Effect of G-Actin on Profilin Binding to Sub-Micellar Concentration of Polyphosphoinositides

Abstract

Profilin is an essential actin binding protein which promotes filament turnover. In the last decade, profilin has been involved in the signalling pathway linking the receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signalling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4.5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (Lu et al., 1996. Biochemistry 35. 14027-34). So far, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P₂ molecules (Goldschmidt-Clermont et al., 1991. Science 251. 1231-3). In the cell, PPI lipids are not organized as they are in micelles or small vesicles therefore their interaction with profilin might be quite different. PI(4.5)P₂ is known to dissociate the actin profilin complex due to overlapping binding site for actin and PI(4,5)P₂ on profilin. However, recent works suggest the presence of two binding regions for PI(4,5)P₂ on profilin. One of these regions allows binding of PI(4,5)P₂ to profilin crosslinked with actin (Skare and Karlsson, 2002, FEBS Letters 522, 119-124). In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(4,5)P₃. We determined the relevant dissociation constant by fluorescence anisotropy when sub-micellar concentrations of fluorescently labelled PPI bind to profilin both in presence and absence of actin. We show that the dissociation constant of profilin with sub-micellar concentrations of PPI is significantly different to that of profilin with micelles or small vesicles. We also show that profilin binds more strongly to sub-micellar concentrations of PI(4,5)P₃ than to sub-micellar concentrations of PI(4,5)P₂. Finally, we demonstrate the existence in solution of a ternary complex between actin, profilin and PPI and the effect of actin on profilin's dissociation constant.