Fig. 5. Tyrosine phosphorylation of FcRIIA expressed in BHK cells is required for translocation of the crosslinked receptor. (A) Comparison of phosphorylation of wild-type (wt) FcRIIA and mutant Y298F FcRIIA in cells after receptor crosslinking at 0°C and after subsequent warming of the cells for 10-20 minutes at 20°C (capping). Distribution of biotin-labeled IV.3 anti-FcRII reflects the amounts of the receptor in the blot, whereas actin labeling demonstrates equal loading of the proteins. On the left, molecular mass standards are shown in kDa. (B) Tyrosine phosphorylation of wild-type FcRIIA, induced by receptor crosslinking (Ctrl), is inhibited by 10 µM PP1 and 1 mM HMA. The blot was reprobed with anti-actin to demonstrate that equal amounts of proteins were loaded on the gel. Arrowheads indicate tyrosine-phosphorylated FcRIIA in A and B. (C) Formation of cap-like structures by crosslinked wild-type FcRIIA and Y298F FcRIIA in BHK transfectants. The results are the means±s.e.m. from four experiments. (D) Colocalization of crosslinked wild-type FcRIIA with tyrosine-phosphorylated proteins and actin filaments during receptor crosslinking (0°C) and formation of cap-like structures (10 minutes at 20°C). Inserts in the `crosslinking' row show a magnified fragment of the cell, which reveals significant colocalization of crosslinked FcRIIA and phosphotyrosine-bearing proteins. (E) Crosslinking of mutant Y298F FcRIIA at 0°C did not induce tyrosine phosphorylation of proteins and no cap-like structures are formed after 10 minutes at 20°C. In D and E, the top panels show cells fixed directly after binding of IV.3 anti-FcRII, which reveals diffuse distribution of the non-crosslinked receptors on the cell surface and low level of phosphotyrosine-bearing proteins inside the cells. Bar, 15 µm.