Figure 8.

Ezrin promotes PTH1R mediated signaling via PLC and PIP2 depletion impedes receptor cell surface expression in HEK293 cells. HEK293 cells grown in either 24-well plates and labeled with ³H-myo-inositol (A) or 96-well plates (B) were transiently transfected with the PTH1R and either LacZ or ezrin, as indicated, at 100 ng for each plasmid/well in A and 20 ng for each plasmid/well in B. A. Cells were treated with either vehicle (acetic acid; white bars) or 100 nM PTH(1-34) (gray bars) for 15 minutes, followed by analysis of total inositol phosphates (mean ±s.d.; n=4; * p < 0.05 from LacZ transfected controls). B. Cells were loaded with Fura2 and PTH-mediated increase of intracellular calcium analyzed as described in the Materials and Methods section (mean ±s.d.; n=4; * p < 0.05 from LacZ transfected controls). C. HEK293 cells transiently transfected with PTH1R-YFP and PIPaseKR were immunostained with HA-tag-specific antibodies. Confocal images of the indicated proteins are shown. D. HEK293 cells grown in 12-well plates were transiently transfected with the PTH1R-YFP and either LacZ, ezrin, PIPase or PIPaseKR, as indicated, at 200 ng for each plasmid/well. The PTH1R cell surface index is reported (mean ±s.d.; n=3; * p < 0.05 from PIPase transfected controls).