Fig. 1. Clustering chimeric receptors expressing wild-type integrin ß cytoplasmic domains induces tyrosine phosphorylation of p130CAS and paxillin. (A) Amino acid sequences of the homologous ß1 and ß3 cytoplasmic domains, as well as the alternatively spliced form ß3B, expressed in the context of chimeric receptors containing the extracellular and transmembrane domains (TM) of the tac subunit of the IL-2 receptor (tac chimeras). The ß4 cytoplasmic domain (not shown) is considerably larger and shares no homology with other integrin ß cytoplasmic domains. (B) Transiently transfected normal human fibroblasts expressing chimeras containing either the ß1, ß3, ß3B or ß4 cytoplasmic domains, or expressing the control receptor lacking a cytoplasmic domain (CR), were incubated in clustering assays for 40 minutes. Lysates (10 µg/lane) were separated by SDS-PAGE, western blotted for phosphotyrosine (upper panel) and reprobed for p130CAS (lower panel). (C) In separate clustering experiments, p130CAS was immunoprecipitated from 200 µg of lysates prepared from human fibroblasts expressing either the control receptor (CR) or the tac-ß1 chimera. Immunoprecipitates (IP) and 10 µg of cell lysates (lys) were separated by SDS-PAGE, western blotted for phosphotyrosine (upper panel) and reprobed for p130CAS and FAK (lower panels). (D) Similarly, paxillin was immunoprecipitated from 300 µg of lysates prepared from REF52 cells expressing either the control receptor (CR) or the tac-ß1 chimera. The IP and 10 µg of cell lysates (lys) were separated by SDS-PAGE, western blotted for phosphotyrosine (upper panel) and reprobed for paxillin (lower panel). P130, p130CAS; pax, paxillin.