A DEVELOPMENTALLY-REGULATED FORM OF INSULIN-LIKE GROWTH-FACTOR RECEPTOR BETA-SUBUNIT IN C2-MYOBLASTS EXHIBITING ALTERED REQUIREMENTS FOR DIFFERENTIATION

Ligand-dependent autophosphorylation and immunoprecipitation have been used to distinguish insulin and insulin-like growth factor-I (IGF-I) receptor beta-subunits in the permissive and inducible subclones of th e C2 myoblast cell line. Permissive myoblasts differentiate spontaneou sly, whereas myoblasts of the inducible subclone require exogenous IGF s to undergo terminal differentiation. Permissive myoblasts contain be ta-subunits of 95 and 101 kilodalton (kDa) mol wt. The 95-kDa subunits are immunoprecipitated with antipeptide antibodies directed against t yrosine kinase (AbP2), juxtamembrane (AbP4), and carboxy-terminal (AbP 5) domains of the insulin receptor and insulin receptor monoclonal ant ibody 29B4. The tryptic phosphopeptide map of the 95kDa band suggests that it contains both insulin and IGF-I receptor beta-subunits. The 10 1-kDa subunit is immunoprecipitated by AbP2, AbP4, and AbP5, because i t forms a hybrid complex with the 95-kDa protein, but it does not reac t directly with AbP4, AbP5, or antibody 29B4. Phosphorylation of the 1 01-kDa subunit is more responsive to IGF-I than to IGF-II or insulin, indicating that it is a second IGF-I receptor beta-subunit, Inducible myoblasts exhibit a single major beta-subunit of 106 kDa mol wt. Its i mmunoreactivity and phosphopeptide map are virtually identical to thos e of the 101-kDa IGF-I receptor beta-subunit from permissive cells. Ho wever, unlike the 101-kDa beta-subunit, phosphorylation of the 106-kDa protein appears to be more responsive to IGF-II than to either IGF-I or insulin. It is lost upon differentiation of myoblasts into myotubes concomittant with the appearance of 95- and 101-kDa beta-subunits. Th ese data demonstrate 1) an alpha2beta2 IGF receptor that has high sens itivity for IGF-II in inducible, but not in permissive, myoblasts; 2) the beta-subunit of this receptor exhibits different migration in sodi um dodecyl sulfate-polyacrylamide gels from either of those found in p ermissive cells; and 3) expression of this beta-subunit is development ally regulated. This suggests that the inducible cell beta-subunit is a component of a stage-specific alpha2beta2 IGF receptor subtype that functions as an IGF-II receptor.