The role of β₁Pix/caveolin-1 interaction in endothelin signaling through G α subunits

Abstract

Endothelin-1 (ET-1) is a potent mitogen that transmits signals through its cognate G protein-coupled receptors to stimulate extracellular signal-regulated kinase Erk1/2. Endothelin-1 receptors (ET-Rs) are known to interact with caveolin-1 and co-localize in caveolae which integrate different receptor and signaling proteins. We have recently shown that β₁Pix binds specifically to ET-Rs. Here, we show that β₁Pix binding to caveolin-1 is dependent on heterotrimeric G proteins activation state. β₁Pix interaction with different G proteins is increased in the presence of the G protein activator AMF. Moreover, extraction of cholesterol with methyl-β-cyclodextrin disrupts the binding of β₁Pix to Gα_q, Gα₁₂ and phospho-Erk1/2 but not the binding of β₁Pix to G_β1. The disruption of β₁Pix dimerization strongly reduced the binding of caveolin-1, Gα_q and Gα₁₂. Constitutively active mutants of Gα_q and Gα₁₂ increased Cdc42 activation when co-expressed with β₁Pix but not in the presence of β₁Pix dimerization deficient mutant β₁PixΔ (602–611). ET-1 stimulation increased the binding of phosphorylated Erk1/2 to β₁Pix but not to β₁PixΔ (602–611). RGS3 decreased ET-1-induced Cdc42 activation. These results strongly suggest that the activation of ET-Rs leads to the compartmentalization and the binding of Gα_q to β₁Pix in caveolae, where dimeric β₁Pix acts as platform to facilitate the binding and the activation of Erk1/2.

Introduction

Endothelin-1 (ET-1) is a potent mitogenic peptide that mediates a wide variety of cell signaling including cell proliferation and gene expression through its G protein-coupled receptors, ET_AR and ET_BR [1] which can activate different G proteins. ET_AR acts through G_q and G_s to stimulate phospholipase C (PLC) and adenylate cyclase respectively. ET_BR stimulates PLC via G_q, and inhibits adenylate cyclase through G_i [2;3]. ET receptors have also been shown to couple to G₁₂ in NIH3T3 cells where G₁₂ mediates the formation of actin stress fiber formation [4]. ET_AR and ET_BR are both localized within caveolae [5;6], invaginations of the plasma membrane containing caveolin proteins which act as scaffolding proteins to cluster different signaling molecules such as G_α subunits, Ha-Ras, PKC and Src [7]. We have shown that ET-1 induces cell proliferation through up-regulation of the Cdc42/Rac guanine nucleotide exchange factor β₁Pix [8]. β₁Pix contains a Src homology 3 (SH3) domain, a Dbl homology (DH) domain, a pleckstrin homology (PH) domain for activation of Rho GTPases and a leucine zipper (LZ) domain responsible for β₁Pix dimerization [9]. ET-1 stimulation of mesangial cells induces β₁Pix translocation to focal adhesion and Cdc42 activation through PKA-dependent phosphorylation of β₁Pix [10]. Moreover, the activation of Gα_s signaling pathway results in the binding of 14-3-3β to β₁Pix through protein kinase A-dependent phosphorylation leading to the inhibition of Rac1 and membrane ruffle formation [11]. β₁Pix regulates mast cell secretion through G_αi-dependent mechanism [12] and β₁Pix/G_αi3 interaction mediates p66Shc activation induced by EGF receptor transactivation [13].

Although several studies have shown that Erk1/2 activation by extracellular stimuli requires small GTPases activation, the mechanisms whereby G protein coupled receptors (GPCRs) regulate this signaling pathway are not fully understood. One mechanism of Erk1/2 activation involves Gα_i-coupled receptor where ligand stimulation induces the release of G_βγ leading to Ras activation. In addition, ET-1 can also activate Erk1/2 through EGF receptor transactivation [14]. In recent years, there has been increasing evidence that the activation of some small GTPases by GPCRs is mediated by the activation of guanine nucleotide exchange factors (GEFs). For example, a family of RhoAGEFs consisting of p115RhoGEF, PDZ-RhoGEF and leukemia-associated RhoGEF contain a regulator of G protein signaling homology domain that directly binds to Gα₁₂ [15;16]. The p63RhoGEF, however, was found to link RhoA to Gα_q/11-coupled receptors by directly interacting with active Gα_q/11 [17].

Materials and Methods

Materials

Cell culture media and supplements were obtained from Invitrogen (Carlsbad, CA). Endothelin-1 was purchased from Calbiochem (La Jolla, CA). Anti-ET_A-R and anti-ET_B-R were from Millipore (Billerica, MD). Anti-Myc, anti-caveolin-1, anti-G_β1 and anti-Gα subunits antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Antiphospho-Erk1/2 was purchased from Cell Signaling (Danvers, MA). RGS3 antiserum was described previously [18].

Cell culture and transfection

Human mesangial cells were cultured in RPMI 1624 (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a 37°C humidified incubator with 5% CO₂. Transient transfection of cells with mammalian expression vectors was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Plasmids

The Myc-tagged β₁Pix and β₁PixΔ (602–611) plasmids have been described [8;11]. Gα_q, Gα_qQ209L, Gα₁₂, Gα_12Q231L were obtained from Missouri S&T cDNA Resource Center. RGS3 was inserted into pcDNA3.1 plasmid as described before [18].

Immunoprecipitation and Western blot analysis

Cells were transfected with the appropriate construct for 24 h. Cells were washed twice in phosphate-buffered saline (PBS) and lysed in lysis buffer containing 20 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. Equal amounts of proteins were separated by using 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore), immunoblotted with the appropriate antibody, and visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Inc.). For immunoprecipitation, antibodies against Myc, caveolin-1 or Gα subunits were added to the cell lysate (500 µg) for 2 h of incubation, followed by addition of protein A- or protein G-agarose beads for an additional hour. The beads were washed three times in PBS. The immunoprecipitated proteins were released from the beads by boiling in 1x sample buffer for 5 min and subsequently analyzed by Western blotting. Total cell lysate (TCL) was run to assess the over-expression of the constructs. Expression of recombinant Myc-β₁Pix and its mutants was verified by immunoblotting with anti-Myc antibody.

Cdc42 activity assay

Cells were transfected with empty vector, Myc-tagged β₁Pix alone or in combination with the indicated Gα subunit for 24 h. Cells were lysed in lysis/wash buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, 1 mM EDTA, 1% glycerol, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). To measure the active GTP-bound form of Cdc42 GTPase in the cell lysates, we performed a pull-down assay (Upstate) using recombinant glutathione S-transferase (GST)-p21 binding domain (PBD). Aliquots (500 µg) of the supernatants were incubated with 10 µg of GST-PBD for 1 h and precipitated by centrifugation. Complexes were boiled in a Laemmli sample buffer and then separated on 15% sodium dodecyl sulfate-polyacrylamide gels. The separated proteins were immunoblotted using a monoclonal anti-Cdc42 antibody.

Results

β₁Pix interacts with caveolin-1 and G proteins

Several studies have implicated caveolin-1 as a scaffolding protein that enables compartmentalization of specific signaling molecules. ET-Rs have been shown to interact with caveolin-1 and to localize to caveolae [5;6]. Recently, we showed that β₁Pix binds to ET-Rs [13], therefore we examined whether β₁Pix interacts with caveolin-1. To evaluate the potential interaction between β₁Pix and caveolin-1 and the role of β₁Pix dimerization in this interaction, we expressed wild type β₁Pix or β₁Pix dimerization deficient mutant, β₁PixΔ (602–611). Using anti-caveolin-1 antibody for immunoprecipitation, we found that wild type β₁Pix interacts with caveolin-1. This interaction was significantly reduced in cells expressing dimerization deficient β₁Pix mutant (Fig. 1A), indicating that dimerization is crucial for caveoiln-1 binding to β₁Pix. Caveolin-1 has been shown to interact with different Gα subunits through caveolin-1 scaffolding domain [19;20]. Furthermore, this interaction was regulated by the activation state of G proteins and was significantly decreased by GTPγS [19;21]. Therefore, we set out to determine the effect of G protein activation by AMF on the interaction between β₁Pix and caveolin-1. Our result showed that GTPγS induced a strong increase in the interaction between β₁Pix and caveolin-1 compared to that obtained with GDP, and this interaction was further increased in the presence of AMF (Fig. 1B).

Figure 1.

The binding of caveolin-1 to β₁Pix depends on G protein activation state and β₁Pix dimerization. (A) Cells were transfected with Myc-β₁Pix or β₁PixΔ (602–611) for 24 h. Cell lysates were immunoprecipitated with anti-caveolin-1 antibody and the bound proteins were analyzed by Western Blot using anti-Myc antibody. (B) Cells were transfected with Myc-β₁Pix for 24 h and lysed in lysis buffer containing either GDP (10 µM), GTPγS (10 µM) or AMF (AlCl₃, 50 µM; MgCl₂, 10 mM; NaF, 5 mM). Anti-caveolin-1 was used for immunoprecipitation followed by Western Blot analysis using anti-Myc and anti-caveolin-1 antibodies, respectively. These data are representative of four independent experiments. (C/D) Cells were transfected with empty vector or Myc-β₁Pix and lysed in the absence or presence of AMF. Lysates were immunoprecipitated using either anti-Gα_q/11 (C) or anti-Gα₁₂ (D) antibodies and the Western Blots were analyzed by anti-Myc antibodies as indicated. Similar results were obtained in three different experiments.

Caveolin-1 is one component of caveolae that functions as scaffolding protein to facilitate signaling between different proteins. G protein α subunits have been identified as caveolin-interacting proteins [19], therefore we were interested to investigate a possible interaction between β₁Pix and G proteins. Cells were transfected with empty vector or wild type β₁Pix and lysed in the presence or absence of heterotrimeric G protein activator AMF. We found that β₁Pix interacts with endogenous Gα_q, Gα₁₂ (Fig.1C, D) and Gα_s (data not shown) and that this interaction was significantly increased in the presence of AMF. Cells transfected with empty vector served as a negative control. To examine whether the interaction between β₁Pix and Gα_q or Gα₁₂ depends on the physical integrity of caveolae, cells were treated with methyl-β-cyclodextrin, a cholesterol-depleting agent that disrupts caveolae. Results showed that methyl-β-cyclodextrin blocked the binding of Gα_q and Gα₁₂ to β₁Pix (Fig. 2A, B), but did not block the interaction between β₁Pix and G_β1 (Fig. 2C).

Figure 2.

The binding of Gα_q and Gα₁₂ to β₁Pix requires caveolae and β₁Pix homodimerization. Cells were transfected with Myc-β₁Pix for 24 h and either left untreated or treated with methyl-β-cyclodextrin (5 mM) for 1 h. Cell lysates were immunoprecipitated with anti-Gα_q/11 (A), anti-Gα₁₂ (B) or anti-G_β1 (C) antibodies. Precipitated proteins were separated by SDS-PAGE and immunoblotted with anti-Myc, anti-Gα_q/11, anti-Gα₁₂ or anti-G_β1 antibodies. The content of Gα subunits, G_β1 and Myc in the total cell lysates indicates the levels of expression of these proteins. These data are representative of similar results obtained in three independent experiments. (D/E) HMCs cells were transfected with Myc-β₁Pix or Myc-β₁PixΔ (602–611) for 24 h. Lysates were immunoprecipitated using anti-Gα_q/11 (D) or anti-Gα₁₂ (E) antibodies and Western blots were analyzed with anti-Myc antibodies. The expression of Myc-β₁Pix, Gα_q/11 and Gα₁₂ was analyzed by Western Blots performed on the total cell lysates. These data are representative of similar results obtained in three independent experiments.

To determine the role of dimerization on β₁Pix binding to G proteins, cells were transfected with β₁Pix(wt) or β₁PixΔ (602–611). Results showed that the binding of β₁Pix to Gα_q and Gα₁₂ is strongly reduced in cells expressing β₁Pix dimerization deficient mutant β₁PixΔ (602–611) (Fig. 2D, E).

Constitutively active Gα_q and Gα₁₂ potentiate β₁Pix-induced Cdc42 activation

We have demonstrated that the interaction between β₁Pix and wild type Gα_q or Gα₁₂ is increased in the presence of AMF. In order to investigate the functional significance of these physical interactions, we tested the effect of constitutively active Gα_q and Gα₁₂ subunits on Cdc42 activity. To measure active Cdc42 (Cdc42-GTP), we utilized the pull-down assay that consists of measuring Cdc42-GTP bound to GST-Pak1-PBD in lysates of transfected HMCs. Expression of either β₁Pix, Gα_qQ209L or Gα_12Q231L alone induced a modest increase in Cdc42 activity (Fig. 3A). The expression of β₁Pix with constitutively active Gα_q or Gα₁₂ induced a significant increase in Cdc42 activity, indicating that the effect of Gα_q or Gα₁₂ and β₁Pix on Cdc42 activity were additive (Fig. 3A).

Figure 3.

Gα_q and Gα₁₂ increase β₁Pix-induced Cdc42 activation. (A) Cells were transfected with empty vector, Myc-β₁Pix, Gα_qQ209L or Gα_12Q231L for 24 h as indicated. (B) Cells were transfected with Myc-β₁Pix, Myc-β₁PixΔ (602–611), Gα_q or Gα₁₂ as indicated. (C) Cells were transfected with β₁Pix alone or in combination with RGS3 before addition of ET-1 (100 nM) for 5 min. The GTP-bound Cdc42 was measured using pull down assay as described under “Materials and Methods”. The total cell lysates were analyzed for the expression of Myc-β₁Pix, Cdc42, Gα_q, Gα₁₂ or RGS3. Graphs at the bottom of each panel shows fold-increase in Cdc42-GTP densitometric values from three independent experiments normalized to vector which was assigned a value of 1. Results are expressed as mean ± S.E.M. Data are representative of at least three independent experiments.

We have shown that β₁Pix binding to Gα_q and Gα₁₂ requires β₁Pix dimerization. To investigate whether the interaction of Gα_q and Gα₁₂ with β₁Pix is functionally relevant to Cdc42 activation, we analyzed the effect of the expression of β₁Pix dimerization deficient mutant β₁PixΔ (602–611), which binding to Gα_q and Gα₁₂ is strongly disrupted, on Cdc42 activity. As shown in Fig. 3B, both β₁Pix and β₁PixΔ (602–611) alone were able to increase the amount of active Cdc42. However, the co-expression of β₁PixΔ (602–611) and Gα_qQ209L induced a smaller increase in Cdc42 activity compared to cells transfected with β₁PixΔ (602–611) alone. The co-expression of β₁PixΔ (602–611) and Gα_12Q231L induced an activation of Cdc42 that is comparable to that obtained in cells expressing β₁Pix (wt) alone (Fig. 3B). These results indicate that the binding of Gα_q and Gα₁₂ to β₁Pix potentiate Cdc42 activation.

RGS3 inhibits ET-1-induced Cdc42 activation

RGS3 is a member of the regulators of G protein signaling (RGS) family. In vitro experiments with purified glutathione S-transferase-fusion RGS3 showed its ability to bind Gα_q [22]. We therefore evaluated the ability of RGS3 to modulate Gα_q-mediated signaling. We first showed that β₁Pix increased Cdc42 activity, which was further increased by ET-1 stimulation (Fig. 3C). This increase was strongly, although not completely, inhibited in cells expressing RGS3, indicating that ET-1 activation of Cdc42 is mediated at least in part by Gα_q.

ET-1 increased phospho-Erk1/2 binding to β₁Pix

To determine whether β₁Pix is involved in Erk1/2 activation, cells were transfected with empty vector, Myc-β₁Pix or Myc-β₁PixΔ (602–611). Cells were stimulated with ET-1 (100 nM for 5 min) and cell lysates were immunoprecipitated with anti-Myc antibody. We found that in unstimulated cells expressing β₁Pix alone phospho-Erk1/2 was bound to β₁Pix and ET-1 stimulation further increased the interaction between phospho-Erk1/2 and β₁Pix (Fig. 4A). Expression of β₁Pix dimerization deficient mutant, β₁PixΔ (602–611), significantly decreased ET-1-induced binding of phopsho-Erk1/2 to β₁Pix (Fig. 6A). The disruption of caveolae with methyl-β-cyclodextrin strongly inhibited ET-1-induced binding of phospho-Erk1/2 to β₁Pix (Fig. 4B).

Figure 4.

ET-1-induced Erk1/2 activation and binding to β₁Pix. (A) Cells were transfected with Myc-β₁Pix or Myc-β₁PixΔ (602–611) as indicated for 24 h before addition of ET-1 (100 nM) for 5 min. (B) Cells were transfected with Myc-β₁Pix and treated with methyl-β-cyclodextrin (5 mM) for 1 h before addition of ET-1. (C) Cells were transfected with Myc-β₁Pix for 24 h. Cells were incubated with Pertussis Toxin (0.2 µg/ml) overnight or with H-89 (20 µM) or Ro31-7549 (20 µM) for 30 min before stimulation with ET-1 (100 nM) for 5 min. Cell lysates were immunoprecipitated with anti-Myc antibodies and Western blots were analyzed as indicated. These results are representative of three independent experiments.

ET-1-induced binding of phospho-Erk1/2 to β₁Pix was strongly inhibited by PKC inhibitor, Ro31-7549 but not by PKA inhibitor, H-89, or pertussis toxin (Fig. 4C). This result further corroborates that Gα_q is involved in ET-1-induced activation and binding of Erk1/2 to β₁Pix, whereas Gα_i/o and Gα_s are not. Altogether, these results suggest that dimerization allows β₁Pix to bind to caveolin-1 and acts as platform to facilitate the formation of a multi-molecular complex between these proteins which results in Erk1/2 activation and it’s binding to β₁Pix.

Discussion

In this study, we have demonstrated that β₁Pix interacts with caveolin-1, Gα_q and Gα₁₂, and that this interaction was significantly higher in the presence of G protein activator AMF. Co-expression of β₁Pix and activated mutants of Gα_q or Gα₁₂, induced an additive increase of the amount of active Cdc42. The disruption of β₁Pix dimerization severely reduced β₁Pix binding to caveolin-1 and G α subunits and prevented the ability of β₁Pix to enhance Gα_q- and Gα₁₂-induced Cdc42 activity. ET-1-induced Cdc42 activation was reduced by RGS3, which block G_q-mediated signaling. ET-1-induced binding of phospho-Erk1/2 to β₁Pix was inhibited by RGS3 and by PKC inhibitor but not by pertussis toxin or PKA inhibitor, H-89. The disruption of caveolae by methyl-β-cyclodextrin completely blocked the binding of β₁Pix to Gα_q and Gα₁₂, and reduced interaction between β₁Pix and phospho-Erk1/2.

Although many members of Rho family small GTPases can be stimulated through G protein-coupled receptors, it remains unclear how the activating signal is transmitted from the receptor to the corresponding GTPase. Active Gα₁₃ has been shown to directly interact and activate p115RhoGEF [16], while another member of p115RhoGEF, LARG, interacts with Gα_q and Gα_12/13 to activate RhoA [15]. These RhoGEFs interact only with active Gα_q and Gα_12/13 through their regulator of G protein signaling (RGS) boxes.

We have recently shown for the first time that β₁Pix, which does not belong to the family of RGS box-containing RhoA-specific GEFs, interacts specifically with Gα_i3 in the presence of AMF [13]. Here, we extend the ability of β₁Pix binding to other G proteins, namely Gα_q, Gα₁₂ and Gα_s (data not shown). Furthermore, we have previously shown that the activation of Gα_s signaling pathway induces the phosphorylation of β₁Pix by PKA resulting in Cdc42 activation [10]. Here, we show that the co-expression of constitutively active Gα_q or Gα₁₂ and β₁Pix resulted in an additive increase in Cdc42 activity. Gα_q and Gα₁₂ were unable to enhance Cdc42 activity in the presence of β₁Pix dimerization deficient mutant indicating that the physical interaction between these Gα_q or Gα₁₂ and β₁Pix is required for Cdc42 activation. The fact that RGS3 partially blocked Cdc42 activation induced by ET-1, confirms the role of G_q-mediated Cdc42 activation in ET-1 signaling [18] in addition to Gα₁₂ mediated effect. Therefore, it is reasonable to suggest that the activation of β₁Pix is induced directly by activated Gα_q through stimulation of the ET-Rs.

ET-1-induced binding of activated Erk1/2 to β₁Pix was partially inhibited by PKC inhibitor and by RGS3, indicating the involvement of Gα_q, but not that of Gα_i/o or Gα_s. This is in agreement with our previous study showing that RGS3 inhibits ET-1-induced Erk1/2 activation [18]. Gα_q-mediated activation of PKC can stimulate Erk1/2 through phosphorylation of C-Raf [23]. Erk1/2 can also be stimulated through Cdc42 activation. The disruption of β₁Pix dimerization or the disruption of caveolae by cholesterol depletion strongly inhibited the binding of Gα_q and activated Erk1/2 to β₁Pix, indicating that this multi-molecular signaling complex requires β₁Pix dimerization and intact caveolae.

The downstream signaling responses initiated by ET-Rs through Gα_12/13-dependent mechanisms have been less clearly defined than those dependent on activation of G_q, G_s and G_i proteins. Many GPCRs that are able to activate Gα_q also couple to Gα_12/13. In membranes of aortic smooth muscle cells, ET-Rs have been shown to couple to Gα_q as well as Gα_12/13. In our study, we show that in the presence of β₁Pix, Gα₁₂ increases ET-1-induced Cdc42 activation. Therefore, we cannot exclude the possibility that Gα₁₂ is also involved in ET-1-induced Erk1/2 activation and binding to β₁Pix.

Different Gα subunits contain caveolin binding motif and directly bind to caveolin-1 through its caveolin scaffolding domain [24]. Our result does not rule out that β₁Pix binds Gα_q and Gα₁₂ through caveolin-1. Interestingly, in this case the role of RGS domain in mediating the binding of Gα subunits to p115RhoGEF [16] will be comparable to the role of the scaffolding domain of caveolin-1 in mediating the interaction between Gα subunits and β₁Pix. Previous studies have indicated that caveolin-1 preferentially interacts with inactive GDP-bound Gα_s and inhibits nucleotide exchange [19]. In our case, β₁Pix was able to associate with Gα_s (data not shown) and the disruption of caveolae completely blocked the interaction between β₁Pix and Gα subunits. Furthermore, activation of heterotrimeric G proteins by AMF showed an increase in the interaction between β₁Pix and caveolin-1, consistent with the increased association found between β₁Pix and AMF-activated Gα_q and Gα₁₂. This strongly suggests that β₁Pix can interact with active Gα subunits while still bound to caveolin-1. The results showing that Gα_q specifically concentrates in caveolae, whereas Gα_s concentrate much more in lipid rafts [20] may explain the sensitivity of inactive Gα_s to bind caveolin-1. T_α has been shown to compete with T_βγ to bind caveolin-1 since caveolin binding motif on T_α overlaps with T_βγ binding site [21]. This finding also implies that the trimer (GDP-bound) T_αβγ cannot bind to caveolin-1 since the binding of T_α and T_βγ to caveolin-1 is mutually exclusive. This is in agreement with our finding showing that the binding of G_β1 to β₁Pix was not blocked by cholesterol depletion. Caveolae have been proposed to provide a microenvironment where different signaling molecules (GPCRs, G proteins, Src family tyrosine kinases) are concentrated. Therefore, localization of ET-Rs to caveolae along with Gα_q, β₁Pix and Erk1/2 may also contribute to allow efficient and specific signaling by ET-R.

Conclusions

Activation of endothelin receptors leads to the compartmentalization and the binding of Gα_q to β₁Pix in caveolae, where dimeric β₁Pix acts as platform to facilitate the binding and the activation of Erk1/2. Our data suggest that the activation of β₁Pix is induced directly by activated Gα_q, the process triggered by stimulation of endothelin receptors.