CHARACTERIZATION OF THE ACTIN-BINDING SITE ON SMOOTH-MUSCLE FILAMIN

We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) a fter digestion with Staphylococus aureus V8 protease. This fragment wa s shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the str ongly conserved 27-mer sequence of alpha-actinin, already implicated a s an actin binding site (Kuhlman, P. A., Hemmings, L., and Critchley, D, R. (1992) FEBS Lett. 304, 201-206), we obtained evidence suggesting that the homologous sequence of filamin (121-147 sequence) is the maj or element in the interaction with actin. In particular, we used enzym e-linked immunosorbent assay experiments, in conjunction with a synthe tic peptide approach, and found that the hydrophobic part of the 27-me r peptide (141-147 sequence) is largely involved in actin binding. Thu s, the filamin sequence 121-147 (or the alpha-actinin sequence 108-134 ) and the actin counterpart composed of residues 112-125 and 360-372 ( we have already implicated) could constitute the main interface betwee n actin and these cytoskeletal proteins. However, the divergent behavi or of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strengt h dependence of the filamin-actin interaction, in contrast to that wit h alpha-actinin, strongly suggests that, besides hydrophobic interacti ons conferred by the 27-mer sequence, more hydrophilic region(s) of fi lamin participate(s) in the binding.