Abstract 859: Direct Interactions with G proteins are Required for the Nongenomic Signaling by Estrogen Receptor α that Governs Endothelial Nitric Oxide Synthase Activity and Monocyte Adhesion to Endothelial Cells
A plasma membrane-associated subpopulation of estrogen receptor α (ERα) mediates G protein-dependent nongenomic activation of endothelial NO synthase (eNOS) by estradiol (E2). In the present study we investigated the molecular mechanisms underlying the functional linkage between ERα and G proteins. Using purified, recombinant proteins in pulldown experiments, we determined that ERα binds directly and dynamically to monomeric Gαi via a 10 amino acid domain that resides within nuclear localization signal 3 of the receptor. Whereas wild-type ERα expressed in COS-7 cells induced rapid src phosphorylation in response to E2, deletion of the Gαi binding domain yielded an inactive receptor. In additional pulldowns, Gαi interaction with wild-type ERα was greater for GDP-bound Gαi versus GTP-bound Gαi and it was enhanced by Gαi myristoylation, which are modifications known to promote Gαi interactions with classical G protein-coupled receptors (GPCR). ERα binding was greater to Gαi associated with Gβγ as a heterotrimer versus monomeric Gαi, and additional studies showed that ERα binds Gβγ directly via a C-terminal domain of the receptor distinct from the Gαi binding domain. E2 caused the release of both Gαi and Gβγ from ERα in the absence of GTP binding to Gαi, which is unique compared to conventional GPCR-G protein dynamics. In COS-7 cells expressing wild-type ERα, the disruption of ERα-G protein interaction with either a dominant negative mutant form of the receptor lacking the Gαi binding domain or a 10 amino acid peptide representing the Gαi binding domain prevented ERα signaling to src; Gβγ sequestration by cotransfection of the β-adrenergic receptor kinase C-terminus had a similar effect. Furthermore, endogenous ERα activation of eNOS in bovine aortic endothelial cells (BAEC) was also prevented by the dominant negative mutant of ERα or the Gαi binding domain peptide. Moreover, E2-induced attenuation of monocyte adhesion to BAEC, which was entirely mediated by eNOS activation, was also blocked by expression of the same dominant negative mutant of ERα. These cumulative findings indicate that novel direct interactions between ERα and G proteins are required for E2-induced nongenomic signaling in endothelial cells of significance to vascular health and disease.