Localization of a Binding Site for Phosphatidylinositol 4,5-Bisphosphate on Human Profilin (*)

Abstract

Profilin is a small 12-15-kDa actin-binding protein, which in eukaryotic organisms is ubiquitous and necessary for normal cell growth and function. Although profilin's interactions with its three known ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP²), and poly-L-proline (PLP)) have been well characterized in vitro, its precise role in cells remains largely unknown. By binding to clusters of PIP², profilin is able to inhibit the hydrolysis of PIP² by phospholipase C1 (PLC1). This ability is the result of profilin's affinity for PIP², but the specific residues of profilin's amino acid sequence involved in the binding of PIP² are not known. Using site-directed mutagenesis, we sought to localize regions of profilin important for this interaction by generating the following mutants of human profilin (named according to the wild-type amino acid altered, its position, and the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53I, R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of L10R, all of the mutants were successfully expressed in Escherichia coli and purified by affinity chromatography on PLP-Sepharose. Only Y6F and K25Q demonstrated moderately less stringent binding to PLP, indicating that most of the mutations did not induce marked alterations of profilin's structure. When tested for their relative abilities to inhibit the hydrolysis of PIP² by PLC1, most of the mutants were indistinguishable from wild-type profilin. Exceptions included D8A, which demonstrated increased inhibition of PLC1, and R88L, which demonstrated decreased inhibition of PLC1. To assess the importance of the region surrounding residue 88 of human profilin, three synthetic decapeptides selected to correspond to non-overlapping stretches of the human profilin sequence were tested for their abilities to inhibit PLC1. We found that only the decapeptide that matched the peptide stretch centered around residue 88 was able to inhibit PLC1 activity substantially and was able to do so at nearly wild-type profilin levels. Taken together with the finding that mutating residue 88 resulted in decreased inhibition of PLC1 activity, these data provide strong evidence that this region of human profilin represents an important binding site for PIP².