Gαs directly drives PDZ-RhoGEF signaling to Cdc42

Abstract

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα₁₃ binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα₁₃ Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gα_s (Gα_sQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by G_s-coupled receptors, but not by those coupled to G_i or G_q, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gα_s-binding region, PRG-linker. Active Gα_s interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with Gα_sQ227L's ability to guide PRG's interaction with Cdc42. Endogenous G_s-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gα_s can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

Keywords: ARHGEF11; Cdc42; DH/PH catalytic module; G protein–coupled receptor (GPCR); GPCR; Galpha-s; Gαs; PDZ-RhoGEF; PDZ-RhoGEF (PRG); Rho (Rho GTPase); Rho GTPases; Rho guanine nucleotide exchange factor (RhoGEF); cell signaling; guanine nucleotide exchange factor (GEF); heterotrimeric G protein.

Figure 1.

In addition to its canonical effect on RhoA, PRG DH/PH activates Cdc42 and promotes filopodia formation. A, hypothetical effects of membrane-anchored RH-RhoGEF DH/PH catalytic modules. B, confocal images of PAE cells showing EGFP-RH-RhoGEF DH/PH-CAAX constructs (green) and their effects on F-actin (red). Zoomed-in areas are shown in the bottom row. Scale bar, 20 μm. C, graph shows the percentage of cells exhibiting filopodia-like structures. Each dot represents the means ± S.E. of at least 30 cells per experiment (n = 3). ***, p < 0.0001; n.s., no significance, one-way ANOVA followed Tukey. D, representative blot shows expression of EGFP-RH-RhoGEF DH/PH-CAAX constructs. E and F, activation of Cdc42 (E) and RhoA (F) by EGFP-RhoGEF DH/PH-CAAX constructs transfected into HEK293T cells was detected by PAK-CRIB and Rhotekin-RBD pulldown (PD), respectively. The graphs represent the means ± S.E. densitometric values (n = 3). **, p = 0.005; ***, p < 0.001; ****, p < 0.0001, one-way ANOVA followed Tukey. G, pulldown of active EGFP-RhoGEF DH/PH-CAAX constructs based on their affinity for nucleotide-free recombinant Cdc42-G15A (left panel) and RhoA-G17A (middle panel). Total cell lysates (TCL) are shown in the right panel. H, interaction between PRG DH/PH and Cdc42-T17N was assayed in transfected HEK293T cells subjected to pulldown assays (PD:GST). D–H, protein expression is confirmed in total cell lysates.

Figure 2.

Gα_s-Q227L binds PRG DH/PH enabling this prototypical RhoA-specific GEF to directly activate Cdc42. A, hypothetic model postulating Gα subunits as potential regulators of PRG DH/PH catalytic module. B and C, the effect of GTPase-deficient Gα subunits on the interaction of EGFP-PRG-DH/PH-CAAX with Cdc42-G15A (B) and RhoA-G17A (C) was analyzed by pulldown (PD) using lysates from HEK293T cells transfected with HA-tagged Gα_s, Gα_i, Gα_q, or Gα₁₃ QL mutants and EGFP-PRG-DH/PH-CAAX. The graph in B represents the means ± S.E. (n = 3). ***, p < 0.001; n.s., no significance, one-way ANOVA followed Tukey. D, to address whether Gα_s-QL detected in the PRG-DH/PH·Cdc42-G15A pulldown was part of a ternary complex, pulldown experiments were done in the presence or absence of PRG-DH/PH. The graph represents the means ± S.E. (n = 3). **, p < 0.0001, t test. E, the potential interaction between active Gα subunits and PRG DH/PH was analyzed in HEK293T cells transfected with GST-PRG-DH/PH and HA-tagged GTPase-deficient Gα subunits subjected to pulldown assays. F, interaction between Gα_s-QL and the catalytic domain of the three RH-RhoGEFs was assayed by pulldown using HEK293T cells transfected with HA-Gα_s-QL and GST-p115-DH/PH, GST-PRG-DH/PH, or GST-LARG-DH/PH. GST and HA-Gα₁₃-QL served as negative controls. G, the effect of Gα_s-QL on the activation of Cdc42 by PRG-DH/PH was assessed by pulldown using lysates of transfected HEK293T cells. The graph represents the means ± S.E. (n = 3). **, p = 0.01, t test. Representative blots show the fraction of active Cdc42 (top panel) and the active fraction of PRG-DH/PH with affinity for Cdc42-G15A (middle panel). H, the effect of Gα_s-QL on the interaction between PRG-DH/PH and Cdc42-T17N was analyzed by pulldown using lysates of transfected HEK293T cells. The graph represents the means ± S.E. (n = 3). *, p = 0.01, t test. I and J, the effect of Gα_s-QL on full-length PRG affinity for Cdc42 was analyzed in HEK293T cells that were transfected with full-length AU1-PRG (I) without or with HA-Gα_s-QL or only with HA-Gα_s-QL to address its effect on endogenous PRG (J). The active fraction of full-length PRG with affinity for Cdc42-G15A was isolated by pulldown and revealed by immunoblotting with anti-PRG antibodies. The graphs represents the means ± S.E. (n = 3). *, p = 0.01 in H and 0.04 in I, t test.

Figure 3.

Gα_s-Q227L binds PRG DH and PH domains and the linker region joining them. A, model showing GST-tagged PRG-DH/PH constructs used to map Gα_s·PRG-DH/PH interaction. B and C, interaction between HA-Gα_s-QL and the indicated GST-PRG-DH/PH constructs was analyzed by pulldown using lysates of transfected HEK293T cells. HA-Gα_s-QL and HA-Gα₁₃-QL (used as control) were revealed with anti-HA antibodies. D, multiple alignment of p115RhoGEF, LARG, and PRG-linker regions. E, structure of PRG-DH/PH·RhoA complex (24). F, model showing Gαs-GTP·PRG-DH/PH complex; hypothetically, active Gαs constrains PRGDH/PH to bind Cdc42. G, model showing the potential inhibitory effect of the EGFP–PRG-linker construct on PRG activation by Gα_s-QL. H, HEK293T cells transfected with EGFP-tagged PRG-linker construct (or EGFP) together with HA-Gα_s-QL and GST-PRG-DH/PH (or GST) were subjected to GST pulldown assays. I, Gα_s-dependent PRG·Cdc42 interaction was analyzed in HEK293T cells transfected with HA-Gα_s-QL (or control plasmid) and AU1-PRG together with EGFP–PRG-linker or EGFP and subjected to Cdc42-G15A pulldown. The graph represents the means ± S.E. (n = 3). **, p = 0.001; ***, p = 0.0001; ns, no significance, one-way ANOVA followed Tukey.

Figure 4.

Agonist-dependent stimulation of G_s-coupled receptors enables PRG to bind Cdc42. A and B, membrane recruitment of endogenous PRG promoted by G_s-coupled GPCR signaling was assessed in PAE (A) and HT29 (B) cells stimulated with 1 μm PGE₂ or butaprost, respectively. PRG in membrane fractions was revealed by Western blotting. GLUT1 and AKT1 were used as membrane and cytosolic markers, respectively. The graphs represent the means ± S.E. (n = 3, *, p < 0.05 in A; and n = 4, **, p < 0.01 in B; t test). C, time course of PRG·Cdc42 interaction was assessed in COS7 expressing G_s-DREADD receptors. The cells were stimulated with 1 µm CNO and subjected to Cdc42-G15A pulldown. The graph represents the means ± S.E. (n = 3). *, p < 0.05, one-way ANOVA followed Tukey. D, the effect of different endogenous heterotrimeric G proteins on PRG affinity for Cdc42 was studied in COS7 cells transfected with AU1-PRG and G_s-, G_i-, or G_q-DREADDs. The cells were stimulated with CNO for 15 min and subjected to Cdc42-G15A pulldown assays. The graph represents the means ± S.E. (n = 3). *, p < 0.05, one-way ANOVA followed Tukey. E, effect of the PRG-linker construct on agonist-stimulated interaction between PRG and Cdc42 was assessed in COS7 cells transfected with Gs-DREADD, AU1-PRG, and EGFP–PRG-linker or EGFP. The cells were stimulated with CNO for 15 and 30 min and subjected to Cdc42-G15A pulldown. The graph represents the means ± S.E. (n = 3). *, p = 0.0342; **, p = 0.0056, t test. F, effect of PRG-linker on agonist-dependent phosphorylation of CREB was assessed in COS7 cells expressing G_s-DREADDs and stimulated with CNO. The graph represents the means ± S.E. (n = 5). **, p < 0.01, t test. G, agonist-dependent phosphorylation of PKA substrates was assessed using PAE cells expressing EGFP or EGFP–PRG-linker and stimulated with butaprost. Lysates from EGFP-PKA-Cα−transfected cells served as control to detect PKA substrates. The graph represents the means ± S.E. (n = 4). ***, p = 0.0009; ****, p < 0.0001; n.s., no significance, one-way ANOVA followed Tukey. H, model depicts the canonical G₁₃-PRG signaling axis to Rho and the emerging GPCR–Gα_s–PRG–Cdc42 pathway based on the current findings. In cells, both systems putatively guide dynamic adjustments on actin-cytoskeleton reorganization.