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. 2001 May 4;276(18):15445-52.
doi: 10.1074/jbc.M008961200. Epub 2001 Feb 6.

Physical and functional interactions of Galphaq with Rho and its exchange factors

S A Sagi  1 , T M Seasholtz, M Kobiashvili, B A Wilson, D Toksoz, J H Brown
Affiliations

Physical and functional interactions of Galphaq with Rho and its exchange factors

S A Sagi et al. J Biol Chem. .

Abstract

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.

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Figures

Fig. 1
Fig. 1. Selective precipitation of Gαq with Lbc
COS-7 cells were transfected with empty vector, plasmid encoding Myc-tagged proto-Lbc, or plasmid for Myc-tagged p115RhoGEF and with empty vector or plasmid encoding the GTPase-deficient Gα subunit of either Gαq, Gα12, or G α13. A, whole cell lysates from the transfected COS-7 cells were prepared and Western blotted for the appropriate Gα subunit, to ensure protein expression. B, cell extracts were immunoprecipitated (IP) with anti-Myc (9E10 mouse monoclonal) antibody and then Western blotted with c-Myc antibody to visualize the epitope-tagged proto-Lbc and p115RhoGEF. C, immunoprecipitates, as in B, were blotted with antibodies against the appropriate Gα subunit. Blots are representative of two to three experiments, each performed in duplicate.
Fig. 2
Fig. 2. Gαs does not coprecipitate with Lbc
COS-7 cells were transfected with empty vector or plasmid encoding Myc-tagged proto-Lbc along with empty vector or plasmid encoding the GTPase-deficient Gα subunit of either G αq or Gαs. A, whole cell lysates from the transfected COS-7 cells were prepared and Western blotted for the appropriate Gα subunit, to ensure protein expression. B, cell extracts were immunoprecipitated (IP) with anti-Myc (9E10 mouse monoclonal) antibody, which recognizes the epitope-tagged proto-Lbc, and then Western blotted with antibodies against the appropriate Gα subunit. Blots are representative of two experiments, each performed in duplicate.
Fig. 3
Fig. 3. Gαq does not induce activation of RhoA
A, COS-7 cells were transfected with hemagglutinin-tagged wild-type RhoA, hemag-glutinin-tagged L63RhoA or empty vector (pCMV5). Cell lysates were prepared, and a portion of this whole cell lysate was subjected to SDS-PAGE and Western blotting with anti-RhoA antibody to measure total Rho (lower panel). The remaining portion of the lysates was affinity-precipitated with the RBD of Rhotekin and Western blotted with anti-RhoA. Only activated Rho binds to the Rhotekin RBD (top panel). The doublet pattern is present because the hemagglutinin-tagged RhoA migrates slower than the endogenous RhoA. B, COS-7 cells were transfected with pCis vector or various Gα subunits. The cell extracts were analyzed as in A. C, after transfection of COS-7 with Gαq, proto-Lbc or both active or total Rho was detected in cell extracts as in A.
Fig. 4
Fig. 4. Gαq synergizes with Rho GEFs to induce Rho-dependent SRE.L-mediated gene expression
COS-7 cells were transfected with an SRE.L-luciferase reporter plasmid along with the empty pCis vector or with pCis containing cDNA for the different Gα subunits as on the x axis. Cells were cotransfected with empty vector (light gray), proto Lbc (medium gray), or p115RhoGEF (black). Luciferase was quantified 24–48 h after transfection. Data are averages ± S.E. of the fold over vector control for three experiments, each performed in triplicate.
Fig. 5
Fig. 5. Activation of endogenous Gαq synergizes with proto-Lbc on SRE.L-mediated gene expression
COS-7 cells were trans-fected with a control vector or proto-Lbc along with an SRE.L-luciferase reporter plasmid. Following transfection cells were maintained in serum-free medium (vehicle) or in serum-free medium containing 100 ng/ml recombinant PMT for 24 h, then harvested and assayed for luciferase activity. Data are representative of three experiments, each performed in triplicate.
Fig. 6
Fig. 6. The synergistic SRE.L responses to Gαq and Lbc in COS-7 cells are Rho-dependent
COS-7 cells were transfected with an SRE.L-luciferase reporter plasmid and the expression plasmids for the proteins indicated on the x axis. Black bars indicate cotransfection with an expression plasmid for C3 exoenzyme; gray bars are vector-transfected controls. Luciferase was quantified 24–48 h after transfection. Data are averages ± S.E. of the fold increase over vector control for three experiments, each performed in triplicate.
Fig. 7
Fig. 7. Lbc selectively synergizes with Gαq to induce astrocytoma cell rounding
1321N1 human astrocytoma cells were microinjected with empty vector (pCis) or with Gα subunits as indicated on the x axis along with control vector or plasmid encoding proto-Lbc. All cells were coinjected with plasmid for nuclear GFP to allow identification of injected cells. Actin was visualized by staining with rhodamine-conjugated phalloidin. Data shown are the percent of microinjected cells that have a rounded appearance. Values are the average ± S.E. from at least three experiments with a minimum of 50 microinjected cells per condition per experiment.
Fig. 8
Fig. 8. Gαq synergizes with Rho to induce SRE.L-mediated gene expression
COS-7 cells were transfected with an SRE.L-luciferase reporter plasmid and the expression plasmids for constitutively active Gα subunits along with control vector or plasmid encoding activated RhoA. Luciferase was quantified 24 h after transfection. Values are the average ± S.E. of the fold increase over vector control for three experiments, each performed in triplicate.
Fig. 9
Fig. 9. SRE.L activation is dependent on PLC activation and independent of PKC
COS-7 cells were transfected with the SRE.L-luciferase reporter plasmid, and luciferase was determined as described. A, cells were transfected with empty pCMV5 vector or one of the following Gαq constructs: GTPase-deficient Gαq (GαqQL), wild-type Gαq (GαqWT), or GTPase-deficient Gαq that cannot activate PLCβ (GαqQL/DNE) along with control vector or proto-Lbc. B, COS-7 cells were transfected with a GTPase-deficient Gαq (GαqRC) and/or Lbc along with control vector plasmid encoding the C terminus of PLCβ1 (PLCβCT). C, COS-7 cells were transfected with a GTPase-deficient Gαq (GαqRC) and/or Lbc. After overnight transfection, cells were treated with vehicle or with 3 μ M GF109203X (to inhibit PKC) for 24 h prior to harvest. Data are average ± S.E. of the fold increase over vector control for two or three experiments performed in triplicate.
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