Figure 4.

Kinetics of recycling back to the plasma membrane of the WT and triple mutant FSHR after agonist-provoked internalization. (A) Cell surface ¹²⁵I-FSH bound to the recycled FSHR throughout the second, 4 h incubation (see Materials and Methods). The relative amount of cell surface ¹²⁵I-FSH/mutant FSHR complex was less compared to that shown by the WT receptor (see Results Section). (B) Total ¹²⁵I-FSH/FSHR complex recycled back to the plasma membrane (cell surface ¹²⁵I-FSH plus TCA precipitable ¹²⁵I-FSH), following exposure of HEK-293 cells transiently expressing the WT or mutant FSHR to agonist. Total recycled FSH/mutant FSHR complex was decreased compared to the FSH/WT FSHR complex (see Results Section). (C) TCA-soluble (degraded) radioactivity recovered from the culture medium throughout the second, 4 h incubation. Less soluble radioactivity was recovered at time 4 h in media from cells transfected with the mutant receptor; meanwhile, more TCA precipitable (undegraded) ¹²⁵I-FSH was recovered in the media from cells expressing the WT FSHR (inset). (D) Cell-associated ¹²⁵I-FSH recovered during the second incubation.