Figure 6. Cooperation of SPCA2 N- and C- terminal domains in Ca²⁺ signaling.

(A) SPCA2 C-terminus was sufficient to activate Ca²⁺ signaling. Function of deletion and point mutants of SPCA2 C-terminal domain were examined by NFAT translocation assay in HEK293 cells. Full length SPCA proteins were HA-tagged, all C-terminus fragments shown were GST-tagged. n = 3. (B) Basal intracellular Ca²⁺ concentrations in HEK293 cells, with the expression of GST-tagged deletion and point mutants of SPCA2 C-terminal domain described in (A). From left to right, n = 49, 45, 46, 48, 40, 54, 59. (C) Effects of N-terminal fragments of SPCA proteins on NFAT translocation induced by SPCA2 full length or C-terminal fragment shown in (A). n = 3. (D) Immunoblot and normalized cell growth in soft agar of MCF-10A cells transduced with vector, SPCA2, GST, and membrane-anchored SPCA2 C-terminus (GST-tagged). n = 3. (E and F) Interaction between Orai1 N- (N: aa1–91)), C- (C: aa255–301) termini, intra- (L1: aa141–177), extracellular (L2: aa198–234) loops and SPCA2 full length or N-terminus. (G) Interaction between Orai1 full-length and subregions of N-terminus (N: aa1–91; N1: aa1–47; N2: aa48–91), C-terminus (C: aa255–301), C-terminus mutation (C-L273S) and SPCA2. Error bars represent standard error (B) or standard deviation (A, C and D).