EXPRESSION AND FUNCTIONAL-ANALYSIS OF THE 66-KDA PROTEIN, A MATRIX ASSEMBLY RECEPTOR OF FIBRONECTIN, DURING MYOGENESIS

Previously, we have identified a 66-kDa protein on the surface of chic k myoblasts that has a binding activity toward the N-terminal 29-kDa f ragment of fibronectin [Moon, K. Y., Shin, K. S., Song, W. K., Chung, C. H., Ha, D. B., and Kang, M. S. (1994) J. Biol. Chem. 269, 7651-7657 ]. In this report, we describe the changes in expression and cellular localization of the 66-kDa protein, fibronectin, and alpha 5 integrin during myogenesis. The effects of an anti-66-kDa antibody on myoblast differentiation were also studied. Immunofluorescence staining of myob lasts with an anti-66-kDa protein antibody revealed dotlike circular a ggregates in which the 66-kDa protein colocalized with alpha 5 integri n and fibronectin. These aggregates are proposed to be initiation site s of fibronectin matrix assembly in early myoblasts. As myoblasts beca me elongated, the dot-like aggregated pattern progressively diminished and the 66-kDa protein and fibronectin redistributed into long fibril lar structures located along the cell periphery. These long fibrillar structures did not contain alpha 5 integrin. Instead, the cells staine d at this stage with an anti-alpha 5 integrin antibody showed small sp ikes in the adhesion plaques. As the cells further matured, the 66-kDa protein and the fibronectin matrix totally disappeared, whereas alpha 5 integrin was still present in myotubes. These results suggest that the 66-kDa protein is responsible for the initiation and elongation of fibronectin matrix assembly, whereas alpha 5 integrin is only involve d in the initiation of fibronectin matrix assembly. Immunoblotting ana lysis showed that the expression of the 66-kDa protein and fibronectin dramatically decreased during myogenesis and then completely disappea red in the late stage of myogenesis. The disappearance of fibronectin matrices is closely correlated to the decrease in the 66-kDa protein. Furthermore, the anti-66-kDa protein antibody suppressed the accumulat ion of fibronectin into the extracellular matrix and promoted the diff erentiation of myoblast in concentration- and treatment time-dependent manners. These results suggest that the 66-kDa protein may regulate m yoblast differentiation by controlling the incorporation of fibronecti n into the extracellular matrix.