While the physiology of membrane-initiated estradiol signaling in the nervous system has remained elusive, a great deal of progress has been made toward understanding the activation of cell signaling. Membrane-initiated estradiol signaling activates G proteins and their downstream cascades, but the identity of membrane receptors and the proximal signaling mechanism(s) have been more difficult to elucidate. Mounting evidence suggests that classical intracellular estrogen receptor-α (ERα) and ERβ are trafficked to the membrane to mediate estradiol cell signaling. Moreover, an interaction of membrane ERα and ERβ with metabotropic glutamate receptors has been identified that explains the pleomorphic actions of membrane-initiated estradiol signaling. This review focuses on the mechanism of actions initiated by membrane estradiol receptors and discusses the role of scaffold proteins and signaling cascades involved in the regulation of nociception, sexual receptivity and the synthesis of neuroprogesterone, an important component in the central nervous system signaling.

Estrogen receptors (ERs) and estrogen-binding proteins have been localized intracellularly and on the cell surface. The membraneassociated proteins initiate signaling that activates a myriad of cellular responses including the modulation of ion channels and ultimately transcription. Although many of the downstream actions of membrane ERs, including ER␣ and ER␤, have been characterized, the mechanisms regulating membrane ER levels have remained elusive in the nervous system. In the present study, we used surface biotinylation to identify and study the estradiol regulation of membrane ER␣ in mixed-sex, cultured hypothalamic neurons from rat. Following surface biotinylation, Western blot analysis revealed full-length 66 kDa ER␣ and several ER␣ splice variants, most notably a biotinylated 52 kDa ER␣-immunoreactive protein. Treatment of the neurons with estradiol caused a rapid and transient increase of the biotinylated 52 kDa and 66 kDa ER␣ proteins in the plasma membrane. Exposure of the neurons to estradiol also significantly increased internalization of 52 kDa and 66 kDa ER␣ membrane proteins, a measure of receptor activation. In the hypothalamus, membrane ER␣ signaling depends on transactivation of metabotropic glutamate receptor-1a (mGluR1a). Estradiol treatment increased the internalization of mGluR1a in parallel with ER␣, a finding consistent with the hypothesis of an ER␣-mGluR1a signaling unit. These results demonstrate that estradiol regulates the amount of ER␣ in the membrane, suggesting estradiol can regulate its own membrane signaling.

17-β-Estradiol (E2) is a steroid hormone involved in numerous bodily functions, including several brain functions. In particular, E2 is neuroprotective against excitotoxicity and other forms of brain injuries, a property that requires the extracellular signal-regulated kinase (ERK) pathway and possibly that of other signaling molecules. The mechanism and identity of the receptor(s) involved remain unclear, although it has been suggested that E2 receptor α (ERα) and G proteins are involved. We, therefore, investigated whether E2-mediated neuroprotection and ERK activation were linked to pertussis toxin (PTX)-sensitive G-protein-coupled effector systems. Biochemical and image analysis of organotypic hippocampal slices and cortical neuronal cultures showed that E2-mediated neuroprotection as well as E2-induced ERK activation were sensitive to PTX. The sensitivity to PTX suggested a possible role of G-protein- and β-arrestin-mediated mechanisms. Western immunoblots from E2-treated cortical neuronal cultures revealed an increase in phosphorylation of both G-protein-coupled receptor-kinase 2 and β-arrestin-1, a G-protein-coupled receptor adaptor protein. Transfection of neurons with β-arrestin-1 small interfering RNA prevented E2-induced ERK activation. Coimmunoprecipitation experiments indicated that E2 increased the recruitment of β-arrestin-1 and c-Src to ERα. These findings suggested that ERα is regulated by a mechanism associated with receptor desensitization and downregulation. In support of this idea, we found that E2 treatment of cortical synaptoneurosomes resulted in internalization of ERα, whereas treatment of cortical neurons with the ER agonists E-6-BSA-FITC [β-estradiol-6-(O-carboxymethyl)oxime-bovine serum albumin conjugated with fluorescein isothiocyanate] and E-6-biotin [1,3,5(10)-estratrien-3,17 β-diol-6-one-6-carboxymethloxime-NH-propyl-biotin] resulted in agonist internalization. These results demonstrate that E2-mediated neuroprotection and ERK activation involve ERα activation of G-protein- and β-arrestin-mediated mechanisms.

In addition to its well-known activational mechanism, the steroid hormone 17-b-estradiol (E2) has been shown to rapidly activate various signal transduction pathways that could participate in estrogen-mediated regulation of synaptic plasticity. Although the mechanisms underlying these effects are not clearly understood, it has been repeatedly suggested that they involve a plasma membrane receptor which has direct links to several intracellular signaling cascades. To further address the question of whether E2 acts directly at the synapse and through membrane-bound receptors, we studied the effects of E2 and of ligands of estrogen receptors on various signaling pathways in cortical synaptoneurosomes. Our results demonstrate that E2 elicits N-methyl-D-aspartate receptor phosphorylation and activates the extracellular signal-regulated kinase and the phosphatidylinositol 3-kinase/Akt signal transduction pathways in this cortical membrane preparation. Furthermore, we provide evidence for the presence of a membrane-bound estrogen receptor responsible for these effects in cortical synaptoneurosomes. Our study demonstrates that E2 directly acts at cortical synapses, and that synaptoneurosomes provide a useful system to investigate the mechanisms by which E2 regulates synaptic transmission and plasticity.

Regulation of sexual reproduction by estradiol involves the activation of estrogen receptors (ERs) in the hypothalamus. Of the two classical ERs involved in reproduction, ERα appears to be the critical isoform. The role of ERα in reproduction has been found to involve a nuclear ERα that induces a genomic mechanism of action. More recent a plasma membrane ERα has been shown to trigger signaling pathways involved in reproduction. Mechanisms underlying membrane-initiated estradiol signaling are emerging, including evidence that activation of plasma membrane ERα involves receptor trafficking. The present study examined the insertion of ERα into the plasma membrane of N-38 neurons, an immortalized murine hypothalamic cell line. We identified, using western blotting and PCR that N-38 neurons express full-length 66 kDa ERα and a 52 kDa ERα spliced variant missing the fourth exon - ERαΔ4. Using surface biotinylation we observed that treatment of N-38 neurons with estradiol or with a membrane impermeant estradiol elevated plasma membrane ERα protein levels, indicating that membrane signaling increased receptor insertion into the cell membrane. Insertion of ERα was blocked by the ER antagonist ICI 182,780 or with the protein kinase C (PKC) pathway inhibitor bisindolylmaleimide (BIS). Downstream membrane-initiated signaling was confirmed by estradiol activation of PKC-theta (PKCθ) and the release of intracellular calcium. These results indicate that membrane ERα levels in N-38 neurons are dynamically autoregulated by estradiol.