Biochemical characterisation of the actin-binding properties of utrophin.
Utrophin is a large ubiquitously expressed cytoskeletal protein that is important for maturation of vertebrate neuromuscular junctions. It is highly homologous to dystrophin, the protein defective in Duchenne and Becker muscular dystrophies. Utrophin binds to the actin cytoskeleton via an N-terminal actin-binding domain, which is related to the actin-binding domains of members of the spectrin superfamily of proteins. We have determined the actin-binding properties of this utrophin domain and investigated its binding site on F-actin. An F-actin cosedimentation assay confirmed that the domain binds more tightly to beta-F-actin than to alpha-F-actin and that the full-length utrophin domain binds more tightly to both actin isoforms than a truncated construct, lacking a characteristic utrophin N-terminal extension. Both domain constructs exist in solution as compact monomers and bind to actin as 1:1 complexes. Analysis of the products of partial proteolysis of the domain in the presence of F-actin showed that the N-terminal extension was protected by binding to actin. The actin isoform dependence of utrophin binding could reflect differences at the N-termini of the actin isoforms, thus localising the utrophin-binding site on actin. The involvement of the actin N-terminus in utrophin binding was also supported by competition binding assays using myosin subfragment S1, which also binds F-actin near its N-terminus. Cross-linking studies suggested that utrophin contacts two actin monomers in the actin filament as does myosin S1. These biochemical approaches complement our structural studies and facilitate characterisation of the actin-binding properties of the utrophin actin-binding domain.
Moores
C A
CA
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Kendrick-Jones
J
J
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002645:Chickens; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014357:Trypsin / Q000494:pharmacology; D049411:Utrophin
D000199:Actins; D003598:Cytoskeletal Proteins; D016189:Dystrophin; D008565:Membrane Proteins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D049411:Utrophin; D002918:Chymotrypsin; D014357:Trypsin