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 seve ral point mutations in bovine profilin I by computer modeling. The recombin ant proteins were analyzed in vitro for their actin-binding properties. Mut ant proteins with a putatively higher affinity for actin were produced by a ttempting to introduce an additional bond to actin, However, these mutants displayed a lower affinity for actin than wild-type profilin, suggesting th at additional putative bonds created this way cannot increase profilin's af finity for actin. In contrast, mutants designed to have a reduced affinity for actin by eliminating profilin-actin bonds displayed the desired propert ies 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 wildtype profilin, was analyzed fu rther in cells. When microinjected into fibroblasts, F59A colocalized with the endogenous profilin and actin in ruffling areas, suggesting that profil ins are targeted to and tethered at these sites by ligands other than actin . Profilin null cells of Dictyostelium were transfected with bovine wild-ty pe profilin I and F59A, Bovine profilin I, although expressed to only appro ximately 10% of the endogenous profilin level determined for wildtype Dicty ostelium, caused a substantial rescue of the defects observed in profilin n ull amoebae, as seen by measuring the growth of colony surface areas and th e percentage of polynucleated cells. The mutant protein was much less effec tive. These results emphasize the highly conserved biological function of p rofilins with low sequence homology, and correlate specifically their actin -binding capacity with cell motility and proliferation.