Fig. 8. The effects of GRP1-PH–GFP mutations on PIP₃ or InsP₄ binding, cellular localization and inhibition of cellular spreading in COS-7 cells. (A) COS-7 (upper) and HEK 293 cells (lower) were transfected with the indicated mutant GRP1-PH–GFP construct and examined by confocal microscopy without stimulation (HEK 293 cells) or after stimulation with peroxyvanadate (30 µM peroxide, 100 µM ortho-vanadate) for 5-10 minutes (COS-7 cells). (B) Binding of recombinant GRP1-PH–GFP mutants to PIP₃ conjugated to agarose beads (sn, unbound fraction in the supernatant; pellet, bound fraction associated with the beads); bars represent the % bound fraction determined by Phosphorimager analysis from two separate experiments (mean±range), one of which is shown as a representative. (C) Binding of InsP₄ to GRP1-PH–GFP mutants. Recombinant proteins were incubated with [³H]InsP₄ in the presence of increasing concentration of unlabeled InsP₄ for 10 minutes. Protein-bound radioactivity was determined as described under Materials and Methods and expressed as % B₀ (means±s.e.m., n=3). (D) Inhibition of cell spreading by the various GRP1-PH–GFP constructs as described in Fig. 4. (mean±s.e.m., n=3). In (B) and (D), the R284C mutant, unable to bind the lipid, is indicated by darker columns. (E) The position of the mutated residues (I307 and K340) within the GRP1-PH domain relative to the PIP₃ binding site in the crystal structure of GRP1-PH (1FHX).