Figure 5.

Cell surface expression of Klotho, the CaSR, and ADAM10. Confocal images of HEK-293 cells expressing Klotho, the CaSR, and ADAM10. Cells were fixed using nonpermeabilizing conditions and triple stained with primary antibodies specific for Klotho (green), the CaSR (red), and ADAM10 (magenta). The top row shows cells in basal conditions (1.6 mM Ca_o), where the three proteins appear together in aggregates (white arrows). The middle row shows cells after 3 hours in 4 mM Ca_o, where the aggregates are reduced in number, size, and intensity, consistent with the experiments in which significant amounts of Klotho are cleaved from the cell membrane. In the bottom row, the cells are treated with 4 mM Ca_o and 1 μM GI245023X (ADAM10 inhibitor) for 3 hours, conditions in which ADAM10 will be inactive and the CaSR will be active. Inhibition of ADAM10 activity prevents Klotho cleavage and causes persistence and possibly increased size of the aggregates (white arrows), implicating trafficking or protein complex formation in the CaSR–ADAM10 activation–Klotho cleavage process. Scale bar, 5 μm.