ABSTRACT: ER?36 is a naturally occurring, membrane-associated, isoform of estrogen receptor ?. The expression of ER?36 is due to alternative splicing and different promoter usage. ER?36 is a dominant-negative effector of ER?66-mediated transactivational activities and has the potential to trigger membrane-initiated mitogenic, nongenomic, estrogen signaling; however, the subcellular localization of ER?36 remains controversial. To determine the cellular localization of ER?36 in estrogen-responsive human uterine smooth muscle (ht-UtSMC) and leiomyoma (fibroid; ht-UtLM) cells, we conducted systematic confocal microscopy and subcellular fractionation analysis using ER?36 antibodies. With Image J colocalizaton analysis plugin, confocal images were analyzed to obtain a Pearson's Correlation Coefficient (PCC) to quantify signal colocalization of ER?36 with mitochondrial, endoplasmic reticulum, and cytoskeletal components in both cell lines. When cells were double-stained with an ER?36 antibody and a mitochondrial-specific dye, MitoTracker, the PCC for the two channel signals were both greater than 0.75, indicating strong correlation between ER?36 and mitochondrial signals in the two cell lines. A blocking peptide competition assay confirmed that the mitochondria-associated ER?36 signal detected by confocal analysis was specific for ER?36. In contrast, confocal images double-stained with an ER?36 antibody and endoplasmic reticulum or cytoskeletal markers, had PCCs that were all less than 0.4, indicating no or very weak signal correlation. Fractionation studies showed that ER?36 existed predominantly in membrane fractions, with minimal or undetected amounts in the cytosol, nuclear, chromatin, and cytoskeletal fractions. With isolated mitochondrial preparations, we confirmed that a known mitochondrial protein, prohibitin, was present in mitochondria, and by co-immunoprecipitation analysis that ER?36 was associated with prohibitin in ht-UtLM cells. The distinctive colocalization pattern of ER?36 with mitochondria in ht-UtSMC and ht-UtLM cells, and the association of ER?36 with a mitochondrial-specific protein suggest that ER?36 is localized primarily in mitochondria and may play a pivotal role in non-genomic signaling and mitochondrial functions.
