Binding of ADAM12, a Marker of Skeletal Muscle Regeneration, to the Muscle-specific Actin-binding Protein, α-Actinin-2, Is Required for Myoblast Fusion

Abstract

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific ␣-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for ␣-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membranedistal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with ␣-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major ␣-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with ␣-actinin-2 is important for ADAM12 function.ADAMs (for "a disintegrin and metalloprotease") 1 are a family of transmembrane glycoproteins encoded by at least 30 genes identified in Caenorhabditis elegans, Drosophila, Xenopus, and various mammalian species. The extracellular portion of ADAMs share a high sequence homology and domain organization with the class III snake venom metalloprotease-disintegrins. Both ADAMs and snake venom metalloprotease-disintegrins contain a metalloprotease-like domain with an associated regulatory prodomain, a disintegrin-like domain, a cysteine-rich domain, and an epidermal growth factor-like domain. In addition, ADAMs contain a transmembrane domain and a cytoplasmic tail (1-4). ADAMS with active metalloprotease are involved in diverse and important cellular processes (5-7). ADAM17 (tumor necrosis factor converting enzyme; TACE), ADAM10 (Kuzbanian), and ADAM9 function in the shedding of the ectodomain of membrane-anchored proteins, such as the cytokine tumor necrosis factor-␣ (8, 9), the tumor necrosis factor receptor, transforming growth factor-␣ (10, 11), the heparin-binding epidermal growth factor-like growth factor (12), and the cleavage of the amyloid precusor protein (13-15). ADAM12 is also an active metalloprotease, which binds to ␣2-macroglobulin in vitro (16), but its physiological substrates have not been identified.The other extracellular domains of ADAMs, the disintegrin and the cysteine-rich domains, also play important roles in cell-cell and cell-matrix adhesion, cell differentiation, and fusion. The disintegrin-like domains of ADAMs are the most highly conserved...

ADAM12 and α9β1Integrin Are Instrumental in Human Myogenic Cell Differentiation

“…In rodents, constitutive muscle expression of ADAM12 starts at the embryonic stage when myotubes are formed (Kurisaki et al, 1998), persists at the neonatal stage (Yagami-Hiromasa et al, 1995;Borneman et al, 2000;Kronqvist et al, 2002), and ceases in adulthood (Borneman et al, 2000;Kronqvist et al, 2002). Adult muscle regeneration after experimental injury is associated with ADAM12 reexpression by fusing myogenic cells and newly formed fibers (Galliano et al, 2000;Kronqvist et al, 2002). Consistently, ADAM12 expression parallels fusion and myogenin expression of C2C12 myogenic cells (YagamiHiromasa et al, 1995).…”

“…For example, ADAM12 cell signaling may positively influence myogenesis, because membrane-bound ADAM12 activates phosphatidylinositol (PI) 3-kinase by mediating its recruitment to the membrane , active PI 3-kinase being crucially involved in terminal differentiation of myogenic cells (Jiang et al, 1998;Li et al, 2000). Moreover, the ADAM12 cytoplasmic tail binds ␣-actinin 1 and 2 (Galliano et al, 2000;Cao et al, 2001), this binding being required for full-blown fusion of C2C12 myogenic cells (Galliano et al, 2000).…”

Novel alternatively spliced ADAM8 isoforms contribute to the aggressive bone metastatic phenotype of lung cancer

“…The proinvasive activity induced by wild-type ADAM8 would require the coordinated activity of cell-matrix adhesion and degradation, cytoskeleton organization and motility. Previous findings have suggested a role for ADAM12, which is closely related to ADAM8, in the extracellular processing of target substrates that favor motility and cytoskeletal remodeling (Kveiborg et al, 2008) such as Src (Stautz et al, 2010), through interactions with key components of cell-matrix adhesion such as actinin-1 (Cao et al, 2001) and actinin-2 (Galliano et al, 2000). Similarly, ADAM8 has been implicated in the processing of adhesion proteins in other systems (Hooper and Lendeckel, 2005).…”

ADAM12 Alleviates the Skeletal Muscle Pathology in mdx Dystrophic Mice

“…For immunohistochemistry on frozen sections, polyclonal antiserum to the Cterminal peptide of mouse ADAM12 (rb 109) was used as previously described. 21 This antiserum reacts to both mouse and human ADAM12. For immunohistochemical staining on paraffin sections, the following antibodies were used: rat anti-mouse macrophage antibody F4/80 (Serotec) at 1:800 and rat anti-mouse CD45 (SRT5, Serotec) at 1:100.…”

“…19,20 Postnatally, the expression of ADAM12 in skeletal muscle ceases, 19 but reappears transiently during regeneration. 21,22 Some studies suggest that ADAM12 is a myogenic factor. 19,23 Here, we generated transgenic mice that continuously express ADAM12 in mature skeletal muscle.…”