FIGURE 9.

Impact of SET binding site mutations on GnRHR subcellular localization and signaling. αT3-1 and CHO cells were transfected with HA-tagged GnRHR construct carrying (mutant) or not (WT) mutations precluding SET binding onto intracellular domains of GnRHR (mutations corresponding to mutant A of Fig. 3A were generated on full-length GnRHR by site-directed mutagenesis). A, expression and subcellular localization of GnRHR were monitored by immunocytochemistry by staining either permeabilized or non-permeabilized cells with monoclonal anti-HA antibody (red). Nuclei were stained with DAPI (blue). Images were acquired using a Zeiss LSM 700 confocal laser microscope. B, CHO cells transfected either with WT or mutant GnRHR were incubated with increasing concentrations of GnRHa, and intracellular calcium was determined as described under “Experimental Procedures.” Results were expressed as the percentage of the maximal response in cells transfected with WT GnRHR. The data are presented as the mean ± S.E. of three independent experiments performed in triplicate.