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Pubmed ID :19207106
Publication Date : //

Characterization of the catalytic activity of the membrane-anchored metalloproteinase ADAM15 in cell-based assays.


ADAM15 (a disintegrin and metalloproteinase 15) is a membrane-anchored metalloproteinase, which is overexpressed in several human cancers and has been implicated in pathological neovascularization and prostate cancer metastasis. Yet, little is known about the catalytic properties of ADAM15. Here, we purified soluble recombinant ADAM15 to test for its ability to cleave a library of peptide substrates. However, we found no processing of any of the peptide substrates tested here, and therefore turned to cell-based assays to characterize the catalytic properties of ADAM15. Overexpression of full-length membrane-anchored ADAM15 or the catalytically inactive ADAM15E-->A together with various membrane proteins resulted in increased release of the extracellular domain of the fibroblast growth factor receptor 2iiib (FGFR2iiib) by ADAM15, but not ADAM15E-->A. This provided a robust assay for a characterization of the catalytic properties of ADAM15 in intact cells. We found that increased expression of ADAM15 resulted in increased FGFR2iiib shedding, but that ADAM15 was not stimulated by phorbol esters or calcium ionophores, two commonly used activators of ectodomain shedding. Moreover, ADAM15-dependent processing of FGFR2iiib was inhibited by the hydroxamate-based metalloproteinase inhibitors marimastat, TAPI-2 and GM6001, and by 50 nM TIMP-3 (tissue inhibitor of metalloproteinases 3), but not by 100 nM TIMP-1, and only weakly by 100 nM TIMP-2. These results define key catalytic properties of ADAM15 in cells and its response to stimulators and inhibitors of ectodomain shedding. A cell-based assay for the catalytic activity of ADAM15 could aid in identifying compounds, which could be used to block the function of ADAM15 in pathological neovascularization and cancer.

Authors : Maretzky Thorsten , Yang Guangli , Ouerfelli Ouathek , Overall Christopher M , Worpenberg Susanne , Hassiepen Ulrich , Eder Joerg , Blobel Carl P ,

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