Differential ligand binding affinities of human estrogen receptor-α isoforms

• Published in: PloS one 2013-04, Vol.8 (4), p.e63199-e63199
• Main Authors: Lin, Amanda H Y, Li, Rachel W S, Ho, Eva Y W, Leung, George P H, Leung, Susan W S, Vanhoutte, Paul M, Man, Ricky Y K
• Format: Article
• Language: English
• Subjects: 17β-Estradiol; Affinity; Binding Sites; Biology; Cell Line; Cell lines; Cell Membrane; Conformation; Drugs; Estradiol - metabolism; Estrogen Receptor alpha - agonists; Estrogen Receptor alpha - antagonists & inhibitors; Estrogen Receptor alpha - genetics; Estrogen Receptor alpha - metabolism; Estrogen receptors; Estrogens; Gene Expression; Humans; Insertion; Isoforms; Kinases; Ligands; Lipoylation; Nitric oxide; Palmitoylation; Phytoestrogens; Phytoestrogens - metabolism; Protein Binding; Protein Isoforms; Protein Processing, Post-Translational; Receptors; Rodents; Sex hormones
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0063199

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.