Abstract
Kremens are high-affinity receptors for Dickkopf 1 (Dkk1) and regulate the Wnt/β-catenin signaling pathway by down-regulating the low-density lipoprotein receptor-related protein 6 (LRP6). Dkk1 competes with Wnt for binding to LRP6; binding of Dkk1 inhibits canonical signaling through formation of a ternary complex with Kremen. The majority of down-regulated clathrin-mediated endocytic receptors contain short conserved regions that recognize tyrosine or dileucine sorting motifs. In this study, we found that Kremen1 is internalized from the cell surface in a clathrin-dependent manner. Kremen1 contains an atypical dileucine motif with the sequence DXXXLV. Mutation of LV to AA in this motif blocked Kremen1 internalization; as reported previously for other proteins, the aspartic acid residue in Kremen1 is not critical. Inhibition of expression of the adaptor protein 2 (AP-2) or inhibition of clathrin by pitstop 2 also blocked Kremen1 internalization. The novel amino acid sequence identified in Kremen1 is similar to the motif previously identified in hydra, yeast, and other organisms known to signal from the trans-Golgi network to the endosomal compartment.
Highlights
Cell signaling networks allow cells to proliferate and differentiate based on environmental conditions
HeLa cells co-transfected with Tac-Kremen1 and either caveolin1-eGFP [22] or clathrin LC-eYFP [23] expression plasmids confirmed that Tac-Kremen1 co-localized with a clathrin structure on the cell surface but not with caveloae (Fig. 1C and D)
Dickkopf 1 (Dkk1) is a secreted glycoprotein that negatively regulates Wnt/b-catenin signaling [4,39] by binding to lipoprotein receptor-related protein 6 (LRP6), a co-receptor of Wnt ligand, prior to internalization by clathrinmediated endocytosis [5]
Summary
Cell signaling networks allow cells to proliferate and differentiate based on environmental conditions. Internalization of cell surface transmembrane proteins is governed by the presence of sorting motifs in the cytosolic tail of the transmembrane protein [1,2]. Some of these sorting motifs are recognized by adaptor proteins present on the membrane facing clathrin-coated pits [1,2]. The Wnt/b-catenin signaling pathway is often required in cellular events such as proliferation, differentiation, migration, and determination of polarity [3]. Kremens are high-efficiency receptors of Dickkopf 1 (Dkk1), a protein that destabilizes Wnt/b-catenin signaling. Dkk and LRP6 form a ternary complex with Kremen2 [4]. It was previously suggested that formation of a ternary complex by Dkk, LRP6, and Kremen negatively regulate Wnt/b-catenin signaling [4]
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