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. 2004 Dec 7;101(49):17126-31.
doi: 10.1073/pnas.0407492101. Epub 2004 Nov 29.

Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha

Qing Lu  1 , David C Pallas, Howard K Surks, Wendy E Baur, Michael E Mendelsohn, Richard H Karas
Affiliations

Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha

Qing Lu et al. Proc Natl Acad Sci U S A. .

Abstract

Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate gene expression. SHRs also mediate rapid, nongenomic cellular activation by steroids. In vascular endothelial cells, the SHR for estrogen, estrogen receptor (ER) alpha, is _targeted by unknown mechanisms to a functional signaling module in membrane caveolae that enables estrogen to rapidly activate the mitogen-activated protein kinase and phosphatidylinositol 3-Akt kinase pathways, and endothelial NO synthase (eNOS). Here we identify the 110-kDa caveolin-binding protein striatin as the molecular anchor that localizes ERalpha to the membrane and organizes the ERalpha-eNOS membrane signaling complex. Striatin directly binds to amino acids 183-253 of ERalpha, _targets ERalpha to the cell membrane, and serves as a scaffold for the formation of an ERalpha-Galphai complex. Disruption of complex formation between ERalpha and striatin blocks estrogen-induced rapid activation mitogen-activated protein kinase, Akt kinase, and eNOS, but has no effect on ER-dependent regulation of an estrogen response element-driven reporter plasmid. These findings identify striatin as a molecular scaffold required for rapid, nongenomic estrogen-mediated activation of downstream signaling pathways. Furthermore, by demonstrating independent regulation of nongenomic vs. genomic ER-dependent signaling, these findings provide conceptual support for the potential development of "pathway-specific" selective ER modulators.

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Figures

Fig. 1.
Fig. 1.
ERα binds directly to striatin via amino acids 183–253 of the ER. (A) Schematic representation of relevant domains of striatin. (B) Striatin coimmunoprecipitates with ERα in a variety of cells, including cells that endogenously express ERα [EAhy926 cells (human endothelial cell line), MCF7 cells (human breast cancer cell line), and GH3B6 cells (rat pituitary cell line)] and ERα-null cells transfected with an ERα expression plasmid [Rad91 cells (human radial artery-derived vascular smooth muscle cell line)]. IP, immunoprecipitation of ERα; NI, nonimmune immunoprecipitation; L, fraction of total cell lysates. (C) Short-term E2 treatment enhances complex formation between ERα and striatin. ERα was immunoprecipitated from Rad91 cells transfected with ERα, with or without exposure to 10–8 M E2 for 20 min. (D) Striatin binds ERα directly. GST fusion proteins were incubated with lysates of Rad91 cells transfected with ERα (Left) or with recombinant ERα (Right). (E) Striatin binds to amino acids 183–253 of ERα. (Upper) Schematic representation of ERα fragments used in GST pulldown experiments. (Lower) GST fusion proteins containing full-length ERα or ERα fragments were incubated with lysates of Cos1 cells.
Fig. 4.
Fig. 4.
Disruption of striatin-ERα binding prevents E2-induced nongenomic, but not genomic, signaling. (A) Overexpression of a peptide consisting of the striatin-binding domain within ERα, ER176–253, prevents complex formation between ERα and striatin. (B) Overexpression of the blocking peptide ER176–253 does not interfere with transcriptional transactivation of ERα by E2. Cos1 cells were transiently transfected with an ERα expression plasmid and an estrogen response element-driven luciferase reporter plasmid, with a plasmid encoding ER176–253 or an empty vector. Cells were treated with vehicle alone (open bars) or 10–8 M E2 (filled bars). E2-induced activation of ERα was somewhat greater in the presence of ER176–253. Bars represent mean ± SE from four independent experiments. *, P < 0.05 vs. empty vector. (C) Overexpression of ER176–253 blocks E2-induced increases in phosphorylation of MAPK. Lysates of Cos1 cells transfected with ERα and a control vector, or ERα and ER176–253, were immunoblotted for phospho-MAPK (pMAPK) or total MAPK, after treatment with E2 for various durations. Bar graphs show the mean ± SE for four independent experiments. *, P < 0.01; **, P < 0.05 vs. time 0. (D) Overexpression of the blocking peptide ER176–271 prevents E2-induced phosphorylation of Akt kinase. EAhy926 cells expressing endogenous ERα were incubated with Tat-GFP (○) or Tat-ER176–253 (▪) for 6 h before E2 treatment. Total protein lysates were then immunoblotted for phospho-AKT (pAKT) normalized for the amount of total Akt. These ratios were normalized to one for the vehicle-treated cells. Results represent mean ± SE derived from four independent experiments. *, P < 0.05 vs. time 0. (E) Overexpression of the blocking peptide ER176–253 prevents E2-induced phosphorylation of eNOS. EAhy926 were incubated with Tat-GFP (○) or Tat-ER176–253 (▪) for 6 h before E2 treatment. Total protein lysates were then immunoblotted for phospho-eNOS (peNOS) normalized for the amount of total eNOS. These ratios were normalized to one for the vehicle-treated cells. Results represent mean ± SE derived from four independent experiments. *, P < 0.05 vs. time 0.
Fig. 2.
Fig. 2.
Striatin _targets ERα to the plasma membrane. (A–C) EAhy926 cells without overexpression of striatin were immunostained for ERα (A) or striatin (B), and the images were merged (C). Under these conditions only minimal amounts of membrane-associated ERα is detected. (D–I) EAhy926 cells transfected with a striatin expression plasmid were immunostained for ERα (A and D), striatin (B, E, and H), or eNOS (G), and the images were merged (C, F, and I). Under these conditions substantial membrane-associated ERα is detected, which colocalizes with striatin, as does eNOS. (J) Overexpression of striatin increases the proportion of ERα detectable in membrane fractions. Lysates of EAhy926 cells transfected with a control vector (–) or a striatin expression plasmid (+) were fractionated by differential centrifugation. The purity of plasma membrane and nuclear fractions was identified by immunoblotting for predominantly membrane proteins [EGFR and IGFR (data not shown)] or the nuclear protein histone.
Fig. 3.
Fig. 3.
Striatin assembles a complex containing ERα and Gαi. (A) E2 induces Gαi to complex with striatin and ERα, and this is blocked by coincubation with PTX or the ER antagonist ICI 182,780 (ICI). Equivalent recovery of Gαi was shown by immunoblotting (data not shown). SFM, serum-free medium. (B) PTX abolishes E2-induced phosphorylation of MAPK. *, P < 0.05 vs. SFM. n = 3 independent experiments. (C)Gαi does not bind directly to ERα. (D)Gαi binds to the C terminus of striatin. GST fusion proteins containing either full-length striatin or the N terminus of striatin (striatin1–203) were incubated with lysates of Cos1 cells transfected with Gαi. (E) Striatin is required for complex formation between ERα and Gαi. GST or GST–Gαi fusion proteins were incubated with recombinant (rER), in the absence or presence of purified full-length striatin or striatin1–203. rERα, recombinant ERα. (F) Striatin forms a complex with eNOS. eNOS was identified by immunoblotting in GST fusion pulldowns and by coimmunoprecipitation of lysates from EAhy926 cells.
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References

    1. Mendelsohn, M. E. & Karas, R. H. (1999) N. Engl. J. Med. 340, 1801–1811. - PubMed
    1. Chambliss, K. L., Yuhanna, I. S., Anderson, R. G., Mendelsohn, M. E. & Shaul, P. W. (2002) Mol. Endocrinol. 16, 938–946. - PubMed
    1. Chambliss, K. L., Yuhannna, I. S., Mineo, C., Liu, C., German, Z., Sherman, T. S., Mendelsohn, M. E., Anderson, R. G. W. & Shaul, P. W. (2000) Circ. Res. E44–E52. - PubMed
    1. Chen, Z., Yuhanna, I. S., Galcheva-Gargova, Z. I., Karas, R. H., Mendelsohn, M. E. & Shaul, P. W. (1999) J. Clin. Invest. 103, 401–406. - PMC - PubMed
    1. Haynes, M. P., Sinha, D., Strong Russell, K., Collinge, M., Fulton D, MoralesRuiz, M. & Bender, J. R. (2000) Circ. Res. 87, 677–682. - PubMed

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