The C-Terminal Domain of Tissue Inhibitor of Metalloproteinases-2 Is Required for Cell Binding but Not for Antimetalloproteinase Activity

Self Cite

131

109

The matrix metalloproteinases (MMPs), 1 a multidomain family of zinc-dependent endopeptidases, degrade all structural components of the extracellular matrix (ECM) and many bioactive molecules, thereby playing essential roles in many physiological and pathological processes (1-4). Based on structural organization and subcellular localization, the MMP family is divided into secreted and membrane-anchored enzymes (1, 5). The membrane type-MMPs (MT-MMPs) comprise six members of plasma membrane-tethered MMPs, which include four type I transmembrane enzymes: MT1-, MT2-, MT3-, and MT5-MMP, and two glycosylphosphatidylinositol-anchored enzymes: MT4-and MT6-MMP (4, 6 -8). Although there is some tissue-specific expression of MT-MMPs, there is also significant overlap both in expression and in function raising the question of how cells command the repertoire of MT-MMPs at their disposition during proteolytic events. A major mechanism for controlling MT-MMP activity in the pericellular space is mediated by the action of tissue inhibitors of metalloproteinases (TIMPs). However, as opposed to soluble MMPs, which are almost equally inhibited by all TIMPs (9, 10), the MT-MMPs are highly selective when it comes to TIMP interactions. For example, the type I transmembrane MT-MMPs are poorly inhibited by TIMP-1 but are relatively well inhibited by TIMP-2, TIMP-3, and TIMP-4. In contrast, the glycosylphosphatidylinositol-anchored MT-MMPs are inhibited by both TIMP-1 and TIMP-2 (11). The differential sensitivity to TIMP inhibition among members of the MT-MMP family may facilitate the control of MT-MMP activity in the pericellular space.In addition to being potent ECM-degrading enzymes, the type I transmembrane MT-MMPs can also initiate a cascade of zymogen activation on the cell surface. MT-MMPs activate the zymogenic form of MMP-2 (pro-MMP-2 or pro-gelatinase A) (6, 8), which in turn can activate pro-MMP-9 (pro-gelatinase B) (12). MT1-MMP also promotes the activation of pro-collagenase 3 (MMP-13) (13), a potent collagenolytic protease. Because the gelatinases are efficient gelatinolytic enzymes, the MT-MMP/ MMP-13/gelatinase axis represents a well adapted proteolytic system designed to promote coordinated and complete collagen degradation in the pericellular space. Although the type I

Section: Methodsmentioning

confidence: 99%

Differential Inhibition of Membrane Type 3 (MT3)-Matrix Metalloproteinase (MMP) and MT1-MMP by Tissue Inhibitor of Metalloproteinase (TIMP)-2 and TIMP-3 Regulates Pro-MMP-2 Activation

Zhao

Bernardo

Osenkowski

et al. 2004

Self Cite

131

109

“…Cell Surface Binding of TIMP-2-Recombinant TIMP-2 was iodinated to a specific activity of 5 ϫ 10 10 dpm/mg as recently described (26). Binding of 125 I-labeled TIMP-2 to COS-1 cells propagated in 24-well dishes was performed in duplicate (10% variation between duplicates).…”

Section: Methodsmentioning

confidence: 99%

“…The residual radioactivity associated with cells in the nonspecific binding experiment (50-fold excess TIMP-2) was subtracted from the total bound fraction (no unlabeled TIMP-2) to give specific binding. Scatchard plot analysis of binding data used best fit curves (26).…”

Section: Methodsmentioning

confidence: 99%

The Propeptide Domain of Membrane Type 1 Matrix Metalloproteinase Is Required for Binding of Tissue Inhibitor of Metalloproteinases and for Activation of Pro-gelatinase A

Cao¹,

Drews

Lee

et al. 1998

Matrix metalloproteinases (MMPs, 1 matrixins) are a large family of neutral zinc endopeptidases, which display homologous structural features consisting of a N-terminal propeptide domain, a zinc-coordinated catalytic domain, and a C-terminal hemopexin-and vitronectin-like domain (1-3). During the process of activation of secreted latent MMPs in the pericellular and extracellular environment, conformational perturbation or limited proteolysis within the N-terminal propeptide domain causes a change in the molecule that disrupts the unpaired Cys-Zn 2ϩ interaction and frees the Zn 2ϩ to participate in proteolytic cleavage. The modified MMP then attacks the peptide sequence downstream of the PRCGVPD sequence in an autolytic manner and cleaves the propeptide, thus producing a lower molecular weight activated enzyme (popularly described as a cysteine switch or velcro mechanism (4)). Activation of MMPs is inhibited by a family of proteins collectively known as tissue inhibitors of metalloproteinases (TIMPs).Membrane-type matrix metalloproteinases (MT-MMPs) represent a newly described group of matrixins (5) that are widely but selectively distributed in tissues. As the name implies, MT-MMPs are localized to the plasma membranes of cells by a stretch of hydrophobic amino acids (transmembrane domain; Ref. 6) followed by a short cytoplasmic sequence. MT-MMPs have been the subject of great interest because of their role in activating a secreted MMP, pro-gelatinase A, at the cell surface (5). Expression of MT1-MMP in embryonic tissue (7) and in malignant tumors has been correlated with the degree of activated gelatinase A in these tissues.The mechanism of pericellular activation of pro-gelatinase A appears to involve the formation of a unique bimolecular complex between TIMP-2 and MT1-MMP (5, 8, 9). Our recent study (10) documented that the N-terminal domain of TIMP-2 binds avidly to the catalytic domain of MT1-MMP, thereby producing a complex on the cell surface. The C-terminal domain of progelatinase A then binds to the available C-terminal domain of TIMP-2 (stabilization site), forming a trimolecular complex (9). According to this theory, a second MT1-MMP molecule (not complexed to TIMP-2) then attacks a single bond in complexed pro-gelatinase A, which is followed by an autolytic cleavage (11,12), thus resulting in pro-gelatinase A activation. Excess TIMP-2 interferes with this activation mechanism by binding and inhibiting all available MT1-MMP molecules.A special feature of MT-MMPs, as well as stromelysin-3 (13), is a 10-residue insert immediately preceding the final processing site between the propeptide and catalytic domain, which harbors a paired basic amino acid cleaving enzyme recognition motif (RXKR) (5, 6). Unlike stromelysin-3, which is a secreted MMP, latent membrane-bound MT1-MMP does not appear to be attacked at the RRKR sequence by the propeptide convertasedependent pathway (furin) in MT1-MMP transformed COS cells (14), but is attacked and converted to the activated form when secreted as a C-terminal truncate...

“…Formation of this complex is required for cell surface activation of MMP-2 by MT1-MMP (8 -12). It has been reported that the binding of the N terminus of TIMP-2, T2N, to the cell surface of HT1080 fibrosarcoma cells was an order of magnitude weaker than that of intact TIMP-2 (7,12). The failure of T2N to completely compete for TIMP-2 binding suggested to us that the C-terminal portion of TIMP-2, T2C, might also be binding to the cell surface.…”

mentioning

confidence: 77%

“…Studies from our laboratory (1) and others (2) have demonstrated that TIMP-2 can inhibit angiogenesis independent of its ability to inhibit MMP activity. Early studies of the growth modulating effects of TIMP-2 sought to determine whether TIMP-2 could bind to the cell surface of various cell lines (3)(4)(5)(6)(7). These studies suggested the existence of at least two potential cell surface receptors: one of higher affinity and at least one of relatively lower affinity.…”

mentioning

confidence: 99%

The Anti-angiogenic Peptide, Loop 6, Binds Insulin-like Growth Factor-1 Receptor

Fernández

Roy

Lee

et al. 2010

Tissue inhibitors of metalloproteinases (TIMPs), the endogenous inhibitors of matrix metalloproteinases, have been shown to possess biological functions that are independent of their ability to inhibit matrix metalloproteinases. We have previously shown that the C-terminal domain of TIMP-2 and, in particular, Loop 6 inhibit capillary endothelial cell proliferation and angiogenesis both in vitro and in vivo. To elucidate the mechanism by which Loop 6 inhibits angiogenesis, we sought to determine whether its biological effects were the result of a known TIMP-2 protein-protein interaction or of a receptor-mediated event. In this study, we identify insulin-like growth factor-1 receptor as a binding partner of Loop 6/TIMP-2 and characterize this interaction on the endothelial cell surface and the consequences of this interaction on downstream receptor signaling. TIMP-2,3 an endogenous regulator of matrix metalloproteinase (MMP) activity, is a multifunctional protein that has been shown to regulate angiogenesis, tumor progression, and metastasis. Studies from our laboratory (1) and others (2) have demonstrated that TIMP-2 can inhibit angiogenesis independent of its ability to inhibit MMP activity. Early studies of the growth modulating effects of TIMP-2 sought to determine whether TIMP-2 could bind to the cell surface of various cell lines (3-7). These studies suggested the existence of at least two potential cell surface receptors: one of higher affinity and at least one of relatively lower affinity. The higher affinity interaction has been shown to involve a trimolecular complex of TIMP-2 with MT1-MMP and pro-MMP-2. Formation of this complex is required for cell surface activation of MMP-2 by MT1-MMP (8 -12). It has been reported that the binding of the N terminus of TIMP-2, T2N, to the cell surface of HT1080 fibrosarcoma cells was an order of magnitude weaker than that of intact TIMP-2 (7, 12). The failure of T2N to completely compete for TIMP-2 binding suggested to us that the C-terminal portion of TIMP-2, T2C, might also be binding to the cell surface.In a structure-function analysis of TIMP-2, we have demonstrated that T2C inhibits angiogenesis in a manner independent of any MMP inhibitory activity. We further demonstrated that Loop 6, a smaller domain of T2C, retained the anti-angiogenic activity (1). We therefore hypothesized that T2C and, more specifically, Loop 6 might bind the endothelial cell surface and that this interaction might mediate the antiangiogenic activity of Loop 6. Until recently, none of the low affinity receptors of TIMP-2 described previously (3-7) had been identified. Seo et al. (2) have now shown that at least one of these interactions involves the integrin ␣3␤1. However, in the course of our studies, we found no evidence of the binding of Loop 6 to ␣3␤1. These results suggested that the anti-angiogenic effects of Loop 6 were mediated by a different mechanism and might be the result of a novel interaction of TIMP-2 at the EC surface.Here, we identify IGF-IR as a novel binding partner of L...