Lack of tyrosine phosphorylation of endothelial ICAM-1 and mutants of murine ICAM-1. (A) The bEnd5 cells were cross-linked with rat antimouse ICAM-1 mAb followed by rabbit-antirat IgG. Immunocomplexes were precipitated by protein G sepharose. Whereas ICAM-1 cross-linking did not lead to tyrosine phosphorylation of endothelial ICAM-1 (i) it did induce activation of SAPK/JNK within the endothelioma cells (ii). (B) Mutants of murine ICAM-1 (M1 to M5) were created by PCR mutagenesis as described in “Materials and methods.” M1 and M5 lack the cytoplasmic tail. M2, M3, and M4 harbor point mutations, changing tyrosines to phenylalanines at the cytoplasmic tail (M3 and M4) or at the border of the transmembrane domain as indicated. The amino acid sequences of the ICAM-1 mutants are shown in comparison with the wild-type sequence. Mutated amino acids are highlighted in dark gray. The ICAM-1/ICAM-2 chimeric construct was obtained by conventional cloning via restricion enzyme digestions. Correct sequences and base exchanges were verified by DNA sequencing.