Figure 4.

P_i-dependent ERK1/2 signaling and PiT1-PiT2 hetero-oligomerization are independent of P_i transport. A, Western blot analysis of ERK1/2 phosphorylation (P-ERK 1/2) in transiently transfected MC615 cells, as indicated. Overexpression of P_i transport–deficient hPiT1 (PiT1^S128A) and/or hPiT2 (PiT2^S113A) was performed in PiT1–, PiT2–, or PiT1-PiT2–depleted cells, respectively. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for 30 min with 1 or 10 mm extracellular P_i concentration. Total ERK1/2 proteins were used as a loading control. B, sodium-dependent and -independent P_i uptake was measured in HEK293T cells transfected as indicated. Data are means ± S.E. (error bars) (*, p < 0.05 versus sodium-dependent, n = 4). C, BRET 50 index was measured from BRET saturation curves obtained after a 10-min stimulation with 1, 3, or 10 mm extracellular P_i concentration from HEK293T co-transfected with hPiT1-eYFP and hPiT2-Rluc in an Na⁺-free medium. Data are means ± S.E. (*, p < 0.05 versus 1 mm P_i condition, n = 4). D, sodium-dependent P_i uptake was measured in HEK293T cells transfected with pcDNA6A plasmid (Ctrl) or with plasmids containing normal (hPiT1 and hPiT2) or P_i transport–deficient mutants (hPiT1^S128A and hPiT2^S113A) with or without BRET chimeric acceptor or donor, as indicated. Data are means ± S.E. (*, p < 0.05 versus P_i-transporting PiTs, n = 4). E, BRET 50 index was measured from BRET saturation curves obtained after 1, 3, or 10 mm extracellular P_i concentration stimulation for 10 min from HEK293T co-transfected with the indicated plasmids. Data are means ± S.E. (*, p < 0.05 versus 1 mm P_i condition, n = 4–5).