Utrophin actin binding domain: analysis of actin binding and cellular targeting.
Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiquitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-action (Kd 19 +/- 2.8 microM), midway between that of the corresponding domains of alpha-actinin (Kd 4 microM) and dystrophin (Kd 44 microM). Moreover, this utrophin domain binds to non-muscle actin with a approximately 4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992) Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted 'spacer' or 'shock absorber' role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.
Winder
S J
SJ
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Hemmings
L
L
Maciver
S K
SK
Bolton
S J
SJ
Tinsley
J M
JM
Davies
K E
KE
Critchley
D R
DR
Kendrick-Jones
J
J
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016189:Dystrophin / Q000096:biosynthesis / Q000302:isolation & purification; D004926:Escherichia coli; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D007150:Immunohistochemistry; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D049411:Utrophin
D000199:Actins; D003598:Cytoskeletal Proteins; D016189:Dystrophin; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049411:Utrophin