Characterization of the extracellular matrix protein, beta-ig, during murine embryogenesis and its putative function as an adhesion molecule at the myotendinous junction

A novel protein named beta-ig has been discovered as the product of a transforming growth factor-β1 (TGF-β1) responsive gene. Consensus features within the amino acid sequence suggest beta-ig may associate with other molecules. One feature is a region of internal repeats similar to repeating sequences found in fasciclin-I, a nerve cell growth cone guidance molecule expressed in developing Drosophila. A second feature is an Arg-Gly-Asp (RGD) sequence near the carboxyl terminus, revealing the possibility that beta-ig may interact with members of the integrin family of cell adhesion molecules. Consistent with this latter possibility is the finding that a substratum composed of beta-ig supported attachment of human dermal fibroblasts (LeBaron, 1995). The spatial and temporal distribution of beta-ig during murine development has been studied here in order to obtain useful information to form hypotheses concerning the physiological function of beta-ig. Based on results showing that anti-beta-ig antibody stains regions where myofibrils attach near regions of developing cartilage and bone, in vitro attachment assays using myoblast cultures and purified, recombinant beta-ig were performed. Results are consistent with the hypothesis that beta-ig supports skeletal muscle cell attachment and possibly plays an adhesive role at developing myotendinous junctions (MTJs). Additional results, using a combination of immunofluorescence microscopy and inhibition assays, suggest skeletal muscle cell attachment to beta-ig: (i) is dose and time-responsive, (ii) divalent-cation dependent, (iii) mediated by the α7β1 integrin receptor, (iv) and promotes focal contacts lacking pp125fak and paxillin but containing α-actinin. Ultrastructure analysis indicates beta-ig is present in the extracellular space between myofibers and reveals the binding of beta-ig to ECM fibers in the intercellular space and to the myogenic cell membrane in vivo . Overall, results suggest that beta-ig serves as an attachment molecule at the MTJ and that the interaction of beta-ig with skeletal muscle cells may be physiologically significant to the development of skeletal muscle and particularly, the myotendinous junction.