Cytoplasmic Y78 is essential for c-Mpl internalization. (A) Human c-Mpl cytoplasmic amino acid sequence is shown with the 2 potential AP2 interaction motifs highlighted in bold. A total of 3 constructs were made with Y → F point mutations at Y8, Y78, and both Y8 and Y78. (B) Biotinylation of membrane proteins demonstrates expression of the larger (approximately 85 kDa) mature form of c-Mpl at the at the cell surface (WCL indicates whole-cell lysate). (C) The effects of Tpo stimulation on receptor surface expression in each of the clones was determined by membrane biotinylation and Western blot analysis. Internalization was quantified by densitometry—comparing expression of membrane-localized c-Mpl (85 kDa) to Tpo-starved cells (0 minutes). The data are representative of 3 independent experiments.
