Although several different membrane estrogen

Although several different membrane estrogen receptors have been reported (Qiu et al., 2008; Revankar et al., 2005), a large percentage of membrane-initiated steroid hormone signaling appears to be performed by a subpopulation of the same receptors that act in the nucleus. Specifically, estrogen receptors (ERs) α and β are found at the plasma membrane and are synthesized from the same transcript as their nuclear counterparts (Razandi et al., 1999). We have known that ERα and ERβ are critical for membrane signaling for at least a decade, as their genetic knockout interferes with rapid estrogen-mediated activation of the MAPK/ERK pathway (Ábrahám et al., 2004). While Ábrahám and colleagues did not examine the subcellular location where ERα and ERβ triggered MAPK signaling, the existence of these estrogen receptors at the plasma membrane has since been observed in many Nemonapride receptor regions using a variety of techniques (Micevych and Mermelstein, 2008; Pedram et al., 2009; Razandi et al., 2003). Though the evidence for rapid, non-nuclear initiated action of ERα and ERβ had been compelling for many years, the mechanism(s) by which these receptors signaled outside the nucleus had remained frustratingly unclear. When our lab set about to investigate the signaling pathways responsible for the effects of membrane-initiated estradiol signaling in the mid-2000s, the fundamental issue was that previous reports had typically examined second messenger signals far downstream of the membrane-initiated event. As such, the literature contained a plethora of descriptive studies examining the impact of estrogens on a wide array of cellular processes. For example, estradiol was reported to attenuate L-type calcium currents (Chaban et al., 2003; Mermelstein et al., 1996) as well as the aforementioned activation of MAPK (Gu and Moss, 1996; Lee et al., 2004; Wade and Dorsa, 2003; Zhou et al., 1996). Hence, we wanted to understand the full signaling pathways that were responsible for multiple effects of estradiol. To do so, we utilized an in vitro assay monitoring phosphorylation of the transcription factor CREB as a measure of cellular activation. We first replicated what others had reported, finding that a brief application of physiological estradiol concentrations increased CREB phosphorylation in CA3-CA1 hippocampal neurons from 1 to 2 day-old female rat pups (Boulware et al., 2005). This effect was dose dependent, rapid, and blocked by MEK inhibitors. We then went on to examine the interaction of estradiol with L-type calcium channel signaling. Increased synaptic activity leads to increased CREB phosphorylation via activation of L-type voltage-gated calcium channels, and this activation can be reproduced in cell culture by K+-mediated depolarization. In this assay, a brief stimulation of cells with 20 mM K+ robustly increases CREB phosphorylation via CaMKIV signaling (Deisseroth et al., 1996; Mermelstein et al., 2000). Pretreatment with estradiol attenuates depolarization-induced CREB phosphorylation, revealing the bidirectional effects of estradiol in its modulation of these two discrete pathways. Our experiments additionally showed that these effects of estradiol are postsynaptic, occur via membrane receptors, and, importantly, do not occur in cultures from male animals (Boulware et al., 2005).
Estrogen receptors activate mGluR signaling pathways In order to distinguish the opposing effects of estradiol on CREB phosphorylation, we turned to a “bottom-up” approach, working backwards from CREB phosphorylation to pharmacologically isolate the two signaling pathways (Boulware et al., 2005). (The signaling pathway described below is summarized in Fig. 1) We first focused on proteins known to influence MAPK, and found that inhibition of PKC and IP3 receptors decreased estradiol-induced CREB phosphorylation. PLC inhibition similarly blocked the enhancement, without affecting estradiol-induced inhibition of L-type calcium channel effects. Because PLC, PKC, and IP3Rs are activated by Gq-coupled GPCRs (Gutkind, 2000), we hypothesized that estradiol may act through a known Gq-linked GPCR to enhance CREB phosphorylation. Our first step in addressing this hypothesis was to simply power through the catalog of pharmacological agents that act as antagonists for individual Gq-coupled receptors. With a bit of luck, the second drug we tried was LY367385, an antagonist for mGluR1a, a group I mGluR. Pretreatment of cells with this drug blocked estradiol enhancement of CREB phosphorylation without altering estradiol attenuation of L-type calcium channel signaling. These results were confirmed with a second mGluR1 antagonist. In addition, direct pharmacological activation of mGluR1a increased CREB phosphorylation, and occluded any further enhancement by estradiol. The mGluR1a agonist DHPG elicited CREB phosphorylation regardless of sex, suggesting that the sex difference in estradiol signaling lies upstream of the mGluR. This also supported the idea that estradiol was not acting directly on the mGluR, but rather relies on the ability of an estrogen receptor to activate its signaling. Indeed, we found that the ERα agonist PPT increased CREB phosphorylation, while the ER blocker ICI 182,780 eliminated the effect of estradiol. These data indicated that ERα was able to solicit mGluR1a signaling in female hippocampal neurons. We later went on to repeat these experiments in female striatal neurons, finding that the same type of ER/mGluR signaling occurs, but ERα pairs with mGluR5 instead of mGluR1a (Grove-Strawser et al., 2010).