Plasma membrane localization and function of the estrogen receptor α variant (ER46) in human endothelial cells

Abstract

Estrogen receptor (ER) ␣ variants have been identified in an array of nonendothelial cells. We previously demonstrated that estrogen rapidly induces nitric oxide release via a phosphatidylinositol 3-kinase͞Akt͞endothelial nitric-oxide synthase (eNOS) pathway in EA.hy926 cells (immortalized human endothelial cells), which express a 46-kDa ER. We now confirm that, due to alternative splicing, the 46-kDa endothelial cell protein (ER46) is an amino-terminal truncated product of full-length ER␣ (ER66). ER46 is expressed in the plasma membrane, cytosol, and nucleus of resting, estrogendeprived cells. Flow cytometric and immunofluorescence microscopic analyses demonstrated that the ER46 C but not N terminus is Ab-accessible in the plasma membrane. Inhibition of palmitoylation with tunicamycin and [ 3 H]palmitic acid labeling demonstrated an estrogen-induced, palmitoylation-dependent plasma membrane ER46 recruitment, with reorganization into caveolae. In reconstituted, estrogen-stimulated COS-7 (ER-null) cells, membrane ER46 more efficiently triggered membrane eNOS phosphorylation than ER66. Conversely, ER66 more efficiently mediated estrogen response element reporter-gene transactivation than ER46. These results demonstrate that ER46 is localized and further dynamically targeted to the plasma membrane in a palmitoylationdependent manner. ER46 more efficiently modulates membraneinitiated estrogen actions, including eNOS activation, than fulllength ER66. These findings may have important implications in vascular-specific targeting of estrogen receptor agonists.

Autoantibodies to estrogen receptor α interfere with T lymphocyte homeostasis and are associated with disease activity in systemic lupus erythematosus

“…To date, the precise location of membrane-associated ER is a matter of debate, i.e., it has not been determined whether the membraneassociated ER binding site is on the inner or outer leaflet of the plasma membrane. Several investigators, using a variety of ER-specific antibodies in intact, nonpermeabilized cells, have hypothesized that the expression of ER occurs at the external leaflet of the cell membrane (38)(39)(40)(41)(42)(43)(44)(45). Conversely, other research groups have pointed to the cytoplasmic tail of membrane-associated ER as a key functional domain (8).…”

The localization and non‐genomic function of the membrane‐associated estrogen receptor in oligodendrocytes

“…Smaller ERa isoforms have also been reported. In the plasma membrane of a human EC line, a truncated 46 kDa receptor was the predominant ERa isoform; palmitoylation induced its translocation to the plasma membrane (Li et al, 2003). At least five splice variant isoforms of the rat ERb gene product also have been reported (Al-Bader, 2006;Petersen et al, 1998;Price et al, 2000;Saji et al, 2001).…”

Aging and substitutive hormonal therapy influence in regional and subcellular distribution of ERα in female rat brain

“…The odds are that nonnuclear ERα is the isoform responsible of downstream activation of PI3K signaling pathway (Gundlah et al 2000). Further investigations point ER47 to have a crucial role in mediating eNOS activation specifically (Li et al 2003). Taking into account these observations, the higher proportion of ER47 in estradiol-treated animals at the end of the study might be favoring PI3K signaling pathway activation, being therefore a hallmark of 17β-estradiol-mediated neuroprotection effectiveness.…”

“…In this sense, non-nuclear estrogen receptor has been widely described as responsible of rapid non-genomic estrogens actions (McCarthy 2008;Vasudevan and Pfaff 2008); raising the possibility that estradiol could play a neuroprotective role through a non-genomic rapid mechanism (Singer et al 1999;Wilson et al 2002). The way of its participation, among many others, could be in the increase of eNOS activity in cerebral blood vessels (McEwen 2001;Milner et al 2001;Toran-Aller et al 2002;Li et al 2003) or the downregulation of GSK3β (Glycogen Synthase Kinase 3β). The upregulation of this enzyme is responsible of the hyperphosphorylation of Tau protein, the main component of Alzheimer's disease amyloid (Goodenough et al 2003).…”