Effects of single amino acid substitutions in the actin-binding site on the biological activity of bovine profilin I.

For a detailed analysis of the profilin-actin interaction, we designed several point mutations in bovine profilin I by computer modeling. The recombinant proteins were analyzed in vitro for their actin-binding properties. Mutant proteins with a putatively higher affinity for actin were produced by attempting to introduce an additional bond to actin. However, these mutants displayed a lower affinity for actin than wild-type profilin, suggesting that additional putative bonds created this way cannot increase profilin's affinity for actin. In contrast, mutants designed to have a reduced affinity for actin by eliminating profilin-actin bonds displayed the desired properties in viscosity assays, while their binding sites for poly(L)proline were still intact. The profilin mutant F59A, with an affinity for actin reduced by one order of magnitude as compared to wild-type profilin, was analyzed further in cells. When microinjected into fibroblasts, F59A colocalized with the endogenous profilin and actin in ruffling areas, suggesting that profilins are targeted to and tethered at these sites by ligands other than actin. Profilin null cells of Dictyostelium were transfected with bovine wild-type profilin I and F59A. Bovine profilin I, although expressed to only approximately 10% of the endogenous profilin level determined for wild-type Dictyostelium, caused a substantial rescue of the defects observed in profilin null amoebae, as seen by measuring the growth of colony surface areas and the percentage of polynucleated cells. The mutant protein was much less effective. These results emphasize the highly conserved biological function of profilins with low sequence homology, and correlate specifically their actin-binding capacity with cell motility and proliferation.