17β-Estradiol-Mediated Neuroprotection and ERK Activation Require a Pertussis Toxin-Sensitive Mechanism Involving GRK2 and β-Arrestin-1

Abstract

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.

G-Protein-Coupled Receptor 30 Mediates Rapid Neuroprotective Effects of Estrogen via Depression of NR2B-Containing NMDA Receptors

“…1 B). Consistent with previous findings (Bae et al, 2000;Dominguez et al, 2009), the nonspecific ER agonist, E2 (1, or 10 nM), attenuated the neuronal death based on the dose induced by NMDA (F (10, 77) ϭ 10.73, p Ͻ 0.05, Tukey's test; p ϭ 0.025, 1 nM E2 vs NMDA; p Ͻ 0.01, 10 nM E2 vs NMDA; Fig. 1 B).…”

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons

“…The higher transfection efficiency may be due to the requirement for plasmid DNAs to penetrate not only the plasma membrane but also the nuclear membrane for effectiveness, while siRNA is effective in the cytoplasm. Efficient knockdown using siRNA in organotypic slice cultures has also been seen in other studies (Murphy et al 2008;Dominguez et al 2009). Two days post-transfection, slices were homogenized and analyzed by Western blotting using anti-Stau2 antibody ( Fig.…”

Estrogen Actions on Neuroendocrine Glia

“…The time course of internalization, a marker for membrane receptor activation [reviewed in [136]], mimicked the pattern of membrane ERα insertion: estradiol increased the amount of internalized ERα at 5 min and remained elevated after 2 h. By 24 h, the amount of internalized ERα was below prestimulation levels. These observations of receptor internalization complement studies using a membrane-impermeable E-6-BSA-FITC [β-estradiol-6-(O-carboxymethyl) oxime-bovine serum albumin conjugated with fluorescein isothiocyanate] construct, which binds to and labels membrane ERs [84,114]. When cells are allowed to interact for a period of time (≥5 min) with the E-6-BSA-FITC, the fluorescent marker is seen within intracellular vesicles thought to be early endosomes, which is suggestive of internalization and is a morphological equivalent of the surface biotinylation experiments (fig.…”

“…Membrane impermeable estradiol constructs (e.g. E-6-BSA) and studies showing ERα protein in the membrane fraction of native or transfected cells indicate an ERα association with the membrane [35,85,99,105,106,112,113,114,115,116,117,118]. Since ERα does not have the typical structure of a classic membrane protein, this association has remained unresolved.…”