Plasma membrane estrogen receptors exist and functions as dimers

A small pool of estrogen receptors (ERalpha and -beta) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17beta-estradiol (E2). Interestingly, in endothelial cells made from ERalpha /ERbeta homozygous double-knockout mice, membrane ERalpha or ERbeta are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERalpha. Only wild-type ERalpha supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsalpha and Gqalpha at the membrane in cells expressing the wild-type, but not the dimer mutant, ERalpha. Intact, but not dimer mutant, ERalpha also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERalpha. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.     

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.     

Kinetic analysis of estrogen receptor homo- and heterodimerization in vitro

The coexistence of ERalpha and ERbeta suggests that active receptor complexes are present as homo- or heterodimers. In addition each of three forms of active receptors may trigger different cellular responses. A real-time biosensor based on surface plasmon resonance was used as instrument to determine binding kinetics of homo- and heterodimerization of estrogen receptor alpha and beta. Partially purified full-length estrogen receptor alpha was expressed intracellularly as a C-terminal fusion to a hexa-histidine tag using the baculovirus-expression system. Purified estrogen receptor alpha and beta without tags were used as partners in the dimerization process. An association rate constant of 3.6 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the homodimer formation of ERalpha and 5.7 x 10(3) to 1.5 x 10(4)M(-1)s(-1) for the heterodimer formation was found assuming a pseudo first-order reaction kinetic. The equilibrium dissociation constant for homodimerization of ERalpha was 2.2 x 10(-8) to 5.4 x 10(-8) and 1.8 x 10(-8) to 2.6 x 10(-8)M for the heterodimer formation. The homo- and heterodimer formation was characterized by a slow association kinetics and kinetic rate constants were within the same range.     

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.