Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employed a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. We examined 67 human PH domain-containing proteins for interaction with PIPs, and found 51% of them to have affinity for PIPs with various specificity, the majority of which had not been reported before. Further investigation of ARHGEF3 revealed structural requirements and functional relevance of its newly discovered PI(4,5)P2 binding. Based on the assay data of the 67 proteins, we generated a recursive-learning algorithm, which identified amino acid determinants of PIP binding throughout the entire PH domain. This recursive functional classification predicted that 46% of 250 human PH domains bind PIPs with some specificity. Collectively, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.