The role of the estrogen receptor 46 hydrophobic domain on endothelial membrane-initiated signaling

The expression of Estrogen Receptor 46 (ER46, 174 –595 aa), an N-terminus truncated isoform of the Estrogen Receptor α, in human endothelial cells (EC), and its critical role in membrane-initiated, rapid responses to 17β-estradiol (E2) have been reported. ER46 is targeted to human EC caveolar membranes, where it is a component of a complex containing c-Src, PI3-Kinase (PI3K), Akt and eNOS. E2 rapidly activates eNOS via c-Src/PI3K/Akt in EC. A highly conserved hydrophobic region of ER46, adjacent to a critical palmitoylation site (Cys447), may be required for its membrane localization and function. We generated a hydrophobic region mutant, “4Gly” (I451G/I452G/L453G/L454G), to evaluate the importance of this domain on ER46 membrane localization, eNOS activation, ER46 dimerization, and protein-protein (ER46-c-Src) interactions. Peripheral plasma membrane staining with cell impermeant E2-horse radish peroxidase (E2-HRP) was observed in COS-7 cells transiently transfected with ER46, but not with 4Gly. E2-BSA (30 nM, 10 min) induced eNOS phosphorylation (Ser1177) in plasma membrane fractions of ER46- but not 4Gly- transfected COS-7 cells. Using Fluorescence Resonance Energy Transfer (FRET), we demonstrated that ER46 and the 4Gly mutant both undergo homodimerization, although 4Gly less efficiently. The protein-protein interaction between ER46 and c-Src was also assessed by FRET. The FRET efficiency between mCerulean-tagged ER46 and EYFP-tagged c-Src was significantly increased after E2-BSA (40 nM, 10 min) stimulation in transiently transfected HeLa cells, indicating an E2-induced ER46-c-Src interaction within 75 Å. The FRET efficiency of c-Src-4Gly was 50% compared to that seen with the wild type ER46 in these reconstitution experiments. These results demonstrate that this conserved hydrophobic region of ER46 is critical for the subcellular localization, for required protein-protein interactions and for consequent membrane-initiated rapid signaling responses to E2.