Def-6, a guanine nucleotide exchange factor for Rac1, interacts with the skeletal muscle integrin chain alpha7A and influences myoblast differentiation.
Integrin alpha7beta1 is the major laminin binding integrin receptor of muscle cells. The alpha7 chain occurs in several splice isoforms, of which alpha7A and alpha7B differ in their intracellular domains only. The fact that the expression of alpha7A and alpha7B is tightly regulated during skeletal muscle development suggests different and distinct roles for both isoforms. However, so far, functional properties and interacting proteins were described for the alpha7B chain only. Using a yeast two-hybrid screen, we have found that Def-6, a guanine nucleotide exchange factor for Rac1, binds to the intracellular domain of the alpha7A subunit. The specificity of the Def-6-alpha7A interaction has been shown by direct yeast two-hybrid binding assays and coprecipitation experiments. This is the first description of an alpha7A-specific and -exclusive interaction, because Def-6 did not bind to any other tested integrin cytoplasmic domain. Interestingly, the binding of Def-6 to alpha7A was abolished, when cells were cotransfected with an Src-related kinase, which is known to phosphorylate Def-6 and stimulate its exchange activity. We found expression of Def-6 was not only restricted to T-lymphocytes as described thus far but in a more widespread manner, including different muscle tissues. In cells, Def-6 is seen in newly forming cell protrusions and focal adhesions, and its localization partially overlaps with the alpha7A integrin receptor. C2C12 myoblasts overexpressing Def-6 show a delay of Rac1 inactivation during myogenic differentiation and abnormal myotube formation. Thus, our data suggest a role for Def-6 in the fine regulation of Rac1 during myogenesis with the integrin alpha7A chain guiding this regulation in a spatio-temporal manner.
Samson
Thomas
T
Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. thomas_samson@med.unc.edu
Will
Carola
C
Knoblauch
Alexander
A
Sharek
Lisa
L
von der Mark
Klaus
K
Burridge
Keith
K
Wixler
Viktor
V
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D039001:Integrin alpha Chains / Q000235:genetics / Q000378:metabolism*; D007797:Laminin / Q000235:genetics / Q000378:metabolism; D051379:Mice; D024510:Muscle Development / Q000502:physiology; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D039001:Integrin alpha Chains; D007797:Laminin; D009479:Neuropeptides; D009687:Nuclear Proteins; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; C479750:DEF6 protein, human; C107953:RAC1 protein, human; C484602:Rac1 protein, mouse; C473249:Def6 protein, mouse; C085725:integrin alpha7