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Molecular Endocrinology, doi:10.1210/me.2006-0360
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Molecular Endocrinology 21 (6): 1370-1380
Copyright © 2007 by The Endocrine Society

Direct Interactions with G{alpha}i and Gß{gamma} Mediate Nongenomic Signaling by Estrogen Receptor {alpha}

Premlata Kumar, Qian Wu, Ken L. Chambliss, Ivan S. Yuhanna, Susanne M. Mumby, Chieko Mineo, Gregory G. Tall and Philip W. Shaul

Departments of Pediatrics (P.K., Q.W., K.L.C., I.S.Y., C.M., P.W.S.) and Pharmacology (S.M.M., G.G.T.), University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Philip W. Shaul, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9063. E-mail: philip.shaul{at}utsouthwestern.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor {alpha} (ER{alpha}). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ER{alpha} binds directly to G{alpha}i and Gß{gamma}. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251–260 and 271–595 of ER{alpha}, respectively. Studies of ER{alpha} complexed with heterotrimeric G proteins further show that estradiol causes the release of both G{alpha}i and Gß{gamma} without stimulating GTP binding to G{alpha}i. Moreover, in COS-7 cells, the disruption of ER{alpha}-G{alpha}i interaction by deletion mutagenesis of ER{alpha} or expression of blocking peptide, as well as Gß{gamma} sequestration with ß-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ER{alpha}-G{alpha}i interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ER{alpha} mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STEROID HORMONE RECEPTORS (SHRs) function classically in the nucleus as ligand-activated transcription factors. More recently, it has become apparent that steroid hormones also initiate a diverse set of important nongenomic cellular responses via the activation of plasma membrane-associated SHRs (1, 2). In particular, this has been elucidated regarding the nonnuclear actions of estrogen, which modify growth and differentiation, migration, and other processes in cell types as diverse as oocytes, osteoblasts, osteoclasts, neurons, breast cancer cells, adipocytes, and endothelial cells (1, 2, 3, 4, 5). The underlying mechanisms are best exemplified by the identification of a caveolae membrane-associated population of the classical estrogen receptor {alpha} (ER{alpha}) in endothelial cells that activates Src family tyrosine kinases, phosphatidylinositol 3 kinase/Akt kinase, and erk1,2 to stimulate nitric oxide (NO) production by the endothelial isoform of NO synthase (eNOS). These pathways are critically involved in estrogen-related cardiovascular protection (6). Activation of these pathways also stimulates phosphorylation of ER{alpha} and its coregulators and S-nitrosylation of the receptor to modify nuclear signaling, indicating that there is additional important cross talk between membrane and nuclear SHR function (3, 7).

In previous studies using endothelial cells, we demonstrated that signal initiation by membrane ER{alpha} is pertussis toxin (PTX) sensitive and that ER{alpha} and G{alpha}i can be coimmunoprecipitated from the plasma membrane (8). These findings and related evidence of heterotrimeric G protein involvement in signaling by ER in other cell types (9) raise the possibility that the most proximal mechanisms underlying membrane SHR actions entail interactions with G proteins. Heterotrimeric G proteins are activated conventionally by members of a family of G protein-coupled receptors (GPCRs), the sequences of which predict structures of seven membrane spans that include binding sites for G proteins. Agonist binding to GPCRs promotes the release of GDP from G{alpha}, thus allowing G{alpha} to bind the more abundant nucleotide in the cell, GTP. A conformational change in G{alpha} accompanies GTP binding, leading to the dissociation of G{alpha} and the high-affinity complex of ß and {gamma} subunits from the GPCR. Liberated G{alpha}-GTP and ß{gamma} subunits are competent to modulate the activity of downstream effectors (10, 11). In contrast to the in-depth knowledge available regarding G protein and GPCR interactions, the molecular basis of the functional linkage between SHRs such as ER{alpha} and G proteins is unknown.

In the present investigation we designed experiments to test the hypothesis that ER{alpha} interacts directly with G{alpha}i. Further studies were performed to address the following questions: 1) Are interactions between ER{alpha} and G{alpha}i required for nongenomic signaling by the receptor? 2) Do other SHR that mediate membrane-initiated signaling interact directly with G{alpha}i? 3) Does ER{alpha} also interact directly with Gß{gamma}? 4) What are the domains of ER{alpha} that interact with G{alpha}i and Gß{gamma}? 5) How does the interaction of ER{alpha} with G proteins initiate signaling? and 6) Do these mechanisms modify the function of endothelial cells, which have well-recognized nongenomic responses to estrogen of importance to cardiovascular protection?


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER{alpha} Interaction with G{alpha}i
To first investigate whether ER{alpha} interacts directly with monomeric G{alpha}i, we performed pull-down experiments using purified myristoylated G{alpha}i-GDP that contained a hexahistidine tag inserted at amino acid position 121 to preserve myristoylation and typical receptor interactions with the G{alpha}i C terminus (His6-G{alpha}i-GDP) (12, 13). Direct protein-protein interactions were evaluated with recombinant ER{alpha} protein in the absence or presence of varying concentrations of 17ß-estradiol (E2). In the absence of ligand, ER{alpha} bound G{alpha}i and the interaction was enhanced by E2 in a dose-dependent manner (Fig. 1AGo). The addition of ICI 182,780 alone blunted the interaction, and it also attenuated the enhancement in interaction prompted by E2 (supplemental Fig. 1AGo, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). In contrast to E2, dexamethasone and dihydrotestosterone had no effect on the ER{alpha}-G{alpha}i interaction (data not shown). ER{alpha} interacted preferentially with GDP-bound vs. GTP{gamma}S-bound G{alpha}i (Fig. 1BGo), and the interaction was enhanced by G{alpha}i myristoylation (Fig. 1CGo).


Figure 1
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  		Fig. 1. ER{alpha} Interacts Directly with G{alpha}i, and the Interaction Is Required for Signaling to src

A, Pull-down experiments were performed with myristoylated His6-G{alpha}i-GDP and recombinant ER{alpha} in the absence or presence of E2 at the indicated concentrations. Immunoblot analyses were performed for ER{alpha} and G{alpha}i. In all pull-down experiments, the input lanes represent 20% of the amount of protein used and the sample lanes contain 50% of the pull-downs. Results shown for all pull-downs are representative of three or more independent studies. B, Pull-downs were performed with myristoylated GDP- vs. GTP{gamma}S-bound His6-G{alpha}i. C, Pull-downs were performed with nonmyristoylated vs. myristoylated His6-G{alpha}i-GDP. D, COS-7 cells expressing ER{alpha} were pretreated with vehicle or PTX (100 ng/ml for 120 min) and incubated with 10–8 M E2 for 0–15 min, and src activation was evaluated by immunoblot analyses of cell lysates using anti-phospho-src (p-src) polyclonal and anti-src monoclonal antibodies. E, Cumulative results for src phosphorylation expressed as percentage in nontreated cells (basal) for three independent studies (mean ± SEM; *, P < 0.05 vs. basal).

 
To determine whether G protein interaction with ER{alpha} mediates nongenomic responses to E2, we evaluated src activation in COS-7 cells expressing ER{alpha} in the presence or absence of PTX. Src activation is an early signaling event in multiple membrane-initiated actions of E2 and ER{alpha} (1, 2, 3, 4, 5), numerous known GPCRs control src activation (14, 15), and we have previously shown that PTX prevents ligand-dependent ER{alpha}-G{alpha}i coimmunoprecipitation from plasma membranes (8). E2 (10–8 M)-induced src phosphorylation was prevented by PTX treatment (Fig. 1Go, D and E), indicating that G{alpha}i interaction with ER{alpha} is a required proximal process in membrane ER{alpha} signaling.

Interactions between Other SHRs and G{alpha}i
Multiple SHRs in addition to ER{alpha} initiate rapid responses upon ligand activation that are independent of the modification of gene transcription (1, 2, 16). To investigate whether the direct interaction observed between ER{alpha} and G{alpha}i is shared by other SHRs for which there is evidence of nongenomic signaling involving G proteins (1, 2, 17, 18, 19), plasma membranes purified from COS-7 cells expressing ERß, androgen receptor (AR), glucocorticoid receptor (GR), or vitamin D receptor (VDR) were tested in the myristoylated His6-G{alpha}i-GDP pull-down assay. Membrane-associated ERß and AR bound G{alpha}i, and binding was enhanced by the relevant SHR ligand (Fig. 2Go, A and B). In contrast, GR and VDR did not bind to His6-G{alpha}i-GDP in the absence or presence of ligand (Fig. 2Go, C and D). To determine whether the membrane-associated ERß and AR interactions with G{alpha}i signify direct protein-protein binding, additional His6-G{alpha}i-GDP pull-down experiments were performed with purified recombinant receptor proteins. Recombinant ERß bound G{alpha}i, and the interaction was enhanced by E2 (Fig. 2EGo). Recombinant AR in truncated form (amino acids 606–902) also bound G{alpha}i, and there was increased interaction in the presence of dihydrotestosterone (Fig. 2FGo). Thus, direct interaction with G{alpha}i is a shared feature of ER{alpha} and select SHRs that initiate signaling at the plasma membrane.


Figure 2
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  		Fig. 2. G{alpha}i Interacts with a Distinct Subset of SHRs

Purified myristoylated His6-G{alpha}i-GDP was used to pull down plasma membrane-associated (designated "m") ERß (A), AR (B), GR (C), and VDR (D) in the absence or presence of vehicle or 10–5 M E2, dihydrotestosterone, dexamethasone, or 1,25-dihydroxy vitamin D3, respectively. Proteins were eluted from the Ni-NTA resin with sample buffer and immunoblot analyses were performed for ERß, AR, GR, VDR, and G{alpha}i. In all experiments, the input lanes represent 20% of the amount of protein employed in the pull-down, and the sample lanes contain 50% of the pull-down. E and F, Pull-down experiments were performed with myristoylated His6-G{alpha}i-GDP and either recombinant ERß, in the absence or presence of 10–5 M E2 (E), or recombinant AR606–902 in the absence or presence of 10–5 M dihydrotestosterone (DHT) (F). All results shown are representative of three independent studies.

 
Domain of ER{alpha} Mediating Interaction with G{alpha}i
To identify the domain of ER{alpha} involved in direct interaction with G{alpha}i, Flag-tagged wild-type and deletion mutant human ER{alpha} proteins (Fig. 3AGo) were expressed and purified from baculovirus-infected Sf9 cells and used in pull-down assays with myristoylated His6-G{alpha}i-GDP. Wild-type ER{alpha} and an N-terminal deletion mutant lacking amino acids 1–175 (ER{alpha}{Delta}1–175) bound G{alpha}i comparably (Fig. 3BGo). A C-terminal deletion mutant lacking amino acids 271–595 (ER{alpha}{Delta}271–595) also interacted with G{alpha}i, but an internal deletion of amino acids 180–268 (ER{alpha}{Delta}180–268) prevented binding with G{alpha}i (Fig. 3CGo). In contrast to ER{alpha}{Delta}180–268, a mutant receptor lacking amino acids 185–251 (ER{alpha}{Delta}185–251) was capable of interaction with G{alpha}i (Fig. 3DGo), implicating amino acids 180–184 and/or 252–268 in binding with the G protein. Further investigation revealed that G{alpha}i interaction does not involve amino acids 261–271, and instead it was found that the region of ER{alpha} between amino acids 250 and 260 mediates direct binding to G{alpha}i (Fig. 3EGo). Moreover, the introduction of a peptide representing amino acids 251–260 of ER{alpha} disrupted the interaction between the wild-type receptor and G{alpha}i, whereas scrambled peptide did not (Fig. 3FGo), confirming the findings made by deletion mutagenesis.


Figure 3
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  		Fig. 3. Amino Acids 251–260 of ER{alpha} Interact Directly with G{alpha}i

A, Schematic of Flag-tagged wild-type ER{alpha} (WT) and ER{alpha} deletion mutants used in myristoylated His6-G{alpha}i-GDP pull-down experiments. The identities of the constructs are designated by lowercase letters on the schematic (A) and in the results shown (B–E). Comparisons were made in simultaneous pull-downs with WT ER{alpha} vs. ER{alpha}{Delta}1–175 (B), WT ER{alpha} vs. ER{alpha}{Delta}180–268 or ER{alpha}{Delta}271–595 (C), WT ER{alpha} vs. ER{alpha}{Delta}180–268 or ER{alpha}{Delta}185–251 (D), or with WT ER{alpha} vs. ER{alpha}{Delta}180–268, ER{alpha}{Delta}261–271, and ER{alpha}{Delta}250–260 (E). The interaction between WT ER{alpha} and G{alpha}i was also tested in the absence or presence of a peptide representing the G{alpha}i binding domain (GBD) comprised of amino acids 251–260 of the receptor or scrambled peptide (S) (F). Results shown are representative of three or more independent studies.

 
The involvement of G{alpha}i binding to amino acids 250–260 of ER{alpha} in nongenomic receptor signaling was then evaluated in studies of src phosphorylation in COS-7 cells. Whereas wild-type ER{alpha} promoted src phosphorylation with E2, no response was evident in cells expressing ER{alpha}{Delta}250–260 (Fig. 4Go, A and B). In parallel, we found that expression of an HA-tagged peptide consisting of only amino acids 251–260 of ER{alpha} mimicked the action of PTX (Fig. 1Go, D and E) and blocked nongenomic signaling by wild-type ER{alpha} to src (Fig. 4Go, C and D). These collective observations identify amino acids 251–260 of ER{alpha} as a G{alpha}i binding domain that is critically involved in nongenomic signaling by the receptor.


Figure 4
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  		Fig. 4. Disruption of ER{alpha} Interaction with G{alpha}i Prevents src Activation by E2

A, COS-7 cells were transfected with plasmid encoding WT ER{alpha} or ER{alpha}{Delta}250–260 and incubated with 10–8 M E2 for 0–15 min, and src activation was evaluated by immunoblot analyses of cell lysates using anti-phospho-src (p-src) polyclonal and anti-src monoclonal antibodies. B, Cumulative results for src phosphorylation expressed as percentage in nontreated cells (basal) for three independent studies (mean ± SEM; *, P < 0.05 vs. basal). C, COS-7 cells were transfected with plasmid encoding WT ER{alpha} and either empty vector or a plasmid encoding His-tagged peptide comprised of amino acids 251–260 of ER{alpha}, incubated with 10–8 M E2 for 0–15 min, and evaluated for src activation. D, Cumulative results for src phosphorylation for three independent studies (mean ± SEM; *, P < 0.05 vs. basal).

 
ER{alpha} Interaction with Heterotrimeric G{alpha}ß{gamma} and Gß{gamma} Dimer
In order for G{alpha}i to interact effectively with classical GPCRs, G{alpha}i is associated with Gß{gamma} as a heterotrimer. In addition, both activated G{alpha}i and Gß{gamma} are capable of modulating the activity of downstream effector molecules (10, 11). Therefore, pull-down experiments were performed using purified components to compare the capacity of ER{alpha} to bind monomeric His6-G{alpha}i-GDP and His6-G{alpha}i-GDP associated with Gß1{gamma}2. In the absence of agonist, ER{alpha} interaction with G{alpha}i was enhanced by Gß{gamma} (Fig. 5AGo). Whereas ER{alpha} interaction with monomeric G{alpha}i was increased by E2, the addition of the ligand diminished the interaction between the receptor and G{alpha}i in heterotrimeric form. These observations raised the possibility that ER{alpha} also binds Gß{gamma} directly. Pull-down experiments employing Flag-tagged ER{alpha} demonstrated that such an interaction occurs, and that it is attenuated by E2 (Fig. 5BGo). The addition of ICI 182,780 reversed the E2-induced decrease in interaction between ER{alpha} and Gß{gamma}, and ICI 182,780 alone actually caused an increase in the interaction (supplemental Fig. 1B, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Furthermore, a decline in the ER{alpha}-Gß1{gamma}2 interaction, and also in the ER{alpha}-G{alpha}i interaction, occurred with the addition of E2 to the Flag-tagged ER{alpha} pull-down of G protein heterotrimer (Fig. 5CGo). Moreover, in heterotrimer experiments ICI reversed the loss of ER{alpha}-G{alpha}i interaction that occurs with E2, but not the decrease in ER{alpha}-Gß{gamma} interaction with E2 (supplemental Fig. 1CGo, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org); in fact, ICI 182,780 alone acted similar to E2 alone and caused a decline in ER{alpha}-Gß{gamma} interaction. Thus, the modulation of ER{alpha}-G protein interactions by the ER{alpha} ligands E2 and ICI 182,780 differs whether or not the G proteins are in heterotrimeric form.


Figure 5
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  		Fig. 5. ER{alpha} Interacts Directly with Gß{gamma}

A, Recombinant ER{alpha} protein was pulled down with myristoylated His6-G{alpha}i-GDP using Ni-NTA in the absence or presence of recombinant Gß1{gamma}2 and 10–8 M E2. Immunoblot analyses were performed for ER{alpha}, G{alpha}i, and Gß. B, Purified Gß1{gamma}2 was pulled down with recombinant Flag-tagged wild-type ER{alpha} in the absence or presence of E2. C, Purified G{alpha}i-GDP and Gß1{gamma}2 were pulled down using Flag-tagged wild-type ER{alpha} bait in the absence or presence of E2. D, Comparisons were made in simultaneous Gß1{gamma}2 pull-downs with recombinant Flag-tagged wild-type ER{alpha} (WT) vs. ER{alpha}{Delta}271–595 vs. ER{alpha}{Delta}180–268, with a nonspecific band noted at 50 kDa in all samples in immunoblots for ER{alpha}.

 
To identify the domain(s) of ER{alpha} involved in direct Gß{gamma} binding, experiments were performed using Flag-tagged wild-type ER{alpha} and deletion mutants of ER{alpha} to pull down Gß{gamma}. Whereas wild-type ER{alpha} and the deletion mutant lacking amino acids 180–268 (ER{alpha}{Delta}180–268) displayed comparable binding with Gß{gamma}, the mutant lacking amino acids 271–595 (ER{alpha}{Delta}271–595) did not interact with Gß{gamma} (Fig. 5DGo). Thus, Gß{gamma} interacts directly with ER{alpha} via a receptor domain(s) that is distinct from the G{alpha}i binding domain. Moreover, the interaction with Gß{gamma} promotes receptor interaction with G{alpha}i, and the complex formed by the receptor and heterotrimeric G{alpha}ß{gamma} is disrupted upon agonist binding to the receptor.

Mechanism Underlying Signal Initiation by ER{alpha} Complexed with G{alpha}ß{gamma}
Having observed that the ER{alpha} interaction with G{alpha}i is required for signal transduction and that the receptor also complexes G{alpha}ß{gamma} directly, the potential ability of ER{alpha} to activate G protein heterotrimers by acting as a guanine nucleotide exchange catalyst was explored. Membranes prepared from Sf9 cells coexpressing heterotrimeric G{alpha}ß{gamma} and either ER{alpha} or the M2 muscarinic receptor were incubated with [35S]GTP{gamma}S to determine the kinetics of G{alpha}i nucleotide binding. In the absence of ligand, the M2 muscarinic receptor did not alter the rate of G{alpha}i-GTP production appreciably (Fig. 6AGo); in contrast, expression of ER{alpha} appeared to stimulate a slow kinetic exchange of guanine nucleotide (Fig. 6BGo). However, whereas carbachol stimulation of the M2 muscarinic receptor promoted more rapid G{alpha}i [35S]GTP{gamma}S binding (Fig. 6AGo), E2 stimulation of ER{alpha} did not result in an increase in [35S]GTP{gamma}S binding (Fig. 6BGo), and ICI 182,780 also did not affect guanine nucleotide exchange (data not shown). These results suggest that the mechanism of E2-induced activation of ER{alpha} and G proteins is more complex than simple regulation of the G{alpha}i guanine nucleotide switch, thus differing significantly from GPCR-induced signaling.


Figure 6
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  		Fig. 6. Signal Initiation by E2 and ER{alpha} Complexed with G{alpha}ß{gamma} Is Mediated by Gß{gamma} Independent of Guanine Nucleotide Exchange

A and B, The kinetics of GPCR and ER{alpha} stimulation of membrane-bound G protein GTP binding differ. A, Membranes from Sf9 cells infected with baculoviruses that express His6-tagged-G{alpha}i, Gß, and G{gamma}, and either no additional virus or the M2 muscarinic receptor virus were incubated with [35S]GTP{gamma}S at 30 C in the absence or presence of 10–6 M carbachol. Aliquots of reaction mixtures were taken at the indicated time points, quenched, and Ni-NTA resin was used to pull down the detergent-extracted G{alpha}i. The amount of His-tagged G protein-bound [35S]GTP{gamma}S was determined by liquid scintillation counting. B, Parallel studies of ER{alpha} were performed in the absence or presence of 10–8 M E2. In A and B, results for G{alpha}ß{gamma} alone are indicated by circles and those for G{alpha}ß{gamma} plus receptor by squares, and open and closed symbols represent findings in the absence and presence of ligand, respectively. Values shown in A and B are means for n = 2, and results were confirmed in three separate experiments. C, COS-7 cells transfected with plasmid encoding wild-type ER{alpha} and either empty vector or the cDNA for the ßARK-ct were incubated with 10–8 M E2 for 0–15 min, and src and erk activation was evaluated by immunoblot analyses of cell lysates using anti-phospho-src (p-src), anti-phospho-erk (p-erk), or anti-src or anti-erk antibodies. Cumulative results are shown for src phosphorylation (D) and erk phosphorylation (E) expressed as percentage in non-E2-treated cells (basal) for three independent studies (mean ± SEM; *, P < 0.05 vs. basal).

 
Because we demonstrated that Gß{gamma} interacts dynamically with ER{alpha} and free Gß{gamma} can mediate subsequent cellular responses in diverse paradigms (10, 11), the possibility that liberated Gß{gamma} modulates downstream signaling by ER{alpha} was investigated. COS-7 cells expressing ER{alpha} were transfected with either empty plasmid or plasmid encoding the ß-adrenergic receptor kinase C-terminal tail (ßARK-ct). Under control conditions, E2 stimulated the phosphorylation of src (Fig. 6Go, C and D). In contrast, in cells expressing ßARK-ct, there was an abrogation of E2-induced src activation. To evaluate another downstream target of activated Gß{gamma}, erk phosphorylation was also examined (Fig. 6Go, C and E). Paralleling the findings for src, E2-stimulated erk activation was also blunted by ßARK-ct. These cumulative findings show that E2 activation of ER{alpha} releases both G{alpha}i and Gß{gamma} without stimulating GTP binding to G{alpha}i, and that Gß{gamma} in turn activates the immediate downstream signaling targets src and erk, which are of known importance to multiple nongenomic ER{alpha} actions (1, 2, 3, 4, 5).

ER{alpha}-G Protein Interactions and E2 Modulation of Endothelial Cell Function
The importance of direct ER{alpha}-G protein interactions to cell function was then addressed in the context of G protein-dependent, plasma membrane-associated ER{alpha} activation of eNOS (8). In bovine aortic endothelial cells (BAEC), stimulation of eNOS by E2 was prevented by ICI 182,780 (Fig. 7AGo). In further experiments, BAEC were transfected with empty vector or plasmid encoding the ER{alpha} mutant ER{alpha}{Delta}250–260, which displayed an inability to interact with G{alpha}i (Fig. 3EGo) and an inability to promote E2 activation of src (Fig. 4Go, A and B). Of note, in this model system ER{alpha} and G{alpha}i are present at endogenous levels, and in previous studies we have shown that the overexpression of wild-type ER{alpha} in endothelial cells enhances eNOS activation by E2 (20). Whereas control cells displayed eNOS activation by E2, the response was absent in cells expressing ER{alpha}{Delta}250–260 (Fig. 7BGo). In contrast, eNOS activation by vascular endothelial growth factor or acetylcholine was not altered by ER{alpha}{Delta}250–260 expression and E2-mediated gene transcription assessed using an estrogen response element-luciferase promoter-reporter construct was also not affected (data not shown), indicating that the mutant has a selective dominant-negative action on nongenomic ER function. Similarly, in cells expressing an ER{alpha} peptide consisting of amino acids 251–260, E2 stimulation of eNOS was fully impaired (Fig. 7CGo) but E2-mediated gene transcription was unchanged (data not shown). To test the requirement for ER{alpha}-G protein interactions in the modulation of an endothelial cell phenotype of relevance to E2-related cardioprotection, the impact of the dominant-negative mutant ER{alpha}{Delta}250–260 on E2-induced attenuation of monocyte adhesion was evaluated (Fig. 7Go, D and E). In BAEC transfected with empty vector, the marked increase in monocyte adhesion caused by lipopolysaccharide (LPS) was fully prevented by E2. The effect of E2 was due to nongenomic activation of eNOS because it was abrogated by nitric oxide synthase (NOS) antagonism with N-nitro-L-arginine methyl ester and the hormone did not alter eNOS enzyme abundance (Fig. 7EGo, inset). In contrast, in cells expressing the dominant-negative mutant ER{alpha} (ER{alpha}{Delta}250–260), the E2-related, NO-dependent decrease in monocyte adhesion was absent. Thus, the direct interactions between ER{alpha} and G proteins are required for E2-induced nongenomic actions in endothelial cells of significance to vascular health and disease.


Figure 7
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  		Fig. 7. Disruption of ER{alpha}-G{alpha}i Interaction in Endothelial Cells Attenuates ER-Dependent E2 Activation of eNOS and Resulting Antagonism of Monocyte Adhesion

A, eNOS activation in BAEC was assessed in the presence of buffer alone (basal), 10–8 M E2, or E2 plus 10–5 M ICI 182,780 for 15 min. B and C, BAEC were transfected with empty vector (control) vs. plasmid encoding ER{alpha}{Delta}250–260 (B), or with empty vector vs. a plasmid that expressed a peptide consisting of amino acids 251–260 of the receptor (ER{alpha}251–260) (C), and E2-stimulated eNOS activity was assessed. In A–C, values are mean ± SEM; n = 6. *, P < 0.05 vs. basal; {dagger}, P < 0.05 vs. no ICI 182,780. D, Monocyte adhesion was assessed in BAEC transfected with an empty vector (upper panels) or a plasmid that expressed ER{alpha}{Delta}250–260 (lower panels) and treated with medium alone (control), LPS (100 ng/ml), LPS plus E2 (10–8 M), or LPS plus E2 plus nitro-L-arginine methyl ester (L-N) (2 mM). Images are representative optical fields. E, Cumulative findings for monocytes adhered per x20 magnification field; mean ± SEM; n = 4–5. *, P < 0.05 vs. control; {dagger}, P < 0.05 vs. LPS alone. Inset shows immunoblot analyses for eNOS and actin in the various treatment groups.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SHRs including ER{alpha}, ERß, AR, GR, and VDR mediate a variety of nongenomic responses that govern the behaviors of multiple cell types, and evidence has accumulated that in many contexts these processes are G protein dependent (16, 17, 18, 19, 21, 22). With a focus on ER{alpha}, we have demonstrated for the first time direct interactions between an SHR and G proteins, and have determined that such interactions are critically involved in nongenomic steroid hormone signaling.

In pull-down experiments with purified recombinant proteins, we first showed that there is a direct protein-protein interaction between ER{alpha} and monomeric G{alpha}i, which is enhanced specifically by E2. We also demonstrated that the interaction is altered by modifications of G{alpha}i that govern its interactions with classical GPCRs (10). Using PTX in studies of src phosphorylation, we further determined that G{alpha}i interaction with ER{alpha} is an essential proximal process in membrane ER{alpha} signaling.

In experiments evaluating whether the interaction observed between ER{alpha} and G{alpha}i is shared by other SHRs capable of nongenomic signaling involving G proteins (1, 2, 17, 18, 19), we found that ERß and AR also display direct binding with G{alpha}i that is enhanced by their respective steroid hormone ligands. These observations are consistent with the parallel capacity of membrane-associated ER{alpha} and ERß to promote signaling to eNOS in cultured endothelial cells (21), and the ability of androgens to mediate PTX-sensitive signaling in cell types as diverse as neurons and skeletal muscle (17, 22). In contrast, we observed that GR and VDR do not bind to G{alpha}i. Thus, direct interaction with G{alpha}i is a shared feature of ER{alpha} and select SHRs that initiate signaling at the plasma membrane. Direct interactions with G{alpha}s or G{alpha}q may be operative in the nongenomic functions of other SHRs such as GR and VDR. Consistent with the latter possibility, it has been demonstrated that G{alpha}q is required for VDR-induced nongenomic signaling during matrix biogenesis by chondrocytes (19).

In further studies of ER{alpha}, pull-down experiments performed with G{alpha}i and mutant receptor proteins revealed that the region of ER{alpha} between amino acids 250 and 260 mediates the direct binding to G{alpha}i. In addition, a peptide representing amino acids 251–260 of ER{alpha} disrupted the interaction between the wild-type receptor and G{alpha}i. Furthermore, the ER{alpha} mutant lacking amino acids 250–260 was incapable of activating src in COS-7 cells, and in cells expressing wild-type ER{alpha} the coexpression of an HA-tagged peptide representing amino acids 251–260 blocked nongenomic signaling to src. Thus, we have identified amino acids 251–260 of ER{alpha} as a G{alpha}i binding domain that is critically involved in nongenomic signaling by the receptor. Because there is negligible homology between these amino acids and the corresponding regions of ERß and AR, which we show also bind directly to G{alpha}i, detailed mutagenesis will now be required to identify the domains within ERß and AR mediating this interaction. Interestingly, the G{alpha}i binding domain of ER{alpha} resides within nuclear localization signal 3 (23), raising the intriguing possibility that there are competitive mechanisms dictating the relative function of the receptor at the plasma membrane and in the nucleus.

Additional pull-down experiments focused on the role of Gß{gamma} in ER{alpha}-G{alpha}i coupling. We found that Gß{gamma} interacts directly with ER{alpha} via a receptor domain(s) residing within amino acids 271–595, which is distinct from the G{alpha}i binding domain, that the interaction with Gß{gamma} promotes receptor interaction with G{alpha}i, and that the complex formed by ER{alpha} and heterotrimeric GDP-bound G{alpha}ß{gamma} is disrupted upon E2 activation of the receptor. We also observed that Gß{gamma} is liberated from ER{alpha} by E2 in the absence of G{alpha}i. In studies of the kinetics of G{alpha}i nucleotide binding, the activation of the M2 muscarinic receptor serving as a positive control promoted rapid GTP{gamma}S binding to G{alpha}i, whereas E2 stimulation of ER{alpha} did not. Therefore, Gß{gamma} is released by ER{alpha} independently of conventional GTP binding to G{alpha}i and the resulting conformational change in G{alpha}i that disassociates the ß{gamma} dimer during signaling by classical GPCRs (10, 11). We postulate that the liberation of Gß{gamma} is mediated alternatively by conformational changes that occur in ER{alpha} upon ligand binding. Similar changes in ER{alpha} conformation are known to modify the interaction of the receptor with nuclear cofactors (24, 25). Evidence that conformational changes may impact on ER{alpha}-G protein interactions lies in our findings in pull-downs with ICI 182,780, which modify ER{alpha} conformation in a manner that is unique compared with E2 (24, 25). Under certain conditions, ICI 182,780 reversed E2 effects on ER{alpha}-G protein interactions, under other conditions the ICI compound independently altered ER{alpha}-G protein interactions, and the modulation of ER{alpha}-G protein interactions by both E2 and ICI 182,780 differed whether or not the G proteins were in heterotrimeric form. Furthermore, experiments in ER{alpha}-expressing COS-7 cells showed that cotransfection with ßARK-ct attenuates E2-induced srk and erk activation, indicating that the liberated Gß{gamma} modulates downstream signaling. Thus, we have identified a novel means of G protein activation that provides greater diversity of function of an SHR.

The importance of direct ER{alpha}-G protein interactions to cell function was addressed in studies of ER{alpha} activation of eNOS in cultured endothelium. This process is critically involved in the vascular actions of E2 that underlie the lower risk of cardiovascular disease in premenopausal women vs. men and the potential of estrogen replacement therapy to be cardioprotective (26). The disruption of ER{alpha}-G{alpha}i interaction prevented E2-induced eNOS activation, which was ER dependent, and it also negated the resulting attenuation of monocyte adhesion that is highly relevant to the initiation of atherosclerosis (26). As such, ER{alpha}-G{alpha}i interaction plays an important role in dictating the phenotype of a cell type with well-recognized responses to E2.

The mechanisms that we have elucidated in which E2 initiates downstream nongenomic responses by liberating Gß{gamma} from G{alpha}i and ER{alpha} upon ligand activation independent of guanine nucleotide exchange add considerably to the processes described to date for nongenomic estrogen signaling. Another example is the potential role of direct estrogen binding to the GPCR GPR30. This event occurs in the endoplasmic reticulum, and it is thought to promote intracellular calcium mobilization and the generation of PIP3 in the nucleus (27). Additional mechanisms include the involvement of adaptor proteins such as the modulator of nongenomic actions of the ER (MNAR) and striatin, which bind to ER to promote the maintenance of a signaling module (28, 29). In the emerging field of nongenomic endocrinology, our discovery of direct ER{alpha}:G protein interactions provides important new understanding of the proximal mechanisms by which proteins classically known to serve as transcription factors exhibit a second fundamental capacity to initiate hormone signaling at the plasma membrane. This work also reveals a new role for G protein signaling outside of conventional GPCR activation. It is anticipated that future efforts in this realm will enable us to continue to reveal the intricacies of SHR biology dictating ultimate cellular responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Production of Recombinant G{alpha}I, Gß{gamma}, ER{alpha}, and mutant ER{alpha}
His-tagged G{alpha}i1 was purified from Escherichia coli that had been transformed with a plasmid encoding rat G{alpha}i1 alone or together with a plasmid encoding yeast N-myristoyltransferase to produce myristoylated G{alpha}i (30). Gß1{gamma}2 dimers were synthesized and purified from Sf9 cells as previously described (31). Baculoviruses encoding Flag-tagged wild-type human ER{alpha} and mutant ER{alpha} truncated proteins were produced and amplified using the Bac-to-Bac Sf9 cell transfection system (Invitrogen, Carlsbad, CA). To create the constructs for Flag-tagged wild-type ER{alpha} and the truncation mutants ER{alpha}{Delta}271–595, ER{alpha}{Delta}185–251, ER{alpha}{Delta}261–271, and ER{alpha}{Delta}250–260, the Flag-tag was first inserted N-terminally into the wild-type and the mutant plasmids in pCDNA3.1 using oligonucleotides encoding the heptapeptide tag MDYKDDDK and the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Using EcoR1 restriction sites, the wild-type and mutant receptor forms with Flag tags were transferred into pFASTBAC1 (Invitrogen) for expression in Sf9 cells. Constructs for Flag-tagged ER{alpha}{Delta}1–175 and ER{alpha}{Delta}185–268 were kindly provided by Dr. W. Lee Kraus (Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY). The sequence of all constructs was verified. To prepare the recombinant proteins, Sf9 cells growing in IPL41 medium were infected with baculovirus for 48 h, pelleted, and homogenized in lysis buffer [20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, and protease inhibitor cocktail (Calbiochem, San Diego, CA)], and lysates were subjected to centrifugation at 12,000 x g at 4 C. Lysates were incubated with Anti-Flag M2 affinity gel (Sigma, St. Louis, MO) at 4 C to isolate the Flag-tagged proteins. After four washes, the proteins were eluted by competition with flag peptide. Purity assessed by SDS-PAGE, and Coomassie blue staining was consistently greater than 95%.

Protein Interaction Analyses Using Pull-Downs
Purified myristoylated His-tagged G{alpha}i1 (300 nM) was incubated in 500 µl of 20 mM HEPES buffer (pH 8.0) containing 150 mM NaCl, 5 mM MgCl2, 4% glycerol, 0.05% C12E10, and protease inhibitor cocktail (Calbiochem), with 30 µM GDP or GTP{gamma}S added for 1 h at 30 C. Purified Flag-tagged ER{alpha} proteins were added, plus or minus E2 at 10–5–10–8 molar concentrations, and reactions were incubated at 4 C for 1 h with gentle agitation. Further incubation was performed for 1 h with Ni-nitrilotriacetic acid (NTA) resin (Qiagen, Valencia, CA) to allow binding of His-tagged G{alpha}i1. Samples were washed with the 20 mM HEPES buffer, and the resin was pelleted and suspended in SDS-PAGE sample buffer. After resolution by 10% SDS-PAGE, immunoblot analyses were performed with the G{alpha}i1/2-specific antiserum B087 (32), and mouse monoclonal antibodies Ab-15 (Labvision, Fremont, CA) or AER320 (Labvision) directed against ER{alpha}. In selected experiments, the impact of ICI 182,780 (10–5 M) was determined. In other studies, a peptide representing amino acids 251–260 of ER{alpha} (MKGGIRKDRR) or scrambled peptide (GRGKRIRDKM) was added to the pull-downs (10x relative to wild-type ER{alpha}). Additional pull-downs were performed with myristoylated G{alpha}i1-GDP and recombinant ERß (Invitrogen) or recombinant AR in truncated form (amino acids 606–902) (Invitrogen). Flag pull-down experiments were performed similarly using the Flag-tagged wild-type ER{alpha} and mutant ER{alpha} proteins, G{alpha}i and/or Gß1{gamma}2 and Anti-Flag M2 affinity gel (Sigma). In additional experiments, protein interactions were evaluated using COS-7 cell plasma membranes. COS-7 cells were transfected with cDNAs for ERß, AR (kindly provided by Dr. Michael McPhaul, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX), GR, or VDR, and 48 h later plasma membranes were isolated as previously described (21). The plasma membranes were then used in pull-down experiments with myristoylated G{alpha}i1-GDP done in the absence or presence of 10–5 M E2, dihydrotestosterone, dexamethasone, or 1,25-dihydroxy vitamin D3, respectively. The Ni-NTA eluted samples were resolved by 10% SDS-PAGE, and immunoblot analyses were performed with receptor-specific antibodies for ERß and VDR (Affinity BioReagents, Golden, CO) and AR and GR (Santa Cruz Biotechnology, Santa Cruz, CA), or with the G{alpha}i1/2 protein-specific antiserum B087.

Cell Culture and Transfection
COS-7 cells (American Type Culture Collection, Manassas, VA) grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum were transfected with cDNA encoding wild-type human ER{alpha} or ER{alpha}{Delta}250–260 in pCDNA3.1 using LipofectAMINE Plus (Invitrogen). In selected studies, cells were cotransfected with either empty vector or pLP-CMV-HA(ER{alpha}251–260), an HA-tagged peptide comprised of amino acids 251–260 of ER{alpha}, or with empty vector vs. cDNA for ßARK-ct kindly provided by Dr. Robert Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC) (33). Primary BAEC were cultured and maintained as previously described and used within seven passages (34). BAEC were transfected with either empty vector vs. ER{alpha}{Delta}250–260, or with empty vector vs. pLP-CMV-HA(ER{alpha}251–260). The sequences of all constructs were verified, and expression was confirmed by immunoblot analyses.

Src and erk Activation by Immunoblot Analyses
To assess src activation, COS-7 cells were treated with 10–8 M E2 for 0–15 min and lysed, and immunoblot analyses were performed using anti-phospho-tyrosine-416 Src polyclonal antibody (Cell Signaling Technology, Danvers, MA) and anti-Src monoclonal antibody (Santa Cruz Biotechnology). To assess erk activation, immunoblotting was performed with anti-phospho-erk polyclonal antibody (Promega, Madison, WI) and anti-erk2 monoclonal antibody (Upstate Biotechnology, Charlottesville, VA).

GTP{gamma}S Binding Studies
Sf9 cells were grown in IPL41 medium and infected with baculoviruses that expressed His6-G{alpha}i, Gß1, and G{gamma}2, and either no additional virus or M2 muscarinic receptor or ER{alpha} baculoviruses. Forty-eight hours later, cell membranes were harvested, homogenized into buffer containing 20 mM HEPES, 150 mM NaCl, 2 mM MgSO4, and 1 mM EDTA (pH 8.0) and used for GTP{gamma}S binding time course studies. [35S]GTP{gamma}S was added to the membranes at 30 C in the presence or absence of ligand (10–6 M carbachol or 10–6 M E2 or 10–5 M ICI 182,780) to initiate the reactions and aliquots were removed at specific time points. Each reaction aliquot was quenched in stop buffer (300 mM MgCl2, 3.0 mM GDP, 3.0 mM GTP) and extracted with 1% sodium cholate for 1 h at 4 C. After centrifugation at 100,000 x g for 20 min, the extracts were adsorbed onto Ni-NTA in a buffer containing 20 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM MgCl4, 10 µM GTP, 0.5% C12E10 (Sigma) to pull down His-tagged G{alpha}i-GTP{gamma}S. The amount of G{alpha}i-GTP{gamma}S (picomoles per microgram of membrane protein) was determined by liquid scintillation counting.

NOS Activation
NOS activation was assessed in intact BAEC by measuring L-[14C]arginine conversion to L-[14C]citrulline using previously reported methods (35). Cells were treated with 10–8 M E2 in the absence or presence of 10–5 M ICI 182,780. Stimulated activity is expressed as a percentage of basal activity, and results were confirmed in three independent experiments.

Monocyte Adhesion Assays
The adhesion of monocytes to BAEC was evaluated as previously described (35). Near-confluent BAEC were treated with medium alone or medium plus LPS (100 ng/ml) for 18 h in the absence or presence of 10–8 M E2 with or without 2 mM nitro-L-arginine methyl ester. U937 cells (1 x 106 per 35-mm plate) were added to each monolayer under rotating conditions, nonadhering cells were removed by gentle washing with PBS, cells were fixed with 1% paraformaldehyde, and the number of adherent cells was counted per x20 magnified field. eNOS and actin abundance was evaluated by immunoblot analyses in additional plates of BAEC treated in an identical manner.

Statistical Analysis
Comparisons were made between multiple groups by ANOVA with Neuman-Keuls post hoc testing. Significance was defined as P < 0.05.


   ACKNOWLEDGMENTS
 
We thank M. Dixon for preparing this manuscript.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grants HD30276 (to P.W.S.) and GM34497 (to Alfred Gilman), by an American Heart Association (Texas Affiliate) Postdoctoral Fellowship (0325033Y to G.G.T.), by the Lowe Foundation (to P.W.S.), and by the Crystal Charity Ball Center for Pediatric Critical Care Research (to P.W.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2007

Abbreviations: AR, Androgen receptor; ßARK-ct, ß-adrenergic receptor kinase C-terminal tail; BAEC, bovine aortic endothelial cell; eNOS, endothelial isoform of nitric oxide synthase; ER, estrogen receptor; GPCR, G protein-coupled receptor; GR, glucocorticoid receptor; LPS, lipopolysaccharide; NO, nitric oxide; NOS, nitric oxide synthase; NTA, nitrilotriacetic acid; PTX, pertussis toxin; SHR, steroid hormone receptor; VDR, vitamin D receptor.

Received for publication August 30, 2006. Accepted for publication March 28, 2007.


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