TRPC7 is regulated by PKC-mediated phosphorylation of serine714. (A and B) wt and s4ko cells have different adherens junction (AJ) composition, the former using OB-cadherin, whereas s4ko MEFs use to N-cadherin (arrowheads). Quantitation of cadherin colocalization with the junction maker p120 catenin is shown in B. Triplicate experiments were performed with n > 100 cells of both types. (C) Cell surface biotinylation shows that N-cadherin was expressed on the surface of both cell types whereas OB-cadherin was expressed only on the surface of wt MEFs. (D and E) Silencing of TRPC7 expression in s4ko MEF led to the restoration of OB-cadherin to adherens junctions (arrowheads), thereby resembling wt MEFs even in the continued absence of syndecan-4. Triplicate experiments were performed with n > 50 cells each. (F) Structure of TRPC channels. The semi-conserved TRP domain following the sixth transmembrane domain is boxed and its sequence is shown to the right. A conserved serine residue immediately C-terminal of the TRP domain is marked with a red box. (G and H) wt (G) and s4ko (H) MEFs were transfected with cDNAs encoding wt (Trpc7), phosphomimetic (Trpc7-S714E), and phospho-resistant (Trpc7-S714A) mutant forms of TRPC7. 50% of the wt cells transfected with Trpc7-S714A failed to form mature focal adhesions and possessed higher cytosolic calcium levels than control cells. Trpc7-S714E expressing cells show no significant elevation in cytosolic calcium. (H) The same constructs transfected into s4ko cells. Calcium was only reduced in cells expressing Trpc7-S714E, with corresponding increase in stress fibers and OB-cadherin usage in adherens junctions. Triplicate experiments were performed. (G) n = 30 cells for each condition (left); n = 45–101 cells per condition (right). (H) n = 32–65 cells per condition (left), >200 cells per condition (middle); and n = 30–100 cells per condition in duplicate (right). Data are the mean ± SEM. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; unpaired t test.