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 from surface iodinated and metabolically labeled G6 cells typically showed a five-fold induction of a beta(1) integrin associated protein upon differentiation. Under non-reducing conditions this beta(1) associated protein migrated as 145 kD. No such beta(1) associated protein was observed in the myogenic L8 rat cell line, before or after differentiation, The beta(1) integrin associated cell surface protein present in G6 myotubes remained associated with the beta 1 subunit in the presence of 1% Triton X-100 and 0.5 M NaCl. Like integrin alpha-chains, the protein dissociated from the beta(1) integrin subunit at low pH. Immunoprecipitation 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 antibodies to alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v integrin chains could not deplete the beta(1) integrin associated protein, indicating that it did not interact with any of these known integrin heterodimers. Upon treatment with reducing agents, the beta(1) integrin associated protein migrated in SDS-PAGE as a 155 kD protein. The decreased 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 protein. Antibodies to alpha 2 and alpha 9 failed to immunoprecipitate proteins from G6 myotubes and Northern blot analysis likewise failed to detect messages for these two integrin alpha-chains, Immunohistochemistry with antibodies to different integrins of fetal thigh muscles showed staining of alpha 6 integrin at the sarcolemma, Antibodies to alpha 2 and alpha 9 stained keratinocytes and other non-muscle cells in the fetal leg, but no signal was detected for alpha 2 and alpha 9 integrin on fetal myotubes. Based on these characteristics, we propose that the induced protein is a hitherto unidentified integrin alpha-chain on 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 early human myotube formation. (C) 1995 Wiley-Liss, Inc.