The Journal of Neuroscience, April 1, 2009, 29(13):4228-4238; doi:10.1523/JNEUROSCI.0550-09.2009
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Cellular/Molecular
17β-Estradiol-Mediated Neuroprotection and ERK Activation Require a Pertussis Toxin-Sensitive Mechanism Involving GRK2 and β-Arrestin-1
Reymundo Dominguez,
Eric Hu,
Miou Zhou, and
Michel Baudry
Neuroscience Program, University of Southern California, Los Angeles, California 90089-2520
Correspondence should be addressed to Michel Baudry, Neuroscience Program, University of Southern California, HNB534, Los Angeles, CA 90089-2520. Email: baudry{at}usc.edu
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 
(ER) 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 ER. These findings suggested that ER 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 ER, 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-3,17β-diol-6-one-6-carboxymethloxime-NH-propyl-biotin] resulted in agonist internalization. These results demonstrate that E2-mediated neuroprotection and ERK activation involve ER activation of G-protein- and β-arrestin-mediated mechanisms.
Received Feb. 2, 2009;
accepted Feb. 24, 2009.
Correspondence should be addressed to Michel Baudry, Neuroscience Program, University of Southern California, HNB534, Los Angeles, CA 90089-2520. Email: baudry{at}usc.edu
