MON-269 - Age-Related Phosphorylation of Estrogen Receptor-Beta (Erβ) Drives Structural and Functional Shifts in Erβ:Gelsolin Interactions

Estrogen Receptor β (ERβ) is neuroprotective through its ability to regulate gene transcription either through direct DNA binding, or multiprotein complex tethering to specific DNA motifs. Our previous studies revealed a novel relationship between ERβ and gelsolin (GSN) in regulating transcription at several gene promoters and facilitating nuclear translocation. GSN is a cytoskeletal protein important for cell integrity, structure and nuclear cytoskeletal organization. However, we were unable to determine if ERβ and GSN interacted directly or were part of a larger transcriptional regulatory complex. Our previous studies also revealed that ERβ:GSN interactions decreased with age and there were concomitant changes to specific ER phosphorylation sites, raising the possibility that age-related changes in ERβ phosphorylation altered its interaction with GSN. In this study we aimed to 1) determine if ERβ:GSN interactions are direct or indirect in neurons, and 2) if the observed age-related phosphorylation changes in ERβ affect ERβ:GSN interactions. To assess if ERβ:GSN interactions are direct or indirect we transfected fluorescently tagged ERβ and GSN constructs into HEK293 cells and SK–N–SH, a human neuroblastoma cell line and then measured ERβ:GSN interactions using fluorescence lifetime imaging microscopy (FLIM) to quantify Förster resonance energy Transfer (FRET). Our results showed that ERβ directly interacted with GSN, as high FRET and calculated distance between fluorophores suggested very close protein proximities. Moreover, these interactions were significantly strengthened in a neuronal microenvironment, indicating that cell-type specific microenvironments can impact ER’s ability to interact within larger transcriptional regulatory networks. We next used site directed mutagenesis to mimic the age-associated in-vivo phosphorylation states of ERβ and used FRET to assess the effect of phosphorylation on ERβ:GSN interactions. Those results revealed site-specific phosphorylation of ERβ, as observed with aging, that induced changes on the structure and affinity of ERβ:GSN complex. Finally, we transfected phosphomimetic/null ERβ to quantify ERβ-mediated repression of the amyloid precursor protein (APP) gene promoter using luciferase assays. Our prior work showed that ERβ repressed APP gene transcription, and these results showed that site-specific phosphomimetic mutations affected ERβ-mediated repression. Together, our results revealed that aging alters ERβ phosphorylation leading to decreased interactions with GSN, which alters ERβ transcriptional regulatory activity. These novel data provide a potential mechanistic explanation to aging associated changes in ERβ-mediated signalling that stems from changes in ERβ phosphorylation and ERβ:protein interactions.