Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha-actinin-2, is required for myoblast fusion

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin an d metalloprotease") family. ADAM12 has been implicated in muscle cell diffe rentiation and fusion, but its precise function remains unknown. Here, we s how that ADAM12 is dramatically up-regulated in regenerated, newly formed f ibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undiffe rentiated myoblasts and is transiently up-regulated at the onset of differe ntiation when myoblasts fuse into multinucleated myotubes, whereas other AD AMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of di fferentiation. Using the yeast two-hybrid screen, we found that the muscle- specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific int eraction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a seco nd binding site was identified in the membrane-distal region. Co-immunoprec ipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 ce lls, whereas expression of a cytosolic ADAM12 lacking the major alpha-actin in-2 binding site had no effect on cell fusion. Our results suggest that in teraction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.