Identification of SAP97 as an intracellular binding partner of TACE Available to Purchase

Tumor necrosis factor alpha converting enzyme (TACE) is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumor necrosis factor α. Using the yeast two-hybrid system, we identified the scaffolding protein synapse associated protein 97(SAP97) as a binding partner of the cytoplasmic domain of TACE. By deletions and site-directed mutagenesis, we demonstrated that this interaction involved the PDZ3 domain of SAP97 and the extreme C-terminal amino-acid sequence of TACE. This interaction as well as the identification of the specific domains involved was confirmed in vitro by affinity purification and in mammalian cells by co-immunoprecipitation and alteration of localization analyzed by immunofluorescence microscopy. In addition, confocal microscopy showed that endogenous TACE and SAP97 colocalized in some intracellular areas of COS-7 cells and CACO-2 cells. Furthermore, overexpression of SAP97, unlike that of a mutant form of SAP97 deleted for its PDZ3 domain, altered the ability of TACE to release its substrates. Altogether, these results demonstrate an interaction between TACE and SAP97, which may have a functional implication for the regulation of TACE shedding activity.