein (OSCP), as reported previously. These data indicate that specific estrogen binding proteins different from classical nuclear estrogen receptor (66 kDa) are present in the cellular membranes of the female rat brain. The ligand blotting technique described here would also be applicable for the identification of other membrane steroid binding proteins/receptors using similar radiolabelled steroid-BSA conjugates.