Cripto forms a complex with activin and type II activin receptors and can block activin signaling

Abstract

Activin, nodal, Vg1, and growth and differentiation factor 1 are members of the transforming growth factor beta superfamily and signal via the activin type II (ActRII/IIB) and type I (ALK4) serine/threonine kinase receptors. Unlike activins, however, signaling by nodal, Vg1, and growth and differentiation factor 1 requires a coreceptor from the epidermal growth factor-Cripto-FRL1-Cryptic protein family such as Cripto. Cripto has important roles during development and oncogenesis and binds nodal or related ligands and ALK4 to facilitate assembly of type I and type II receptor signaling complexes. Because Cripto mediates signaling via activin receptors and binds directly to ALK4, we tested whether transfection with Cripto would affect the ability of activin to signal and/or interact with its receptors. Here we show that Cripto can form a complex with activin and ActRII/IIB. We were unable to detect activin binding to Cripto in the absence of ActRII/IIB, indicating that unlike nodal, activin requires type II receptors to bind Cripto. If cotransfected with ActRII/IIB and ALK4, Cripto inhibited crosslinking of activin to ALK4 and the association of ALK4 with ActRII/IIB. In addition, Cripto blocked activin signaling when transfected into either HepG2 cells or 293T cells. We have also shown that under conditions in which Cripto facilitates nodal signaling, it antagonizes activin. Inhibition of activin signaling provides an additional example of a Cripto effect on the regulation of signaling by transforming growth factor-beta superfamily members. Because activin is a potent inhibitor of cell growth in multiple cell types, these results provide a mechanism that may partially explain the oncogenic action of Cripto.

Figure 1

Domain structure of mouse Cripto. Conserved domains of Cripto are shown including the N-terminal signal peptide, EGF-like domain (EGF), CFC domain, and C-terminal hydrophobic region containing the site of glycosylphosphatidylinositol (GPI) attachment. The sites of three tandem point mutations are also indicated (mEGF1, N69G and T72A; mEGF2, R88G and E91G; mCFC, H104G and W107G).

Figure 2

Covalent crosslinking of [¹²⁵I]activin-A to type II activin receptors, Cripto, and ALK4. (A) 293T cells were transfected with the following constructs: lane 1, vector; lane 2, ActRII-myc; lane 3, ActRII-myc + ALK4; lane 4, ActRII-myc + Cripto; lane 5, ActRII-myc + Cripto mCFC; lane 6, ActRII-myc + Cripto ΔEGF; lane 7, ActRII-myc + ALK4 + Cripto; lane 8, ActRII-myc + ALK4 + Cripto mCFC; lane 9, ActRII-myc + ALK4 + Cripto ΔEGF. (B) 293T cells were transfected with the same constructs as described for A but with ActRIIB instead of ActRII-myc. (C) 293T cells were transfected with vector (lane 1), ALK4 + Cripto (lane 2), ActRII + ALK4 (lane 3), or ActRII + ALK4 + Cripto (lane 4). Cells were subjected to crosslinking with [¹²⁵I]activin-A as described in Materials and Methods. Crosslinked complexes were isolated by immunoprecipitation by using an anti-myc antibody (targeting ActRII-myc) (A), an ActRIIB antibody (B), or an anti-ALK4 antibody (C). Immunoprecipitated proteins were resolved by SDS/PAGE and visualized by autoradiography as described in Materials and Methods. (D) 293T cells transfected with vector (lane 1), ActRIIB + ALK4 (lane 2), or ActRIIB + ALK4 + Cripto (lane 3) were solubilized and subjected to SDS/PAGE and Western blot analysis as described in Materials and Methods.

Figure 3

Effects of Cripto on activin-A and BMP-7 signaling in HepG2 cells. HepG2 cells were transfected with either empty vector or Cripto as described in Materials and Methods and then treated with the indicated doses of either activin A (A) or BMP-7 (B). Luciferase activities were normalized relative to β-galactosidase activities, and data are presented as the fold increase in luciferase activity of cells treated with activin-A or BMP-7 relative to untreated cells.

Figure 4

Effects of wild-type Cripto and Cripto mutants on activin-A signaling in 293T cells. 293T cells were transfected with the indicated constructs as described in Materials and Methods and then treated with vehicle or 1 nM activin-A. Luciferase activities were normalized to β-galactosidase activities, and data are presented as the fold increase in luciferase activities relative to untreated cells.

Figure 5

Effects of Cripto on activin-A and nodal signaling in 293T cells. 293T cells were transfected with either empty vector or nodal and the indicated amount of Cripto DNA as described in Materials and Methods and then treated where indicated with 1 nM activin-A. Luciferase values were normalized to β-galactosidase activities, and data are presented as the fold increase in luciferase activities relative to untreated cells.

Figure 6

Model of the proposed mechanism by which Cripto antagonizes activin. (A) Activin signals by binding ActRII/IIB and then recruiting ALK4. ActRII/IIB phosphorylates (P) the GS domain of ALK4, thereby activating the ALK4 kinase and initiating downstream signaling. Nodal does not bind activin receptors and therefore does not signal in the absence of Cripto. (B) Cripto antagonizes activin signaling by forming a complex with activin and ActRII/IIB. We propose that this complex precludes the formation of a functional activin–ActRII/IIB–ALK4 complex and therefore blocks signaling. Nodal binds directly to Cripto, leading to the assembly of ActRII/IIB and ALK4 followed by ALK4 phosphorylation and downstream signaling.