THE TIMP2 MEMBRANE TYPE-1 METALLOPROTEINASE RECEPTOR REGULATES THE CONCENTRATION AND EFFICIENT ACTIVATION OF PROGELATINASE-A - A KINETIC-STUDY

We have used C-terminal domain mutants to further define the role of i nteractions of progelatinase A and membrane type 1 matrix metalloprote inase (MT1 MMP) in the binding of TIMP2 and in the cell-associated act ivation of progelatinase A. Soluble constructs of MT1 MMP were used to demonstrate that binding with TIMP2 occurs primarily through N-termin al domain interactions, leaving the C-terminal domain free for interac tions with progelatinase A. The rate of autolytic activation of progel atinase A initiated by MT1 MMP cleavage could be potentiated by concen tration of the proenzyme by binding to heparin, Residues 568-631 of th e progelatinase A C-terminal domain are important in formation of the heparin binding site, since replacement of this region with the corres ponding stromelysin-1 sequence abolished binding to heparin and the po tentiation of activation. The same region of gelatinase A was required for binding of latent and active enzyme to TIMP2, but residues 418-47 4 were not important. A similar pattern was seen using cell membrane-a ssociated MT1 MMP; residues 568-631 were required for binding and acti vation of progelatinase A, whereas residues 418-474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of en dogenous TIMP2, resulted in potentiation of progelatinase A activation , This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and ef ficient generation of functionally active gelatinase A.