RSPO-LGR4 functions via IQGAP1 to potentiate Wnt signaling

Abstract

R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO-LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO-LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO-LGR4's interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO-LGR4 to the Wnt signaling complex through enhanced IQGAP1-DVL interaction following RSPO stimulation. In this configuration, RSPO-LGR4-IQGAP1 potentiates β-catenin-dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as β-catenin-independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO-LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling.

Keywords: adhesion; cell signaling; migration; receptor activation.

Fig. 1.

RSPO–LGR4 has RNF43/ZNRF3-independent function. (A) Wnt3a and RSPO3-induced β-catenin activity as measured by the TOPflash assay in HEK293T cells transfected with vector control or LGR4 in the presence of scrambled or ZNRF3 siRNA. The baseline arrow indicates no Wnt3a or RSPO3, and Wnt3a arrow indicates a fixed dilution of Wnt3aCM added to the rest of the samples. (B) Dose-dependent response to RSPO1 WT and Q71A mutant in HEK293T cells transfected with vector control or LGR4 in the TOPflash assay. (C) Dose-dependent response to RSPO1 WT and Q71A mutant in HEK293T cells transfected with scrambled or ZNRF3 siRNA in the TOPflash assay. (D and E) HEK293T cells overexpressing ZNRF3–ECDTM in the presence or absence of LGR4 show an enhanced response in the TOPflash assay when treated with serial dilutions of Wnt3aCM (D), but failed to respond to RSPO1 (E). (F) WB analysis of the effect of overexpressing ZNRF3–ECD–TM on interaction of LGR4 with LRP6. HEK293T cells transfected with HA-LGR4 and LRP6 were cotransfected with vector or Myc-ZNRF3-ECD-TM and treated with vehicle or Wnt3a+RSPO1 (WR). IP was performed with anti-HA beads and proteins were detected with Abs as indicated.

Fig. 2.

IQGAP1 binds to LGR4 and mediates Wnt/β-catenin signaling. (A) Co-IP analysis of full-length LGR4 or LGR4–ECDTM with IQGAP1. HEK293T cells were transfected with HA-LGR4 or HA-LGR4-ECD-TM, immunoprecipitated with anti-HA beads, and blotted with IQGAP1 or HA Ab. (B) Co-IP of endogenously expressed LGR4 and IQGAP1 from HeLa cells. Cell lysates were immunoprecipitated with LGR4 Ab (7E7; described in detail in ref 25) or control IgG and blotted with LGR4 or IQGAP1 Ab. (C) Effect of IQGAP1 and IQGAP3 KD on RSPO1-induced Wnt/β-catenin activity in the TOPflash assay. HEK293T cells were transfected with scrambled siRNA or siRNA of IQGAP1 or IQGAP3, or both. (D) Overexpression of mouse IQGAP1 rescued IQGAP1 KD effect in HEK293T cells using the TOPflash assay. (E) Effect of IQGAP1 KD on RSPO1 and Wnt3a-induced LRP6 phosphorylation. HEK293T cells were transfected with scrambled or IQGAP1 siRNA, treated with RSPO1 +Wnt3a for the indicated periods of time as indicated, and probed for LRP6 phosphorylation (S1490) and levels of IQGAP1. All error bars are SEM (n ≥ 3 for all samples).

Fig. 3.

IQGAP1 bridges RSPO–LGR4 to the Wnt signalosome via DVL and promotes MEK1/2-mediated LRP6 phosphorylation. (A) A schematic diagram of IQGAP1 domain structure and deletion/truncation mutants. CHD, calponin homology domain; IR, IQ repeats; GRD, rasGAP-related domain; RGCT, C-terminal domain of RasGAP-related proteins. **Putative DVL binding region. (B) Co-IP analysis of various IQGAP1 mutants with LGR4. HEK293 cells were cotransfected with FLAG-tagged IQGAP1 WT or mutants and HA-LGR4, immunoprecipitated with anti-HA beads, and probed with FLAG or HA Abs. *Specific bands that coimmunoprecipitated with LGR4. (C) Effect of overexpressing IQGAP1 WT or mutants on RSPO1-potentiated Wnt/β-catenin signaling in the TOPflash assay. (D) Effect of MEK1/2 inhibitor U0126 on RSPO1 potentiation of Wnt/β-catenin signaling in the TOPflash assay. (E) WB analysis of the effect of U0126 on Wnt3a (W), RSPO1 (R), or both (WR)-induced LRP6 phosphorylation. (F) Co-IP analysis of IQGAP1 with p-LRP6, LRP6, DVL2, and MEK1/2 with or without LGR4 overexpression. (G) KD of DVL2 and DVL3 reduced complex formation of LGR4 with Wnt signalosome. HEK293T cells were cotransfected with siRNAs targeting DVL1–3, FLAG‐IQGAP1, HA‐LGR4, and LRP6. Cell lysates were immunoprecipitated with anti‐FLAG beads and probed with anti‐HA (LGR4), ‐LRP6, ‐DVL2, and -DVL3 Abs.

Fig. 4.

RSPO–LGR4-recruited IQGAP1 interacts with Wnt signalosome and F-actin assembly components. (A) Co-IP analysis of LGR4–IQGAP1 with Wnt and FA complex molecules with or without Wnt3a+ RSPO1 (WR) costimulation. (B) Confocal microscopy analysis of RSPO3-induced colocalization of IQGAP1 with LGR4 at the leading edge of MDCK cells. MDCK cells transfected with IQGAP1–EGFP (green) and HA-LGR4 were starved and treated with vehicle or RSPO3 for 30 min. Cells were stained with Alexa 594-labeled anti-HA (red) and imaged. The leading edge is highlighted by the white box and in Bottom.

Fig. 5.

RSPO3–LGR4–IQGAP1 signaling regulates the formation of FA and migration in lung cancer cells. (A) Effect of LGR4 KD on Wnt signaling. A549 cells stably expressing four distinct shRNA constructs (nos. 39–42) were generated and probed for levels of LGR4 and Wnt signaling markers. P, parental cells. (B) Effect of RSPO3 KD on Wnt signaling. A549 cells stably expressing two distinct shRNA constructs of RSPO3 (nos. 63 and 67) were generated and probed for levels of RSPO3 and Wnt signaling markers. V, vector control. (C) Effect of KD of RSPO3 or IQGAP1 on FA and cytoskeletal structures. A549 cells stably expressing RSPO3 (no. 63), IQGAP1 (no. 85), or control shRNA were costained with anti-paxillin (green) and rhodamine-labeled phalloidin (red) and viewed by confocal microscopy. (D) WB analysis of Wnt signaling and FA assembly markers in A549 cells stably expressing RSPO3 (no. 63), IQGAP1 (no. 85), or control shRNA. (E) Migration results of A549 cells with KD of LGR4, RSPO3, or IQGAP1. (F) Invasion results of A549 cells with KD of LGR4 or RSPO3.

Fig. 6.

A schematic diagram illustrating the dual mechanism of RSPO3–LGR4 signaling. In the absence of RSPO, RNF43/ZNRF3 ubiquitinates the FZD receptors for degradation, resulting in low Wnt signaling activity. In the presence of RSPOs, LGR4 recruits IQGAP1 and increases its affinity toward DVL, leading to the formation of a supercomplex with the Wnt signalosome through IQGAP1–DVL interaction. IQGAP1-bound MEK1/2 then phosphorylates LRP5/6, which binds Axin and inhibits its activity in organizing β-catenin phosphorylation. RSPO–LGR4-bound IQGAP1 can also interact with noncanonical Wnt signalosome to coordinate actin dynamics due to IQGAP1’s direct binding to actin polymerization machinery, leading to enhanced FA assembly and cell migration.