Identification of K507 and W508 as the residues responsible for the inhibitory function of the KWQFP motif on the unliganded TpoR. (A) Proliferation assays of the TpoR mutants containing alanine substitutions were performed with Ba/F3 cells expressing equal GFP levels. Cells were treated with 5 ng/mL Tpo where indicated or grown in the absence of any cytokines. Cell numbers were counted at day 9. Results shown here reflect averages of triplicates ± SD from 1 representative experiment. (B) Model of inhibition by the cytoplasmic KWQFP motif of the activity of TpoR. In the presence of Tpo, the receptor adopts a conformation where the KWQFP motif cannot bind to membranes or membrane proteins and interdict signaling. (C) Colony formation induced by Tpo-activated AWQFP and KAQFP point mutants of the TpoR. Colonies were assayed as in Figure 2C.