Abstract
The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK.
Highlights
The structural basis of RECK function is currently under extensive investigation
RECK Physically Interacts with membrane type 1 matrix metalloproteinase (MT1-matrix metalloproteinases (MMPs)) and Competitively Inhibits Its Proteolytic Activity—To understand the mechanism underlying the suppression of MT1-MMP activity by RECK, we first attempted to determine whether RECK physically interacts with MT1-MMP
We found that the introduction of RECK into HT1080 cells significantly attenuated the autocatalytic degradation of MT1-MMP (Fig. 1A)
Summary
Roglobulin, which is a major plasma inhibitor of metalloproteases (8). As compared with soluble MMP inhibitors represented by tissue inhibitor of metalloproteinases (TIMPs) (8), the most distinguishing feature of RECK is its ability to covalently anchor to the membrane surface via a post-translational modification (glycosylphosphatidylinositol (GPI) anchor) that is conserved among species (1). Recent studies indicate that MT1MMP internalization is controlled by a clathrin- or caveolaedependent endocytic pathway or by a combination of these two pathways (12–17) These mechanisms contribute to the selective internalization of MT1-MMP from a specific compartment of the cell membrane and its sorting to endosomes for subsequent degradation or recycling (9, 10). This machinery appeared to be implicated in the control of proMMP-2 maturation and cell motility (14, 15). 1 integrin is co-localized with MT1-MMP on the surface of human endothelial cells This interaction appeared to modulate the mechanism of MT1MMP internalization when the cells were exposed to 1 integrindependent extracellular matrices (18).
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