1. Frizzled-7 turnover at the plasma membrane is regulated by cell density and the Ca(2+) -dependent protease calpain-1.
- Author
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Struewing IT, Barnett CD, Zhang W, Yadav S, and Mao CD
- Subjects
- Amino Acid Sequence, Animals, Calcium pharmacology, Calpain antagonists & inhibitors, Cattle, Cell Count, Cell Line, Cell Membrane drug effects, Cell Movement drug effects, Culture Media, Conditioned, Cytoplasmic Vesicles drug effects, Cytoplasmic Vesicles metabolism, Endocytosis drug effects, Endothelial Cells cytology, Endothelial Cells drug effects, Frizzled Receptors chemistry, Humans, Molecular Sequence Data, Mutation genetics, Peptide Fragments metabolism, Protein Structure, Tertiary, Protein Transport drug effects, Receptors, G-Protein-Coupled chemistry, Tetradecanoylphorbol Acetate pharmacology, Wnt Proteins metabolism, Calpain metabolism, Cell Membrane metabolism, Frizzled Receptors metabolism, Receptors, G-Protein-Coupled metabolism
- 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 Delta95-295-Eps15 dominant-negative prevented only PMA-induced Frizzled-7 cleavage. Frizzled-7 C-terminus cleavage was increased with cell density and by the Ca(2+) 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.
- Published
- 2007
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