The C-terminal domain of tissue inhibitor of metalloproteinases-2 is required for cell binding but not for antimetalloproteinase activity.
We have generated a C-terminally-truncated form of recombinant tissue inhibitor of metalloproteinases-2 (designated rTIMP-2 delta) in which the region of the inhibitor extending from residue 128 to 194 and including 3 of the 6 disulfide bonds is deleted. rTIMP-2 and rTIMP-2 delta had similar inhibitory activities toward interstitial collagenase and inhibited the activation of the precursor form of matrix metalloproteinase-2 (proMMP-2). rTIMP-2 also bound with high affinity (Kd 0.99 nM) to HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate. However deletion of the C-terminal domain of TIMP-2 significantly lowered the cell surface binding affinity, with competition experiments indicating a 2 order of magnitude difference between rTIMP-2 and rTIMP-2 delta in the concentrations needed to displace 125I-labeled rTIMP-2 binding. These data indicate that the C-terminal domain of TIMP-2 is not required for the antimetalloproteinase activity but plays a major role in the high affinity cell surface binding of the inhibitor.
Ko
Y C
YC
Childrens Hospital Los Angeles and the Department of Pediatrics, University of Southern California, Los Angeles 90027, USA.
Langley
K E
KE
Mendiaz
E A
EA
Parker
V P
VP
Taylor
S M
SM
DeClerck
Y A
YA
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2
D018076:DNA, Complementary; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D008666:Metalloendopeptidases