ABSTRACT
We cloned and characterized the cDNA and the expression pattern of a novel member of the murine 'Disintegrin and Metalloprotease-Domain Family' (ADAM). The predicted protein sequence reveals highest homology to the testase-subgroup, composed of ADAM 24, ADAM 25 and ADAM 26 and is therefore called testase 4. Reverse transcription-polymerase chain reaction showed a strong expression of testase 4 in the adult testis, but not in any other organ or embryonic stage tested. Careful characterization by in situ hybridization confirmed specific expression of testase 4 in maturing sperm cells of 6-week-old mice, whereas no specific hybridization pattern was detectable in testes of 2.5-week-old mice. These data indicate a correlation between testase 4 expression and spermatogenesis and/or fertilization.
Subject(s)
Disintegrins/genetics , Metalloendopeptidases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression , In Situ Hybridization , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Testis/metabolismABSTRACT
Regulation of Notch1 activity is critical for cell fate decisions and differentiation of skeletal myoblasts. We have employed the skeletal myoblast cell line C2C12 to study posttranslational regulation of Notch1 protein levels during myogenesis. Although the major degradation pathway of the activated intracellular Notch1 fragment appears to involve ubiquitination and degradation by the 26 S proteasome, we provide evidence for an alternative catalytic pathway where the endogenous, transmembrane form of Notch1 is targeted to the lysosomal compartment. Immunoprecipitation analysis revealed ubiquitin-dependent accumulation of transmembrane Notch1 protein after treatment with the lysosomal inhibitor chloroquine but not after treatment with various proteasome inhibitors. This finding was supported by the observation that the transmembrane form of Notch1 was tyrosine-phosphorylated and specifically coprecipitated with the ubiquitin ligase c-Cbl. Our data suggest a regulatory mechanism down-regulating Notch1 protein levels already at the cellular surface, possibly with consequences for Notch-dependent signal transduction during terminal differentiation processes.
