The ligand binding properties of human membrane estrogen receptors

Abstract

Estrogen (17b-estradiol) exerts rapid non-genomic effects in various cell types. Surface binding sites for estrogen has been demonstrated using confocal microscopy. However, the identity of the putative membrane estrogen receptor (mER) and how well they bind with estrogen remained elusive. The present study aims to determine the binding affinities of estrogen to the mER candidates recently identified and to investigate the importance for mERs to be transported to the membrane for estrogen binding. Moreover, the relative binding affinities of the mERs with various estrogenic chemicals and phytoestrogens were evaluated. Human estrogen receptor-a66 (ER66), estrogen receptor-a46 (ER46), estrogen receptor-a36 (ER36) and G protein-coupled receptor 30 (GPR30) were cloned and expressed using cell-free expression systems in the presence of nanolipoprotein molecules as the membrane substitute. Expressed receptor proteins were used in radioactive binding assay. ER66 and ER46 have similar binding affinities towards estrogen (KdG79 pM), whereas ER36 and GPR30 displayed no specific binding. ER66 and ER46 expressed in prokaryotic system have lower binding affinities than the receptors expressed in eukaryotic system. Removal of nanolipoprotein molecules also reduced the binding affinities of ER66 and ER46. Our results suggested that post-translational modification and membrane trafficking of mERs are important for proper conformation for binding. Moreover, relative binding affinities to ER66 and ER46 are similar for the compounds tested, except for the estrogen antagonist, ICI 182 780.