The type III TGFβ receptor regulates filopodia formation via a Cdc42-mediated IRSp53–N-WASP interaction in epithelial cells

Abstract

Synopsis Cell adhesion and migration are tightly controlled by regulated changes in the actin cytoskeleton. Previously we reported that the TGF-β superfamily coreceptor, the type III TGF-β receptor (TβRIII/betaglycan), regulates cell adhesion, migration and invasion and suppresses cancer progression in part, through activation of the small GTPase, Cdc42, and Cdc42-dependent alterations to the actin cytoskeleton. Here we demonstrate that TβRIII specifically promotes filopodial formation and extension in MCF10A and HMEC mammary epithelial cells. Mechanistically, cell surface TβRIII and Cdc42 colocalize to filopodial structures and co-complex in a β-arrestin2 dependent, and a TβRI/TβRII independent manner. The β-arrestin2-mediated interaction between TβRIII and Cdc42 increases complex formation between the Cdc42 effectors, IRSp53 with N-WASP, to increase filopodial formation. We demonstrate a function link between filopodial structures and epithelial cell adhesion as regulated by the TβRIII-Cdc42 interaction. These studies identify TβRIII as a novel regulator of IRSP53/NWASP via Cdc42 to regulate filopodial formation and cell adhesion.

Altering the Proteoglycan State of Transforming Growth Factor β Type III Receptor (TβRIII)/Betaglycan Modulates Canonical Wnt/β-Catenin Signaling

“…All values were normalized to the IgG sample. B, conditioned medium from COS-7 cells transiently expressing full-length T␤RIII and Wnt3a-HA was immunoprecipitated using anti-T␤RIII and immunoblotted using anti-HA and anti-T␤RIII to analyze Wnt3a-T␤RIII interactions as described under "Experimental Procedures" and previously (21,27,77). C, OVCA429 cells transiently expressing GFP (control), full-length T␤RIII, or T␤RIII-⌬GAG were immunoprecipitated using anti-T␤RIII and immunoblotted (IB) using anti-Wnt3a and anti-T␤RIII to analyze Wnt3a-T␤RIII interactions using the Wnt pulldown assay described under "Experimental Procedures" and previously (13) All values were normalized to the GFP sample, and all figures represent at least two independent biological trials.…”

“…Immunoprecipitation and Western Blotting-Immunoprecipitation and Western blotting were performed using standard techniques as described previously (21,27,77). For co-immunoprecipitation in COS-7 cells, T␤RIII-expressing cells were transfected with the indicated Wnt3a-HA construct, and the culture medium was collected 48 h after transfection under serum-free conditions.…”

“…Mechanistically, T␤RIII regulates these pathways either by altering the actin cytoskeleton, via T␤RIII/␤-arrestin2 cytoplasmic interactions (24), or by GAG chain interactions with FGF2 (23). Overall, T␤RIII also acts as a tumor suppressor in prostate (19), lung (25), pancreatic (18), and breast cancer (16,21,26,27) but has been shown to promote metastasis in specific mesenchymal stem-like breast cancers (28), indicating its complex roles for T␤RIII in cancer.…”

“…Mechanistically, T␤RIII regulates these pathways either by altering the actin cytoskeleton, via T␤RIII/␤-arrestin2 cytoplasmic interactions (24), or by GAG chain interactions with FGF2 (23). Overall, T␤RIII also acts as a tumor suppressor in prostate (19), lung (25), pancreatic (18), and breast cancer (16,21,26,27) but has been shown to promote metastasis in specific mesenchymal stem-like breast cancers (28), indicating its complex roles for T␤RIII in cancer.Although the T␤RIII core can bind TGF-␤ superfamily members with high affinity (22,29,30) the extracellular domain also contains two sites of heparan and chondroitin sulfate GAG chain modifications, resulting in T␤RIII existing in multiple forms in vivo (30 -32). Given that Wnt glycoproteins have a high affinity for both heparan and chondroitin GAG chains on proteoglycans (13, 33), we initiated studies to determine the possible role of T␤RIII in canonical Wnt3a signaling.…”

Decision letter: Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo

“…DOI: 10.7554/eLife.05279.011 membrane deformation and includes actin binding sites (Yamagishi et al, 2004). The protein also contains a partial CRIB motif for Cdc42-GTP mediated activation (Govind et al, 2001) and an SH3 domain for interaction with additional filopodia regulators such as Mena (Krugmann et al, 2001), Eps8 (Vaggi et al, 2011), mDia1 (Goh et al, 2012), WAVE2 (Goh et al, 2012), N-WASP (Oh et al, 2013), Lin7 (Crespi et al, 2012) and WIRE (Misra et al, 2010) ( Figure 6A). We found that the expression pattern of irsp53 mRNA in zebrafish embryos spans the relevant early stages during which PGCs migrate.…”