© 2001 Annual Reviews

Estrogen's direct genomic effects are mediated by the nuclear form of the estrogen receptors ERα or ERβ which associate with the Estrogen Response Element (ERE) or the fos/jun heterodimers that bind AP1 sites. Indirect genomic mechanisms include activation of ER-linked second messenger systems such as AC/PKC, cAMP/PKA and MAPK/ERK. Ras activates Raf, leading to sequential phosphorylation and activation of MAPK/ERK which interacts directly with nuclear transcription factors and indirectly through interaction with intermediary proteins. Non-genomic effects at high concentrations involve antioxidant effects not mediated by known ERs. (Reprinted with permission, Ann Rev Phamacol Toxicol 41:569–91, 2001)

Classic theories on steroid hormone action have been undergoing considerable revision owing to cellular effects that occur too quickly to be mediated by gene transcription. Estrogen is no exception and plays a leading role as the story unfolds. A growing acceptance that estrogen receptors exist on the...


Szego and colleagues reported increases in cyclic AMP within 15 seconds of estrogen application as early as 1967.1 The finding challenged convention for two reasons: The time course was too fast to be a result of transcription, and the product was a known second messenger for receptors residing in the cell membrane.

The results were "not very favorably received," says Szego. "It was not confirmed in detail until 1994.2 That's a lot of years." Though vindicated, she says she's indignant that the 1994 paper is the one that's cited.

In the 1970s, Szego's group reported the first evidence of estrogen-specific binding sites on the membrane.3 Around that time, brain researchers had been reporting estrogen's rapid effects on processes such as electrophysiology and neocortical development.

Martin Kelly, professor of pharmacology and physiology at Oregon Health and Sciences University in Portland, says his work in that decade also met with skepticism. Others claimed Kelly's results were "a pharmacological response, not a physiological response." That is, higher concentrations of estrogen can induce oxidative effects not mediated by receptors.

In the late 1990's, work on membrane-initiated estrogen effects reached a crescendo, spurred by evidence that the classical nuclear receptors can be expressed in the plasma membrane.4 Ellis Levin, professor of biochemistry and pharmacology, and his colleagues at the University of California, Irvine, transfected CHO (Chinese hamster ovary) cells, which don't normally express estrogen receptors, with both types of known ERs, ER-α and ER-β. They found ERs in both membrane and nuclear cell fractions.


But just because the classical estrogen receptors can mediate membrane effects in vitro doesn't mean they do so in vivo. Evidence for novel receptor types has provoked "a raging controversy," says Dominique Toran-Allerand, professor of anatomy and cell biology at Columbia University. Says Kelly, "It's divided into two camps": Those who believe that rapid responses are all ER-α and ER-β mediated, and those who believe there are other receptors out there. Both Toran-Allerand and Kelly have demonstrated unique pharmacological profiles of estrogen receptors in their laboratories.

Toran-Allerand uses neuronal cultures from newborn mice and showed evidence for a novel receptor she calls ER-X.5 Receptor binding occurs at low ligand concentration and levels off when receptors are all occupied, satisfying pharmacologic requisites of affinity and saturability. Compared to ER-α, ER-X has a lower molecular weight, a greater response to 17-α-estrodiol (the typically inactive stereoisomer of 17-β-estradiol), and an opposite effect on mitogen-activated protein (MAP) kinase.

Toran-Allerand says that other investigators may be misidentifying ER-X as ER-α. The confusion results, she says, because antibodies to ER-α also cross-react with ER-X.

Kelly uses whole-cell recording techniques to study the electrophysiology of hypothalamic neurons. Using a tamoxifen analog, he has characterized an estrogen receptor that is distinct from ER-α, ER-β, and ER-X.6 Tamoxifen is a breast cancer chemotherapeutic drug with anti-estrogen activity. STX, the analog, lacks affinity for classical ERs, but proved more potent than estrogen in his bioassay.

Many investigators choose a conservative approach when broaching the possibility of novel ER receptors. They await confirmation from independent laboratories, or the gold standard of identification: "the sequencing of the protein," says Toran-Allerand, "which we're in the process of doing."


Membrane estrogen receptors function like G protein-coupled receptors, a superfamily of membrane proteins that includes adrenergic and opioid receptors. "As a result of activating various G proteins," says Levin, estrogen induces "all the expected downstream signaling that you find in the typical G protein-coupled receptor." Indeed, a perusal of recent papers supplies a laundry list of signal transduction mediators: calcium, phospholipase C, inositol phosphate, protein kinase C, protein kinase A, MAP kinase, tyrosine kinase, and ion channels.

Membrane physiologist Eric Smart at University of Kentucky Medical School in Lexington says, "Different ligands induce very different physiological outputs, [but] they use all of the same machinery in between," that is, signal transduction proteins. "Just pick one, say, MAP kinase: MAP kinase is activated by everything. And then it phosphorylates everything. So, you can't turn on all the MAP kinases. You'd need divine intervention to get the right one."

Rather than call on the heavens, the cell uses specialized domains within the plasma membrane. This finding has overturned classical notions of the fluid mosaic model for lipid bilayers. "The membrane does not freely diffuse, and it doesn't interchange with all of the other compartments," says Smart.

In endothelial cells, membrane ERs are found in anatomical structures called caveolae. "You can see them" with electron microscopy, says Toran-Allerand. "They're like indentations in the plasma membrane." (The term caveolae means little cave.)

Structural proteins called caveolins anchor receptor proteins and signaling proteins to this membrane domain. "And everybody and his brother are in there," says Toran-Allerand. Signaling pathways are cascades, and in caveolae, all the components of the cascade are congregated in one spot. So when estrogen binds its receptor, she says, "Everybody's there and you can get this chain reaction. You don't have to go all over the cell."

"It's basically the concept of compartmentalization," says Smart. "If that receptor is prepositioned to a certain signal transduction pathway, to a certain output, then you can use all the machinery in between and still have specificity at the very beginning and at the very end."


Specificity may begin earlier. Estradiol travels through the bloodstream bound to proteins, including high density lipoprotein (HDL). Now, there is evidence that HDL may actually help deliver estrogen to its receptors. HDL binds to scavenger receptors, class B type I (SR-BI), which in endothelial cells are localized to plasma membrane caveolae along with estrogen receptors and nitric oxide synthase. Smart and his colleagues demonstrated that stimulation of nitric oxide synthase occurred with HDL isolated from females but not from males and was dependent on the presence of SR-BI.7

The scenario has SR-BI snagging HDL from the bloodstream and, in females, delivering estrogen to its membrane receptor to induce nitric oxide synthase and vasore-laxation. By linking two factors associated with decreased risk of cardiovascular disease, HDL levels and gender, the findings also provide a mechanism for the relative cardioprotection observed in premenopausal women.

At the same time that nuclear and membrane mechanisms of estrogen are being teased apart, researchers are studying the coordination between the two sites of action, for example, estrogen's two-pronged effects on nitric oxide synthase. Given repeatedly, estrogen increases transcription of the enzyme. With short-term exposure, a so-called spike, the enzyme undergoes posttranslational modification. It's not a new concept, says Smart, "One ligand, in this case estrogen, can both modulate the activity of existing protein and then modulate the amount of protein that's actually there at the nuclear level."

In addition, cross-talk with other signaling molecules, including hormones, growth factors, and neurotransmitters, invites inquiry. "The ability of estrogen to activate multiple pathways may be a way for the cell to integrate other signals into a cell's response to estrogen," says Michael Wang of the University of Michigan, Ann Arbor.

Researchers anticipate more plot twists. Citing recent evidence for a mitochondrial pool of estrogen receptors, Levin says, "It may get a lot more complicated before it gets simple."

Jill U. Adams juadams@verizon.net is a freelance writer in Albany, NY.

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