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Abstract: Frizzled are seven-transmembrane domain G-protein coupled receptors involved in cell polarity and Wnt signaling. The mechanisms regulating their turnover at the plasma membrane remain unclear. We have identified a regulated C-terminus cleavage of Frizzled-7 in endothelial cells using ectopic expression of N- and C-termini-tagged Frizzled-7 proteins. This specific cleavage produced a 10 kDa C-terminus fragment that remained associated with intracellular vesicles and was localized within the 3rd intracytoplasmic loop using N-terminal sequencing and targeted mutagenesis. Frizzled-7 mutated forms displaying reduced C-terminus cleavage were also defective for dvl2 translocation at the plasma membrane. PMA, an activator of PKC and endocytosis, but not Wnt13A and Wnt5A, increased the appearance of Frizzled-7 C-terminus-containing vesicles and Frizzled-7 cleavage. Concanavalin-A, an inhibitor of receptor internalization decreased both constitutive and PMA-induced Frizzled-7 cleavage, while inhibition of the endocytic pathway with Δ95–295-Eps15 dominant-negative prevented only PMA-induced Frizzled-7 cleavage. Frizzled-7 C-terminus cleavage was increased with cell density and by the Ca2+ ionophore ionomycin and was decreased by specific calpain inhibitors, by the expression of DN-calpain-1 and the down-regulation of calpain-1 levels by siRNAs. Altogether, our findings pinpoint calpain-1 as a regulator of Frizzled-7 turnover at the plasma membrane and reveal a link between Frizzled-7 cleavage and its activity. [Copyright &y& Elsevier]

Wnt proteins control cell fate and differentiation during development. Alterations of the Wnt/beta-catenin signaling pathway and changes in wnt gene expression are clearly associated with leukemia. The expression of human wnt13/wnt2b is complex as it involves alternative promoters and RNA splicing giving rise to Wnt13A, -B and -C mRNA isoforms, which encode proteins with different intracellular localizations and functions. We investigated the expression of the human wnt13 in relation to leukemic cell differentiation. Differentiated endothelial cells expressed the highest levels of Wnt13 mRNA isoforms among various endothelial and leukemic cell lines. The differentiation of U937 cells towards monocyte/macrophages resulted in an increase of Wnt13B and -C mRNAs while Wnt13A mRNAs were decreased. The differentiation of K562 cells towards megakaryocytes was accompanied with the up-regulation of all Wnt13 mRNA isoforms. In the two differentiation systems, Wnt13B and -C expression correlated with the expression of the MAF-B transcription factor. Our data demonstrate the differential regulation of wnt13 promoters and pinpoint a Wnt13 isoform switch during differentiation of the leukemic U937 cells towards the monocyte/macrophage lineage, thereby suggesting new players in this process.