Functional analyses of FGFR1 mutants. A. Endoglycosidase analysis of mutant FGFR1s. COS-1 cells were transiently transfected with myc-tagged WT or mutated FGFR1 cDNA. EV= empty vector. Cell lysates were subjected to PNGase (PNG, upper panel) or EndoHf (EH, lower panel) digestion. The overall expression of the G48S, R209H and E670A was not significantly decreased as compared to WT (PNGase-treated bands). Receptor maturation patterns are shown in lower panel. The G48S, R209H and E670A mutants have a similar maturation pattern as WT receptor. B. Cell-surface expression of FGFR1 mutants. COS-1 cells were transiently transfected with myc-tagged WT or mutated FGFR1 cDNA. EV = empty vector. Cell-surface expression levels were determined from fixed cells using an anti-myc primary antibody. Absorbancies were detected at 450 nm. The values on the Y-axis represent fold inductions as compared to the level elicited by EV. The WT, G48S, R209H and E670A have similar cell-surface expression levels. C. MAPK signaling analysis of FGFR1 mutants. L6 myoblasts were transiently transfected with myc-tagged WT or mutated FGFR1 cDNA and treated with FGF2 for 0/2/10/30 min. Phospho-specific antibodies (phospho-p44/42 MAPK) were used to determine phosphorylation of MAPK. To control for equal loading, blots were reprobed using an anti p44/42 MAPK antibody. WT and R209H show clear phosphorylation of MAPK after 10 and 30 minutes of FGF2 treatment. With the mutant receptors G48S and E670A, no clear phosphorylation of MAPK was seen in any of the indicated time points. For comparison of baseline activities, all untreated samples (0 min) were also run on the same gel.