Figure 1

Mutations in the paxillin-binding region of the α4 cytoplasmic domain reduce paxillin binding A) The amino acid sequence of the cytoplasmic domains of the α4 integrins expressed in PC12 cells, starting at the conserved juxtamembrane GFFKR sequence. In all cases, the transmembrane and extracellular domains were wild-type human α4 integrin. The different mutations in the paxillin binding domain are underlined, and the cytoplasmic domain deletion (α4del) is also shown. B) The left panel shows a streptavidin peroxidase/ECL blot of 1% Triton X-100 lysates of biotin-labelled PC12 cells expressing the different α4 constructs following immunoprecipitation with anti-human α4 extracellular domain antibodies and SDS-PAGE on 7% gels. Note the similar expression of the different mutants. The right panel then shows the same immunoprecipitates western blotted with biotin-labelled anti-paxillin antibodies so as to examine paxillin binding to each integrin cytoplasmic domain in the PC12 cells. Note that the single amino acid mutations (α4E983A and α4Y991A) reduce binding, the double mutation (α4EY) even more so, and that no binding is seen following deletion of the α4 cytoplasmic domain (α4del).