Collagenase-3 Binds to a Specific Receptor and Requires the Low Density Lipoprotein Receptor-related Protein for Internalization

Abstract

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptorrelated protein. Ligand blot analysis shows that 125 Icollagenase-3 binds specifically to two proteins (ϳ170 kDa and ϳ600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wildtype mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization. Collagenase-3 (MMP-13)1 is a member of the matrix metalloproteinase family of enzymes, which participates in extracellular matrix remodeling (1). Members of this family have a number of structural and functional features in common. In addition to sharing a similar domain structure, all are synthesized in inactive form, function at neutral pH, and require intrinsic zinc and calcium ions for their activity. Collagenase-3 is a highly regulated enzyme that cleaves native fibrillar collagens of types I, II, and III. The 57-kDa proenzyme is converted to its active 52-kDa form by the plasmin activation cascade, as well as by cathepsin B, stromelysin, and plasma kallikrein (2, 3). Collagenase-3 activity is inhibited by a family of tissue inhibitors of metalloproteinases (4, 5). A range of hormones and agents can also regulate expression of collagenase-3 (6). Parathyroid hormone is one of the hormones participating in this process. Osteoblastic cells respond to parathyroid hormone by increasing collagenase-3 synthesis (6 -8), plasminogen activator activity (9), and tissue inhibitors of metalloproteinases expression (8). In addition, experiments with UMR 106-01 rat osteosarcoma cells showed that over 80% of exogenous rat collagenase-3 was removed from the medium after 8 h of incubation (10). This rapid removal of rat collagenase-3 from the medium suggested the existence of a specific receptor that represents another level of regu...

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