A putative Src homology 3 domain binding motif but not the C-terminal dystrophin WW domain binding motif is required for dystroglycan function in cellular polarity in Drosophila.
The conserved dystroglycan-dystrophin (Dg.Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg.Dys complex is poorly understood in any cell type. We now find that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. We confirm this hypothesis by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (K(d) = 7.6 and 81 microM, respectively) and Drosophila (K(d) = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg.
Yatsenko
Andriy S
AS
Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
Gray
Elizabeth E
EE
Shcherbata
Halyna R
HR
Patterson
Larissa B
LB
Sood
Vanita D
VD
Kucherenko
Mariya M
MM
Baker
David
D
Ruohola-Baker
Hannele
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D016764:Cell Polarity / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D049030:Dystroglycans / Q000235:genetics / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains / Q000235:genetics
D029721:Drosophila Proteins; D016189:Dystrophin; D049030:Dystroglycans