Functional roles of plasma membrane localized estrogen receptors

A series of emerging data supports the existence and importance of plasma membrane localized estrogen receptors in a variety of cells that are targets for the steroid hormone action. When estradiol (E2) binds to the cell surface protein, the ensuing signal transduction event triggers downstream signaling cascades that contribute to important biological functions. Aside from the classical signaling through nuclear estrogen receptors, we have provided evidence for the functional roles of an estrogen receptor localized in the plasma membrane. This review highlights some of the recent advances made in the understanding of the genomic/non-genomic actions of plasma membrane localized estrogen receptors.     

Rapid actions of aldosterone: lymphocytes, vascular smooth muscle and endothelial cells.

The genomic theory of steroid action has been the unquestioned dogma for the explanation of steroid effects over the past four decades. Despite early observations on rapid steroid effects being clearly incompatible with this theory, only recently has nongenomic steroid action been recognized more widely and led to a critical reappraisal of unsolved questions about this dogma. Evidence for nongenomic steroid effects come from all fields of steroid research now, and mechanisms of agonist action are studied with regard to membrane receptors and second messengers involved. A prominent example of a receptor/effector-cascade for nongenomic steroid effects has been described for rapid aldosterone effects in various cell types, including lymphocytes, cultured vascular smooth muscle, and endothelial cells involving nonclassical membrane receptors with a high affinity for aldosterone, but not for cortisol, and phosphoinositide turnover. As another important second messenger, [Ca2+]i is consistently increased by aldosterone within 1-2 min. In vascular smooth muscle cells, calcium is released from perinuclear stores, while in endothelial cells a predominant increase of subplasmalemmal calcium is seen. Effects are half maximal at physiological concentrations of free aldosterone (0.1 nmol/L), while cortisol is inactive up to 0.1 micromol/L; the classical mineralocorticoid antagonist canrenone is ineffective in blocking the action of aldosterone. The data show that intracellular signaling for nongenomic aldosterone effects also involves calcium, but pathways of cell activation may vary between different cell types. Future research will have to target the cloning of the first membrane receptor for steroids, and the evaluation of the clinical relevance of these rapid steroid effects.     

ent-Steroids: Novel tools for studies of signaling pathways

Membrane receptors are often modulated by steroids and it is necessary to distinguish the effects of steroids at these receptors from effects occurring at nuclear receptors. Additionally, it may also be mechanistically important to distinguish between direct effects caused by binding of steroids to membrane receptors and indirect effects on membrane receptor function caused by steroid perturbation of the membrane containing the receptor. In this regard, ent-steroids, the mirror images of naturally occurring steroids, are novel tools for distinguishing between these various actions of steroids. The review provides a background for understanding the different actions that can be expected of steroids and ent-steroids in biological systems, references for the preparation of ent-steroids, a short discussion about relevant forms of stereoisomerism and the requirements that need to be fulfilled for the interaction between two molecules to be enantioselective. The review then summarizes results of biophysical, biochemical and pharmacological studies published since 1992 in which ent-steroids have been used to investigate the actions of steroids in membranes and/or receptor-mediated signaling pathways.     

Progestin, estrogen and androgen G-protein coupled receptors in fish gonads

The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.     

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.