Characterization of the beta-dystroglycan-growth factor receptor 2 (Grb2) interaction.
The beta-dystroglycan/Grb2 interaction was investigated and a proline-rich region within beta-dystroglycan that binds Grb2-src homology 3 domains identified. We used surface plasmon resonance (SPR), fluorescence analysis, and solid-phase binding assay to measure the affinity constants between Grb2 and the beta-dystroglycan cytoplasmic tail. Analysis of the data obtained from SPR reveals a high-affinity interaction (K(D) approximately 240 nM) between Grb2 and the last 20 amino acids of the beta-dystroglycan carboxyl-terminus, which also contains a dystrophin-binding site. A similar K(D) value (K(D) approximately 280 nM) was obtained by solid-phase binding assay and in solution by fluorescence. Both Grb2-SH3 domains bind beta-dystroglycan but the N-terminal SH3 domain binds with an affinity approximately fourfold higher than that of the C-terminal SH3 domain. The Grb2-beta-dystroglycan interaction was inhibited by dystrophin in a range of concentration of 160-400 nM. These data suggest a highly regulated and dynamic dystrophin/dystroglycan complex formation and that this complex is involved in cell signaling.
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Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, V. le Regina Elena 299, Rome, 00161, Italy.
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Macchia
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Rosa
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Brancaccio
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Petrucci
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D000818:Animals; D001665:Binding Sites; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D049030:Dystroglycans; D016189:Dystrophin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D051383:GRB10 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013997:Time Factors; D018909:src Homology Domains
D003598:Cytoskeletal Proteins; D016189:Dystrophin; D008024:Ligands; D008562:Membrane Glycoproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049030:Dystroglycans; D051383:GRB10 Adaptor Protein; D011392:Proline; D005982:Glutathione Transferase