LOCALIZATION OF A BINDING-SITE FOR PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE ON HUMAN PROFILIN

Profilin is a small 12-15-kDa actin-binding protein, which in eukaryot ic organisms is ubiquitous and necessary for normal cell growth and fu nction. Although profilin's interactions with its three known ligands (actin monomers, phosphatidylinositol 4,5-bisphosphate (PIP2), and pol y-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 C gamma 1 (PLC gamma 1). This ability is the result of profilin's affin ity for PIP2, but the specific residues of profilin's amino acid seque nce 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, a nd the amino acid substituted in its place): Y6F, D8A, L10R, K25Q, K53 I, R74L, R88L, R88L/K90E, H119D, G121D, and K125Q. With the exception of L10R, all of the mutants were successfully expressed in Escherichia coil and purified by affinity chromatography on PLP-Sepharose. Only Y 6F and K25Q demonstrated moderately less stringent binding to PLP, ind icating that most of the mutations did not induce marked alterations o f profilin's structure. When tested for their relative abilities to in hibit the hydrolysis of PIP2 by PLC gamma 1, most of the mutants were indistinguishable from wild-type profilin. Exceptions included D8A, wh ich demonstrated increased inhibition of PLC gamma 1, and R88L, which demonstrated decreased inhibition of PLC gamma 1. To assess the import ance of the region surrounding residue 88 of human profilin, three syn thetic decapeptides selected to correspond to non-overlapping stretche s of the human profilin sequence were tested for their abilities to in hibit PLC gamma 1. We found that only the decapeptide that matched the peptide stretch centered around residue 88 was able to inhibit PLC ga mma 1 activity substantially and was able to do so at nearly wild-type profilin levels. Taken together with the finding that mutating residu e 88 resulted in decreased inhibition of PLC gamma 1 activity, these d ata provide strong evidence that this region of human profilin represe nts an important binding site for PIP2.