Characterization of the actin binding site on smooth muscle filamin.
We have isolated an NH2-terminal fragment of filamin (M(r) = 70,000) after digestion with Staphylococus aureus V8 protease. This fragment was shown to interact with filamentous actin in cosedimentation assays. Using cross-reactive anti-peptides antibodies directed against the strongly conserved 27-mer sequence of alpha-actinin, already implicated as 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 major element in the interaction with actin. In particular, we used enzyme-linked immunosorbent assay experiments, in conjunction with a synthetic peptide approach, and found that the hydrophobic part of the 27-mer peptide (141-147 sequence) is largely involved in actin binding. Thus, 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 between actin and these cytoskeletal proteins. However, the divergent behavior of filamin and alpha-actinin toward conformational changes of actin argues in favor of distinctive interfaces. Finally, the ionic strength dependence of the filamin-actin interaction, in contrast to that with alpha-actinin, strongly suggests that, besides hydrophobic interactions conferred by the 27-mer sequence, more hydrophilic region(s) of filamin participate(s) in the binding.
Lebart
M C
MC
Centre National de la Recherche Scientifique, U 249 Institut National de la Santé et de la Recherche Médicale, Ecole Pratique des Hautes Etudes, Université de Montpellier I, France.
Méjean
C
C
Casanova
D
D
Audemard
E
E
Derancourt
J
J
Roustan
C
C
Benyamin
Y
Y
D000185:Actinin / Q000737:chemistry / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003285:Contractile Proteins / Q000737:chemistry / Q000378:metabolism*; D064448:Filamins; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism; D009130:Muscle, Smooth / Q000737:chemistry; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry; D012995:Solubility
D000199:Actins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D000185:Actinin