The PECAM-1 ITIMs are required for attenuation of calcium mobilization in transfected B cells.
DT40 cells transfected with (A) wild-type PECAM-1 or (B) an ITIM-less form of PECAM-1, in which the tyrosine residues at positions 663 and 686 were changed to phenylalanine, were loaded with Fura-2AM and preincubated for 10 minutes at room temperature with murine antichicken BCR together with the indicated concentration of F(ab′)2 fragments of PECAM-1.3 Ab (αPECAM-1 mAb). Cells were washed free of unbound Ab and then added to the warmed (37°C) chamber of an SLM 8100 spectrofluorometer. Calcium mobilization was induced by cross-linking of surface-bound Abs on addition of F(ab′)2 fragments of GAM mouse IgG. The intracellular calcium concentration was assessed every 3 seconds over a period of 120 seconds. Note that co-ligation of the ITIM-less form of PECAM-1 with the BCR fails to attenuate calcium mobilization in DT40 cells.
