Point mutations in the split PLC-γ1 PH domain modulate phosphoinositide binding

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH₂-terminal (PH ₁) and a split PH domain (nPH₂ and cPH₂). We previously reported that the split PH domain of PLC-γ1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P ₂, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-γ1 nPH₂ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-γ1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-γ1 molecules showed reduced PI(4,5)P₂ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH₁ and nPH₂ domains are responsible for membrane-targeted translocation of PLC-γ1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro⁵⁰⁰ and His503 are critical for binding of PLC-γ1 to one of its substrates, PI(4,5)P₂ in the membrane.