FIGURE 7.
Alterations of an extracellular loop 2 aspartate residue reciprocally modulate constitutive activity in human and murine FFA3. Reciprocal hFFA3-D158N and mFFA3-N154D mutants were generated and Flp-InTM T-RExTM 293 cells harboring C-terminally eYFP-tagged forms of these produced. Membranes were prepared from both uninduced and Dox-induced cells as well as from the corresponding wild type forms, and [35S]GTPγS binding assays were performed with varying concentrations of C3 with hFFA3 (A), hFFA3-D158N (B), mFFA3 (C), and mFFA3-N154D (D). The point on each of these curves corresponding to a log[C3] of −8 represents the data from basal unstimulated membranes. In E the relative level of constitutive activity of each receptor is displayed (***, p < 0.001 compared with appropriate −Dox control), whereas F shows the relative expression level of each receptor as measured by eYFP fluorescence.