Selectivity of antibodies to estrogen receptors alpha and beta (ERalpha and ERbeta) for detecting DNA-bound ERalpha and ERbeta in vitro

Antibodies are widely used to detect estrogen receptor (ER) in ER-DNA complexes in electrophoretic mobility shift assays (EMSA). We compared the specificity of antibodies raised to different regions of ERalpha or ERbeta for detecting recombinant human ERalpha (rhERalpha) and recombinant rat ERbeta (rrERbeta) when bound to a consensus estrogen response element (ERE). ERalpha-specific antibodies specifically slowed the migration of the ER-ERE complex by 32 to 84% and inhibited rhERalpha-ERE binding by 17 to 75%. None of antibodies to ERbeta supershifted rhERalpha-ERE complex. Some ERalpha-specific antibodies increased whereas some decreased rrERbeta-ERE binding. Anti-ERbeta antibodies supershifted different amounts of the rrERbeta-ERE complex. Our results indicate that supershift and inhibition of ER-ERE interaction with a specific antibody are equally reliable in the detection of rhERalpha and rrERbeta. ERalpha antibody Ab10, antisera G20 and AT3B, and ERbeta-antiserum Y19 offered the best discrimination between ERalpha and ERbeta. Comparison of the peptide sequences against which various antibodies were raised indicate directions for new ERalpha and ERbeta- specific antibody development. We conclude that a cognate ER antibody that retards the migration of the ER-ERE complex by at least 40% or inhibits ER-ERE interaction by at least 8% provides a reliable detection of a specific ER isoform in EMSA.     

Interaction of tetrahydrocrysene ketone with estrogen receptors alpha and beta indicates conformational differences in the receptor subtypes

Estrogen receptors (ER) alpha and beta bind estradiol (E2) and other estrogenic ligands with different affinities. To measure the rate of E2 association with ERa and ERbeta, we employed tetrahydrocrysene ketone (THCK), a fluorescent ligand that is an agonist with ERalpha and an antagonist with ERbeta. We report that THCK binds E2-liganded and unliganded ERalpha and ERbeta, indicating a THCK binding site(s) other than the E2 binding pocket. THCK fluorescence was greater for ligand-occupied ERbeta than ERalpha, suggesting differences in the microenvironment of the THCK binding site(s). THCK fluorescence was also significantly greater for E2-, 4-hydroxytamoxifen-, and tamoxifen aziridine-liganded versus unliganded ER, allowing calculations of E2 association rate constants (ka) of 7.60 +/- 0.75 and 5.12 +/- 0.30 x 10(5) M(-1) s(-1) for E2-ERalpha and E2-ERbeta, respectively. THCK did not affect ERalpha binding to estrogen response element (ERE) DNA, but decreased ERbeta-ERE binding. We conclude that THCK binding site(s) on ERalpha versus ERbeta are different and important for ER function.     

Nature of functional estrogen receptors at the plasma membrane

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.