Identification of MMP Substrates in the Mammary Gland
Annual summary rept. 10 Jun 2002-9 Jun 2005
CALIFORNIA UNIV SAN FRANCISCO
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Yeast two-hybrid system was used to identify MMP interacting proteins as potential novel substrates. The hemopexin domain of MMP-3 interacted with the C-terminus of Wnt5b in a two-hybrid assay. A deletional analysis of this clone revealed a minimal MMP-3 binding domain of 55 amino acids. A hingeblade swapping approach with MMP-10 further revealed that the hinge region and 3 of the 4 MMP-3 hemopexin domain blades are required for binding of Wnt5b. The C-terminus of Wnt5a is 84 identical to Wnt5b and is the only other Wnt expressed in the mammary gland that interacts with MMP-3 in the two-hybrid assay. Recombinant MMP-3 catalytic domain can cleave both soluble Wnt5b as well as cell bound Wnt5a and b immunoprecipated from RIPA cell lysates. Cleavage seems to occur within the minimal MMP-3 interacting domain as judged by mobility shift on SDS-PAGE and can be blocked by the MMP inhibitors GM-6001 and EDTA. These observations suggest that both Wnt5a and b are MMP-3 substrates, but only Wnt5b is co-expressed with MMP-3 in the ductal microenvironment during mammary gland development. A novel lentiviral based method is being developed to analyze the role of Wnt5a and b and their MMP-3 cleavage products during mammary gland development andor carcinogenesis.
- Anatomy and Physiology
- Medicine and Medical Research