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. 1998 Oct 27;95(22):12973-6.
doi: 10.1073/pnas.95.22.12973.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein alpha subunit Galpha13

J Mao  1 , H Yuan, W Xie, D Wu
Affiliations

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein alpha subunit Galpha13

J Mao et al. Proc Natl Acad Sci U S A. .

Abstract

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein alpha subunits were characterized in transfection systems. Galphaq, Galpha12, and Galpha13, but not Galphai, activate SRF through RhoA. When Galphaq, alpha12, or alpha13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Galpha13, but not Galphaq or Galpha12, showed synergistic activation of SRF with GEF115. The synergy between Galpha13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Galpha13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Galpha13- and Galpha12-induced, but not GEF115 itself- or Galphaq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Galpha12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Galpha12 and Galpha13. Thus, the inhibition of Galpha12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Galpha13 and GEF115 indicates that GEF115 mediates Galpha13-induced activation of Rho and SRF.

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Figures

Figure 1
Figure 1
Regulation of SRF-mediated gene transcription by G proteins and guanine nucleotide exchange factors. (A) NIH 3T3 cells were cotransfected with 0.1 μg of SRE.L-luciferase reporter plasmid, 0.1 μg of GFP expression construct, and 0.1 μg of activated Cdc42V12 or RhoV14, Lbc, GEF115, or 0.01 μg of various activated forms of G protein subunits in the presence (solid bar) or absence (open bar) of 0.02 μg of C3 expression plasmid. (B) NIH 3T3 cells were cotransfected with 0.1 μg of SRE.L-luciferase reporter plasmid, 0.1 μg of GFP expression construct, and 0.01 μg of activated Gαq, Gα12 or Gα13 in the presence (solid bar) or absence (open bar) of 0.05 μg of GEF115 (GEF). (C) NIH 3T3 cells were cotransfected with 0.1 μg of SRE.L-luciferase reporter plasmid, 0.1 μg of GFP expression construct, and 0.01 μg of activated Gα12 or Gα13 in the presence (solid bar) or absence (open bar) of 0.05 μg of Lbc. (D) NIH 3T3 cells were cotransfected with 0.1 μg of SRE.L-luciferase reporter plasmid, 0.1 μg of GFP expression construct, and 0.01 μg of activated Gαq, Gα12, or Gα13 in the presence (solid bar) or absence (open bar) of 0.05 μg of GEFΔN. LacZ (z) expression plasmid was used to make the total amount of DNA equal (0.5 μg per well) in all transfection. One day later, cells were lysed, and GFP levels and luciferase activity were determined. The luciferase activities presented are normalized against the levels of GFP expression. Data show similar tendencies with or without normalization. Experiments were carried out in triplicate and repeated at least twice. The representative experiments are shown, and error bars represent standard derivations. (E) Schematic representation of GEF115 and its mutants.
Figure 2
Figure 2
Effect of GEFΔDH on G protein-mediated SRF activation. NIH 3T3 cells were cotransfected with 0.1 μg of SRE.L-luciferase reporter plasmid, 0.1 μg of GFP expression construct, and 0.05 μg of various wild-type forms of G protein subunits in the presence (solid bar) or absence (open bar) of 0.2 μg of GEFΔDH (A) or LbcΔDH (B). LacZ (z) expression plasmid was used to make the total amount of DNA equal (0.5 μg per well) in all transfections. Assays were carried out as described in Fig. 1. The expression of Gα13 was detected by an antibody specific to Gα13 (C), and GEF115 and its mutants were detected with an anti-myc antibody that recognizes the myc-tag in these proteins (D).
Figure 3
Figure 3
Inhibition of receptor-mediated SRF activation by GEFΔDH. NIH 3T3 cells were cotransfected with 0.1 μg of SRE.L-luciferase reporter plasmid, 0.1 μg of GFP expression construct, and 0.2 μg of GEFΔDH (ΔDH) (A) or GEF115N (B) with or without 0.02 μg of C3 expression plasmid as indicated. LacZ (z) expression plasmid was used to make the total amount of DNA equal (0.5 μg per well) in all transfections. The next day, cells were lysed 6 hr after the addition of ligands of thrombin (10 units/ml) or 5 μM LPA. Data are processed and presented as described in Fig. 1.
Figure 4
Figure 4
N-terminal part of GEF115 contains a RGS motif. (A) NIH 3T3 cells were cotransfected with 0.15 μg of SRE.L-luciferase reporter plasmid, 0.15 μg of GFP expression construct, and 0.2 μg of LacZ, Axin or RGS12 in the presence or absence of 0.05 μg of wild-type Gα12 or Gα13 subunits. One day later cells were lysed, and GFP levels and luciferase activity were determined as in Fig. 1. (B) NIH 3T3 cells were cotransfected with 0.15 μg of CRE-luciferase reporter plasmid, 0.15 μg of GFP expression construct, and 0.2 μg of LacZ, GEF115ΔDH or RGS expression plasmids. Isoprenaline (10 μM) was added the next day for 6 hr. Then GFP levels and luciferase activity were determined as in Fig. 1. (C) The amino acid sequences of GEF115 and RGS12 were compared by using a computer program macaw. Homologous regions were shown. Identical amino acids between RGS12 and GEF115 are shown in boxes.
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