Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors.Interactions between Dll1 and Notch in trans activate the Notch pathway, whereas Dll1 binding to Notch in cis inhibits Notch signaling. Dll1 undergoes proteolytic processing in its extracellular domain by ADAM10. In this work we demonstrate that Dll1 represents a substrate for several other members of the ADAM family. In co-transfected cells, Dll1 is constitutively cleaved by ADAM12, and the N-terminal fragment of Dll1 is released to medium. ADAM12-mediated cleavage of Dll1 is cell density-dependent, takes place in cis orientation, and does not require the presence of the cytoplasmic domain of ADAM12. Full-length Dll1, but not its N-or C-terminal proteolytic fragment, co-immunoprecipitates with ADAM12. By using a Notch reporter construct, we show that Dll1 processing by ADAM12 increases Notch signaling in a cell-autonomous manner. Furthermore, ADAM9 and ADAM17 have the ability to process Dll1. In contrast, ADAM15 does not cleave Dll1, although the two proteins still co-immunoprecipitate with each other. Asn-353 present in the catalytic motif of ADAM12 and other Dll1-processing ADAMs, but absent in ADAM15, is necessary for Dll1 cleavage. Dll1 cleavage is reduced in ADAM9/12/15 ؊/؊ mouse embryonic fibroblasts (MEFs), suggesting that the endogenous ADAM9 and/or ADAM12 present in wild type MEFs contribute to Dll1 processing. Finally, the endogenous Dll1 present in primary mouse myoblasts undergoes cleavage in confluent, differentiating myoblast cultures, and this cleavage is decreased by ADAM12 small interfering RNAs. Our findings expand the role of ADAM proteins in the regulation of Notch signaling.
ADAM12 has recently emerged as a Candidate Cancer Gene in a comprehensive genetic analysis of human breast cancers. Three somatic mutations in ADAM12 were observed at significant frequencies in breast cancers: D301H, G479E and L792F. The first 2 of these mutations involve highly conserved residues in ADAM12, and our computational sequence analysis confirms that they may be cancer-related. We show that the corresponding mutations in mouse ADAM12 inhibit the proteolytic processing and activation of ADAM12 in NIH3T3, COS-7, CHO-K1 cells and in MCF-7 breast cancer cells. The D/H and G/E ADAM12 mutants exert a dominant-negative effect on the processing of the wild-type ADAM12. Immunofluorescence analysis and cell surface biotinylation experiments demonstrate that the D/H and G/E mutants are retained inside the cell and are not transported to the cell surface. Consequently, the D/H and G/E mutants, unlike the wild-type ADAM12, are not capable of shedding Delta-like l, a ligand for Notch receptor, at the cell surface, or of stimulating cell migration. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface. ' 2008 Wiley-Liss, Inc.Key words: proteolytic processing; cell surface; endoplasmic reticulum; intracellular trafficking Metalloprotease Disintegrin 12 (ADAM12) is highly expressed in cancer of the breast, 1,2 prostate, 3 liver, 4 stomach, 5 colon, 3 bladder 6 and in glioblastoma. 7 ADAM12 appears to be selectively upregulated in cancer cells, and its expression is nearly undetectable in normal breast, prostate, colon and liver epithelium. [1][2][3][4][5][6] In mouse models of breast, prostate and colon cancer, ADAM12 is found in a subpopulation of stromal cells adjacent to epithelial tumor cells. 2,3 Although this localization suggests that ADAM12 may play a role in stromal-tumor crosstalk, the molecular mechanism of ADAM12 action during cancer progression is not known.Studies utilizing mouse models of breast and prostate cancers suggested that ADAM12 promotes tumor progression. Forced expression of ADAM12 in breast tumors of MMTV-PyMT mice (carrying the polyomavirus middle T oncogene under mouse mammary tumor virus promoter and developing multifocal mammary adenocarcinomas) accelerated tumor progression. 2 Knocking out ADAM12 expression in prostate tumors of W10 mice (expressing SV40 large T-antigen under control of probasin promoter and developing prostate carcinomas) resulted in smaller and better differentiated tumors. 3 Recently, ADAM12 has been identified as one of the Candidate Cancer Genes in a comprehensive mutational analysis of human breast cancers. 8 Three mutations were found to be associated with breast cancer: D301H in the metalloprotease domain, G479E in the disintegrin domain and L792F in the cytoplasmic tail (Fig. 1a). The metalloprotease domain of ADAM12 is catalytically active and cleaves several protein substrates, including insulin growth factor-binding protein (IGFBP...
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