Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex.
The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy-terminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in beta-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to beta-dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin beta-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca(2+)-dependent binding of calmodulin. The remaining two motifs found in the carboxy-terminus (H1-H2) mediate the formation of utrophin-dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.
Tommasi di Vignano
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A
Department of Biology 'Enrico Calef', University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy.
Di Zenzo
G
G
Sudol
M
M
Cesareni
G
G
Dente
L
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D016384:Consensus Sequence; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D049030:Dystroglycans; D016189:Dystrophin / Q000737:chemistry / Q000378:metabolism*; D049029:Dystrophin-Associated Proteins; D020832:EF Hand Motifs; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D049411:Utrophin
D002147:Calmodulin; D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; C490915:DTNA protein, human; D016189:Dystrophin; D049029:Dystrophin-Associated Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C483600:Utrn protein, mouse; D049411:Utrophin; C099589:dystrophin-related protein 2; D049030:Dystroglycans; D002118:Calcium