Figure 2
Stimulation-dependent versus autonomous signaling of the LMP2A-ITAM. (A) Schematic representation of CD8- and CD72-based transmembrane chimeras containing the cytoplasmic LMP2A N-terminus in type-I or type-II orientation, respectively. (B) DT40 transfectants, which constitutively express CD8/LMP2A were left unstimulated (lane 1) or stimulated for 3 min with either anti-IgM or anti-CD8 antibodies (lanes 2 and 3, respectively) and analyzed for PTK substrate phosphorylation by anti-phosphotyrosine immunoblotting as described in Figure 1C. Identification of Syk and SLP65 phosphoproteins was performed in separate experiments (data not shown). Relative molecular masses of marker proteins are indicated on the left in kDa. (C) Indo-1-loaded CD8/LMP2A transfectants were stimulated with anti-CD8 antibodies (arrow) and mobilization of Ca2+ ions was monitored by flow cytometry. (D) DT40 transfectants, which were left uninduced (lanes 1-2) or treated with 4-HT for 6 h to induce expression of CD72/LMP2A (lanes 3-4) were analyzed for PTK substrate phosphorylation without stimulation (lanes 1 and 3) or after stimulation (lanes 2 and 4) with antibodies against IgM or CD72, respectively, by anti-phosphotyrosine immunoblotting as described in Figure 1C.