Abstract
The extracellular domain of the mature form of ADAM12 consists of the metalloprotease, disintegrin, cysteine-rich, and epidermal growth factor (EGF)-like domains. The disintegrin, cysteine-rich, and EGF-like fragments have been shown previously to support cell adhesion via activated integrins or proteoglycans. In this study, we report that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 myoblasts even in the absence of exogenous integrin activators. This adhesion was not mediated by beta1 integrins or proteoglycans, was myoblast-specific, and required the presence of both the metalloprotease and disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggested that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differ from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain (but not the metalloprotease domain or the disintegrin/cysteine-rich/EGF-like fragment alone) decreased the expression of the cell cycle inhibitor p21 and myogenin, two markers of differentiation, and inhibited C2C12 myoblast fusion. Thus, the novel protein-protein interaction reported here involving the extracellular domain of ADAM12 may have important biological consequences during myoblast differentiation.
Highlights
Ions or activating antibodies, to promote significant levels of cell adhesion (8 –15)
Integrin-mediated adhesion requires pre-activation of cellular integrins with Mn2ϩ ions or integrin-activating antibodies (8 –15), suggesting low affinity interactions between integrins and their ADAM ligands. Such low affinity interactions with integrins are observed for recombinant ADAM domains expressed in E. coli, which most likely lack the correct arrangement of disulfide bonds, and for ADAM domains produced in eukaryotic cells, where the formation of disulfide bonds is enabled
We have recently shown that the disintegrin and cysteine-rich (DC) domain of ADAM12 produced in insect cells interacts with integrin ␣71 and that, in line with reports for other ADAM proteins, this interaction requires integrin activation [14]
Summary
Ions or activating antibodies, to promote significant levels of cell adhesion (8 –15). We report that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 myoblasts even in the absence of exogenous integrin activators.
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