Recombinant Mouse Dkk-4 Protein

Dickkopf related protein 4 (Dkk-4) is a member of the Dickkopf protein family that includes Dkk-1, -2, -3, and -4 and a related protein, Soggy (1). Expression of Dkk-4 has only been shown with sensitive PCR techniques during early embryonic development in mice (2), in differentiated human ES cells (3), or in mice that express dominant-active beta -catenin elevating Wnt signaling in the forebrain (4). Dkk proteins are secreted proteins that are synthesized as precursors with an N-terminal signal peptide; all have two conserved cysteine-rich domains separated by a linker region which contains a potential furin type proteolytic cleavage site. The domains contain 10 cysteines each; prokineticin and colipase families show a configuration of cysteines similar to the second motif (5). Mouse Dkk-4 shows 91%, 72% and 71% amino acid (aa) identity with rat, human and canine Dkk-4, respectively, and 40-46% aa identity with other mouse Dkk proteins. Dkk-4 is predicted as a 25 kDa protein, but transfection of 293T cells produces a shorter (15-17 kDa) form containing only the second cysteine-rich domain, as well as longer (30-32 and 40 kDa) forms that do not appear to be glycosylated or form covalent multimers (1). Of the four Dkk proteins, Dkk-4 is the most like Dkk-1. Both are unequivocal antagonists of the canonical Wnt signaling pathway (1, 6), which is activated by Wnt protein engagement of a receptor complex composed of the Frizzled proteins and one of two low-density lipoprotein receptor-related proteins, LRP5 or LRP6 (7). Dkk-1 and Dkk-4 antagonize Wnt by direct high-affinity binding to LRP5/6, forming ternary complexes of LRP5/6 with the Kremens protein Krm2. Internalization of the complex is triggered, making LRP5/6 unavailable for interaction with Wnt ligands (6-9).