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

Abstract

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.

Rgf1 GEF activity toward Rho1 defines a new actin-dependent signal to determine growth sites independently of microtubules and Tea1

“…Rgf1 is a large (~150 KDa) multi-domain protein, including a pleckstrin homology (PH) domain (34). PH domains could act as a "membrane-targeting device" by anchoring GEFs to phosphoinositides and directing them towards their partner GTPases on the cellular cortex (42,43). To test the ability of Rgf1 to bind different membrane lipids, we fused the Rgf1 protein to GST (without its carboxi-terminal CNH domain to purify it more easily) and…”