title: Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells.
abstract: Estrogen receptor (ER) alpha variants have been identified in an array of nonendothelial cells. We previously demonstrated that estrogen rapidly induces nitric oxide release via a phosphatidylinositol 3-kinase/Akt/endothelial 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 alpha (ER66). ER46 is expressed in the plasma membrane, cytosol, and nucleus of resting, estrogen-deprived 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 palmitoylation-dependent manner. ER46 more efficiently modulates membrane-initiated estrogen actions, including eNOS activation, than full-length ER66. These findings may have important implications in vascular-specific targeting of estrogen receptor agonists.
authors.affiliation: Section of Cardiovascular Medicine, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: M Page
authors.initials: MP
authors.lastname: Haynes
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Bender
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D047628:Estrogen Receptor alpha; D017930:Genes, Reporter; D014644:Genetic Variation; D006801:Humans; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III