FIGURE 3.

HCG stimulation of HLHCGR induces ARF6 but not ARF1 activation in HEK-HLHCGR cells. A, HEK 293 cells transfected with either pcDNA3 or Myc-HLHCGR were serum starved and treated with or without 10 IU/ml HCG for 30 min at 37 °C. The cells were lysed, and the cell lysates incubated with GST-GGA3 PBD resin to analyze the levels of endogenous ARF1-GTP and ARF6-GTP. Total ARF1 or ARF6 expression in the cell lysates as well as the active ARF1 and ARF6 bound to the resin were detected by immunoblotting using anti-ARF1 rabbit pAb and anti-ARF6 mouse mAb, respectively. B and C, analysis of concentration- (B) and time-dependent (C) increase in ARF6 activation in HCG-stimulated HEK-HLHCGR cells. HEK-HLHCGR cells stimulated without or with 1–1000 IU/ml HCG for 30 min or 10 IU/ml HCG for up to 60 min were lysed and subjected to GST-GGA3 PBD pulldown. Total ARF6 and ARF6-GTP were detected by immunoblotting using an anti-ARF6 mouse mAb. Densitometric analysis of the ARF6-GTP is shown as a histogram, after normalizing to the expression of total ARF6 present in the sample. D, effect of the Myr-ARF6 inhibitory peptide on ARF6 activation in HCG-stimulated HEK-HLHCGR cells. HEK-HLHCGR cells preincubated for 2 h at 37 °C without (first lane) or with 2.5 μm penetratin (second lane) or penetratin coupled Myr-ARF1 (third lane) or Myr-ARF6 (fourth lane) peptides were subsequently stimulated with 10 IU/ml HCG for 30 min, lysed and subjected to GST-GGA3 PBD pulldown to quantify endogenous ARF6-GTP. ARF6-GTP and total ARF6 levels were each assessed by immunoblotting. Densitometric analysis of ARF6-GTP is shown as a histogram, after normalizing to the expression of total ARF6 present in the sample.