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Figure 2. FAK was required for maximal surface expression of FGFRs and for phosphorylation of FGFRs, and FRS2. A, B) FGFR surface expression was reduced in the absence of FAK. FGFR surface expression was determined by [125I]FGF-2 binding assay as described in Material and Methods. Percent [125I]FGF-2 bound per cell of FAK –/– MEFs was compared to FAK-expressing MEFs: FAK +/+, P < 0.05 (A) or DA2 (hygro), P < 0.05 (B). FAK –/– MEFs bound 50% less [125I]FGF-2 compared to MEFs containing FAK (FAK +/+ and DA2). Values are means from 3 independent experiments ± SD. C) FGFR1 phosphorylation was decreased in the absence of FAK. FGFR tyrosine phosphorylation was determined in FAK +/+ and FAK –/– MEFs that were serum-starved and incubated with or without 50 ng/ml FGF-2 and 5 µg/ml heparin for 10 min. Cell lysates were immunoprecipitated with 1, rabbit anti FGFR1 or 2, nonspecific rabbit IgG. Chemiluminescence was visualized on Kodak Biomax MS film. 1: In response to FGF/h, FAK +/+ MEFs had significantly more FGFR1 phosphorylation than FAK –/– MEFs. Experiment was performed 2 times with similar results. D) FRS2 phosphorylation was absent in FAK –/– MEFs. FAK +/+ and FAK –/– MEFs were serum-starved and incubated with or without 20 ng/ml FGF-2 and 5 µg/ml heparin for 10 min. Cell lysates were immunoprecipitated with 1, rabbit anti-FRS2 or 2, nonspecific rabbit IgG. Chemiluminescence was visualized on a Kodak Image Station 440 CF. Experiment was performed 3 times with similar results.
