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...

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).The testis and the ovary arise from a common bipotential gonad during mammalian embryogenesis. During fetal life, at 11 days postcoitum (dpc) in the mouse, the indifferent gonad develops as a narrow band of tissue close to the kidney. The testis-determining gene Sry, located on the Y chromosome, acts dominantly to trigger differentiation of testes from the indifferent gonads that would otherwise develop as ovaries (22,29,48). Once the gonads begin to differentiate as testes, they secrete factors, notably anti-Müllerian hormone and testosterone, which determine further sexual development and are required for normal reproductive function in the adult individual. Disturbances in the initial sex-determining switch or in the subsequent differentiation of the testis will lead to incomplete sexual development in XY individuals who would otherwise develop as males.In addition to the master switch in sex determination, Sry, several genes have been identified that are involved in the formation of the indifferent gonad or subsequent differentiation of the testis. These include genes encoding, e.g., the transcription factors Sf1, Wt1, Emx1, Lhx9, M33, and Dmrt1 and the signaling molecules Fgf9, Wnt7a, Wnt4, and Dhh (reviewed in references 7 and 50). Despite the characterization of these genes, it is clear that key factors in gonad and testis development are lacking, and no genes that are directly regulated by the transcription factor Sry have been characterized.

Testatin has been implicated in fetal testis development due to its restricted expression in pre-Sertoli cells immediately after the onset of Sry gene expression. However, testatin knockout mice showed normal testis development and fertility. We investigated the spatial and temporal expression pattern of the Cres/testatin subgroup of genes, including the novel gene Cstl1/Cres4, in fetal mouse gonads and in adult testis, epididymis and ovary. The genes are related to the family 2 cystatins of protease inhibitors. Using real-time PCR and in situ hybridization we could show that 4 subgroup genes, testatin, CstSC, CstTE-1/Cres3 and Cres are expressed in fetal testis. We also confirmed the expression of testatin, CstE2, CstSC, CstTE-1/Cres3, Cres, CstT and Cstl1/Cres4 in adult testis and CstE2, CstTE-1/Cres3, Cres and CstE1/Cres2 in adult epididymis. In testatin knockout animals, the expression of CstE2 was heavily downregulated in adult testis, but not in adult epididymis, compared to wildtype controls. In conclusion, an explanation for the lack of phenotype in testatin knockout mice could be functional redundancy with another member of the Cres/testatin subgroup. The most likely candidate/s would be CstSC, CstTE-1/Cres3 or Cres as they are expressed in the fetal testicular tubules in early testis differentiation together with testatin.