Interaction of integrin alpha 7 beta 1 in C2C12 myotubes and in solution with laminin.
The dimer of integrin alpha 7 and beta 1 is a major laminin-binding receptor in skeletal muscle. We studied interactions of integrin alpha 7 beta 1 with the extracellular matrix protein laminin in solution and in intact cells. Integrin alpha 7 beta 1 bound to EHS laminin (laminin-1, composed of alpha 1, beta 1, and gamma 1 chains), but not to endogenous laminin expressed in C2C12 myotubes. Northern blot analysis demonstrated that C2C12 myotubes synthesized laminin-1 alpha, beta, and gamma subunits mRNAs. C2C12 laminin was, however, immunologically distinct from EHS laminin; it was not recognized by 5D3 anti-laminin-1 monoclonal antibody, whereas 5A2 and LT3 antibodies reacted equally well with C2C12 and EHS laminins. Following deglycosylation of EHS laminin, separation of the subunits by SDS-PAGE, Western blotting, and partial amino acid sequencing of the protein bands, the epitope recognized by 5D3 antibody was localized to the gamma 1 laminin chain. Following binding in vitro, the complex of EHS laminin and integrin alpha 7 beta 1 was subject to chemical cross-linking. The two proteins did not undergo cross-linking at the cell surface, consistent with the fact that in intact, resting myotubes integrin alpha 7 beta 1 interacted poorly with EHS laminin, which may reflect a limited accessibility of integrin alpha 7 beta 1 in the membrane to laminin or an inactive state of the integrin.
Zolkiewska
A
A
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland 20892-1590, USA.
Thompson
W C
WC
Moss
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D003902:Detergents; D000939:Epitopes / Q000187:drug effects / Q000276:immunology; D016023:Integrins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007797:Laminin / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D008565:Membrane Proteins / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis; D017474:Receptors, Laminin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012995:Solubility
D003902:Detergents; D000939:Epitopes; D016023:Integrins; D007797:Laminin; D008565:Membrane Proteins; D012333:RNA, Messenger; D017474:Receptors, Laminin; C082908:integrin alpha7beta1