Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and its distribution during early mouse development

Abstract

cripto is the original member of the family of EGF-CFC genes, recently recognized as novel extracellular factors essential for vertebrate development. During the early stages of mouse gastrulation, cripto mRNA is detected in mesodermal cells; later, cripto mRNA is detected only in the truncus arteriosus of the developing heart. Here we describe the in vivo distribution of Cripto protein throughout mouse embryo development and show that cripto mRNA and protein colocalize. By means of immunofluorescence analysis and biochemical characterization, we show that Cripto is a membrane-bound protein anchored to the lipid bilayer by a glycosylphosphatidylinositol (GPI) moiety. We suggest that presentation of Cripto on the cell surface via a GPI-linkage is important in determining the spatial specificity of cell-cell interactions that play a critical role in the early patterning of the embryo.

Cripto-1: a multifunctional modulator during embryogenesis and oncogenesis

“…Related genes include mouse cryptic (Cfc1) and human cryptic (CFC1) (Shen et al, 1997;Bamford et al, 2000;Shen and Schier, 2000;de la Cruz et al, 2002). EGF-CFC family members contain an NH 2 -terminal signal peptide, a modified EGF-like region, a conserved cysteine-rich domain (CFC motif) and a short hydrophobic COOH-terminus that contains additional sequences for glycosylphosphatidylinositol (GPI) cleavage and attachment (Minchiotti et al, 2000) (Figure 1). In addition, all EGF-CFC proteins contain a consensus O-linked fucosylation site within the EGF-like motif that is necessary for their ability to function as coreceptors for the TGFb-related proteins, Nodal or Vg1/GDF-1 (Schiffer et al, 2001;Yan et al, 2002).…”

“…Most of the EGF-CFC proteins are cell-associated glycoproteins that contain from 171 to 202 amino acids and that range from 18 to 21 kDa in size. However, the native mouse and human Cripto-1 proteins are 24, 28 and 36 kDa in size and additional proteins ranging from 14 to 60 kDa have also been identified and are the result of differential glycosylation at N-and O-linked asparagine and serine residues Kenney et al, 1996;Seno et al, 1998;Niemeyer et al, 1999;Minchiotti et al, 2000;Schiffer et al, 2001). The mouse Cr-1 protein can be released by treatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) (Minchiotti et al, 2000).…”

“…However, the native mouse and human Cripto-1 proteins are 24, 28 and 36 kDa in size and additional proteins ranging from 14 to 60 kDa have also been identified and are the result of differential glycosylation at N-and O-linked asparagine and serine residues Kenney et al, 1996;Seno et al, 1998;Niemeyer et al, 1999;Minchiotti et al, 2000;Schiffer et al, 2001). The mouse Cr-1 protein can be released by treatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) (Minchiotti et al, 2000). Removing the COOH-terminal stretch of residues where GPI linkage occurs generates soluble forms of biologically active mouse and human Cripto-1 (Xu et al, 1999;Minchiotti et al, 2001;Bianco et al, 2002bBianco et al, , 2003Yan et al, 2002).…”

Nodal-dependant Cripto signaling in ES cells: from stem cells to tumor biology

“…All EGF-CFC proteins contain a signal sequence for extracellular secretion, a characteristic EGF-like domain, a second cysteine-rich region called the CFC domain, and a hydrophobic C-terminus (Shen and Schier, 2000). Initially described as secreted molecules, members of this family are extracellular membrane proteins, anchored to the lipid bilayer through a glycosilphosphatidylinositol (GPI) moiety (Minchiotti et al, 2000).…”

“…Molecular genetic experiments in mice, Xenopus and Zebrafish point to a strong functional link between EGF-CFC protein and the TGF-b molecule, Nodal (Gritsman et al, 1999;Minchiotti et al, 2001;Reissmann et al, 2001;Yeo and Whitman, 2001). For instance, mouse cripto mutant embryos share some phenotypic similarities to Nodal mutants; however, cripto mutants are not severely affected as Nodal mutants, thus suggesting that Cripto does not mediate all Nodal signaling (Ding et al, 1998).…”