Frizzled-7 turnover at the plasma membrane is regulated by cell density and the Ca(2+) -dependent protease calpain-1.
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.
Struewing
Ian T
IT
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Lexington, KY 40536, USA.
Barnett
Corey D
CD
Zhang
Wei
W
Yadav
Satya
S
Mao
Catherine D
CD
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D002452:Cell Count; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D017077:Culture Media, Conditioned; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D051157:Frizzled Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051153:Wnt Proteins / Q000378:metabolism
D017077:Culture Media, Conditioned; C115810:FZD7 protein, human; D051157:Frizzled Receptors; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D051153:Wnt Proteins; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium