Palmitoylation of estrogen receptors is essential for neuronal membrane signaling

Abstract

In addition to activating nuclear estrogen receptor signaling, 17β-estradiol can also regulate neuronal function via surface membrane receptors. In various brain regions, these actions are mediated by the direct association of estrogen receptors (ERs) activating metabotropic glutamate receptors (mGluRs). These ER/mGluR signaling partners are organized into discrete functional microdomains via caveolin proteins. A central question that remains concerns the underlying mechanism by which these subpopulations of ERs are targeted to the surface membrane. One candidate mechanism is S-palmitoylation, a posttranscriptional modification that affects the subcellular distribution and function of the modified protein, including promoting localization to membranes. Here we test for the role of palmitoylation and the necessity of specific palmitoylacyltransferase proteins in neuronal membrane ER action. In hippocampal neurons, pharmacological inhibition of palmitoylation eliminated 17β-estradiol-mediated phosphorylation of cAMP response element-binding protein, a process dependent on surface membrane ERs. In addition, mutation of the palmitoylation site on estrogen receptor (ER) α blocks ERα-mediated cAMP response element-binding protein phosphorylation. Similar results were obtained after mutation of the palmitoylation site on ERβ. Importantly, mutation of either ERα or ERβ did not affect the ability of the reciprocal ER to signal at the membrane. In contrast, membrane ERα and ERβ signaling were both dependent on the expression of the palmitoylacyltransferase proteins DHHC-7 and DHHC-21. Neither mGluR activity nor caveolin or ER expression was affected by knockdown of DHHC-7 and DHHC-21. These data collectively suggest discrete mechanisms that regulate specific isoform or global membrane ER signaling in neurons separate from mGluR activity or nuclear ER function.

Figure 1.

Inhibition of palmitoylation blocks 17β-estradiol-induced CREB phosphorylation. A, Confocal images of cultured female hippocampal neurons immunolabeled with MAP2 (green) and CREB phosphorylated at serine 133 (red). Administration of 2-BR blocked 17β-estradiol-induced CREB phosphorylation. Treatments: top left, vehicle; top right, 17β-estradiol (17βE); bottom left, 2-BR; bottom right, 17β-estradiol and 2-BR. Scale bar corresponds to 25 μm. B, Quantification of immunofluorescence demonstrating that 2-BR blocks 17β-estradiol-induced CREB phosphorylation. C, 2-BR also blocked 17β-estradiol-mediated attenuation of L-type calcium channel-dependent CREB phosphorylation, triggered by treatment with 20 mM K⁺ (20K⁺). Different lower case letters within each bar graph indicate statistically different groups.

Figure 2.

The ER palmitoylation site regulates membrane estrogen receptor signaling. A, Hippocampal neurons transfected with EYFP exhibit characteristic 17β-estradiol modulation of CREB phosphorylation. 17β-Estradiol-mediated CREB phosphorylation is dependent on ERα, whereas attenuation of L-type calcium channel-dependent CREB phosphorylation is mediated by either ERα or ERβ. Receptor specificity was determined using the ERα agonist PPT and the ERβ agonist DPN. B, Expression of EYFP-ERα C452A blocked membrane ERα but not ERβ signaling. C, Expression of EYFP-ERβ C354A eliminated membrane ERβ but not ERα signaling. D, EYFP-ERα and EYFP-ERα C452A were equally effective in stimulating nuclear ER function, as measured by ERE-dependent luciferase expression. RLU, relative light units. E, Expression of EYFP-ERα does not compromise 17β-estradiol-induced membrane signaling. Different lower case letters within each bar graph indicate statistically different groups.

Figure 3.

DHHC-7 and DHHC-21 are both necessary for ERα- and ERβ-mediated 17β-estradiol modulation of CREB phosphorylation. A, Transfection of hippocampal neurons with siRNA against no known target did not affect expression of the palmitoylacyltransferases DHHC-7, DHHC-21, or DHHC-10) compared with that for nontransfected neurons. B, In addition, no target (NT) siRNA had no effect on 17β-estradiol regulation of CREB phosphorylation. C, Transfection with siRNA against DHHC-7 knocked down DHHC-7 mRNA but not DHHC-21 or DHHC-10. D, siRNA against DHHC-7 completely eliminated 17β-estradiol regulation of CREB phosphorylation. E, Transfection with siRNA against DHHC-21 reduced DHHC-21 mRNA but not DHHC-7 or DHHC-10. F, siRNA against DHHC-21 also abolished the effects of 17β-estradiol on CREB phosphorylation. G, Transfection of siRNA against DHHC-10 decreased DHHC-10 mRNA, without affecting DHHC-7 or DHHC-21 expression. H, DHHC-10 knockdown did not affect 17β-estradiol modulation of CREB phosphorylation. Different lower case letters within each bar graph indicate statistically different groups.

Figure 4.

siRNA knockdown of either DHHC-7 or DHHC-21does not affect mGluR signaling, caveolin expression, or ER expression. A–C, mGluR1a-mediated CREB phosphorylation and mGluR2-mediated attenuation of L-type calcium channel–dependent CREB phosphorylation in hippocampal neurons are unaffected by transfection of siRNAs against no known target (A), DHHC-7 (B), or DHHC-21 (C). mGluR1a was activated using the group I mGluR agonist DHPG; mGluR2 was activated using the group II agonist APDC. D and E, siRNA against DHHC-7 and DHHC-21 had no effect on mRNA expression of either caveolin 1 (D) or caveolin 3 (E). F, Western blot and band analysis indicating that knockdown of DHHC-7 or DHHC-21 did not affect caveolin 1 protein. Flotillin was used as the loading control. G and H, siRNA against DHHC-7 and DHHC-21 had no effect on mRNA expression of either ERα (G) or ERβ (H). I, Western blot and band analysis indicating that knockdown of DHHC-7 or DHHC-21 did not affect ERα protein. GAPDH was used as the loading control. Different lower case letters within each bar graph indicate statistically different groups. NT, no target.