UP-REGULATION OF A NOVEL INTEGRIN ALPHA-CHAIN (ALPHA(MT)) ON HUMAN FETAL MYOTUBES

Integrin expression and distribution was studied in cloned human fetal G6 myoblasts and myotubes. Immunoprecipitation of beta(1) integrins f rom surface iodinated and metabolically labeled G6 cells typically sho wed a five-fold induction of a beta(1) integrin associated protein upo n differentiation. Under non-reducing conditions this beta(1) associat ed protein migrated as 145 kD. No such beta(1) associated protein was observed in the myogenic L8 rat cell line, before or after differentia tion, The beta(1) integrin associated cell surface protein present in G6 myotubes remained associated with the beta 1 subunit in the presenc e of 1% Triton X-100 and 0.5 M NaCl. Like integrin alpha-chains, the p rotein dissociated from the beta(1) integrin subunit at low pH. Immuno precipitation of G6 myotubes further indicated the presence of alpha 1 , alpha 3, alpha 5, and alpha v integrins, and small amounts of alpha 4 and alpha 6 integrins. Immunodepletion with integrin alpha-chain ant ibodies to alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v in tegrin chains could not deplete the beta(1) integrin associated protei n, indicating that it did not interact with any of these known integri n heterodimers. Upon treatment with reducing agents, the beta(1) integ rin associated protein migrated in SDS-PAGE as a 155 kD protein. The d ecreased mobility in SDS-PAGE upon reduction is a feature shared with alpha 1, alpha 2, and alpha 9 integrin alpha-chains. Antibodies to oil immunoprecipitated an integrin heterodimer distinct from the 155 kD p rotein. Antibodies to alpha 2 and alpha 9 failed to immunoprecipitate proteins from G6 myotubes and Northern blot analysis likewise failed t o detect messages for these two integrin alpha-chains, Immunohistochem istry with antibodies to different integrins of fetal thigh muscles sh owed staining of alpha 6 integrin at the sarcolemma, Antibodies to alp ha 2 and alpha 9 stained keratinocytes and other non-muscle cells in t he fetal leg, but no signal was detected for alpha 2 and alpha 9 integ rin on fetal myotubes. Based on these characteristics, we propose that the induced protein is a hitherto unidentified integrin alpha-chain o n myotubes that we name alpha(mt). Our finding of a novel integrin or- chain that is induced upon myogenic differentiation supports the idea that different integrins are important for myogenesis during different developmental stages, and we suggest that alpha(mt) takes part in ear ly human myotube formation. (C) 1995 Wiley-Liss, Inc.