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Fig. 4. Role of a juxtamembrane region of SHPS-1 in trans-endocytosis of CD47. (A) Schematic representation of mouse SHPS-1 mutants. Black, gray and white boxes indicate the extracellular (Ex), transmembrane (TM) and cytoplasmic (Cyto) regions of SHPS-1, respectively. WT, wild type. Y and F indicate the positions of tyrosine and phenylalanine, respectively. (B) Lysates (15 µg of protein) of CHO-Ras cell lines expressing wild-type or mutant forms of SHPS-1 were subjected to immunoblot analysis with the p84 mAb to SHPS-1. (C) CHO-Ras cells expressing wild-type or mutant forms of SHPS-1 were cocultured with CHO-CD47 cells, fixed, and stained as in Fig. 1J-L. The numbers of surface, internalized, and total CD47-positive vesicles in SHPS-1-expressing cells located adjacent to CHO-CD47 cells were determined by assay B. The extent of CD47 trans-endocytosis in each cell line was determined as the percentage of the total number of CD47-positive vesicles (N_total) that were present at the cell surface (N_sur) or intracellularly (N_in). Data are means ± s.e.m. from three separate experiments. ^**P<0.01 versus the corresponding value for cells expressing wild-type SHPS-1.

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