FIGURE 5.

Identification of the TACE-cleavage site in Ptprz. A, amino acid sequence of the common extracellular membrane-proximal region in Ptprz-A and Ptprz-B. Amino acid numbers refer to the sequence of rat Ptprz-A. Three synthetic peptides are shown under the sequence. The arrowhead indicates the TACE cleavage site deduced by in vitro peptide digestion as below. TM, transmembrane segment. B–D, synthetic peptides only (left panels) or synthetic peptides with recombinant TACE (right panels) were incubated for 30 h at 37 °C, and the products were analyzed by mass spectrometry. The combination of peptides is shown at the top left corner of each panel. Peaks are labeled with arrowheads as in A. The expected m/z value for the N-terminal fragment when cleaved at the Ile-Leu bond of Zejm(G/I) is indicated by an open arrow (C, right panel). The figures are representative of two separate experiments. E, CHO-WT and CHO-M2 were transiently transfected with expression constructs for wild-type or G1631I mutant of Ptprz-B. Twenty-four hours after transfection cells were washed and incubated in fresh serum-free medium with or without PMA for 1 h. The cell extracts (upper panel) and conditioned media (lower panel) were analyzed by Western blotting (WB) with anti-Ptprz-S. The figure is representative of three separate experiments, and the results of the densitometric analyses are shown in supplemental Fig. S3. The designations are shown in Fig. 1, A and E.