Figure 14. Unlike the PLCγ₂ deletion mutant Δ20–22, PLCγ₂S707Y is sensitive to stimulation by EGF.

COS-7 cells were transfected with vectors encoding either wild-type PLCγ₂ (WT) (500 ng per well, left panel), PLCγ₂Δ20–22 (Δ20–22) or PLCγ₂S707Y (S707Y) (both at 150 ng per well). Eighteen hours after transfection, the cells were incubated for a further 24 h as indicated at either 31° C or 37° C with myo-[2-³H]inositol and 10 mM LiCl in the absence of serum and then treated for 60 min at the same temperatures in the presence of 10 mM LiCl with 100 ng/ml EGF, followed by determination of inositol phosphate formation. Background inositol phosphate formation in response to addition of EGF was determined in parallel on cells transfected with empty vector and subtracted from the individual values, with appropriate consideration of error propagation [47]. Additional experiments showed that the stimulatory effect of EGF on wild-type PLCγ₂ activity was concentration-dependent with half-maximal and maximal effects at approximately 10 ng/ml and 50 ng/ml, respectively, and was almost completely blocked (~95%) by the EGFR inhibitor cetuximab (not shown).