title: Cell surface-mediated activation of progelatinase A: demonstration of the involvement of the C-terminal domain of progelatinase A in cell surface binding and activation of progelatinase A by primary fibroblasts.
abstract: We report that the isolated C-terminal domain of progelatinase A is inhibitory to the activation of this proenzyme by primary skin fibroblast plasma membranes but is unable to inhibit organomercurial-induced self-cleavage and activation. Ligand binding studies demonstrate that fibroblasts stimulated with concanavalin A to activate progelatinase A have a significantly enhanced level of cell surface-associated progelatinase A. Tissue inhibitor of metalloproteinases-2 (TIMP-2), an effective inhibitor of membrane-mediated progelatinase A activation, is able to abolish the enhanced level of cell surface-associated progelatinase A that occurs following stimulation. TIMP-1, a poor inhibitor of membrane activation, is unable to inhibit the cell surface binding of progelatinase A. The enhancement in the binding of 125I-progelatinase A to fibroblasts following concanavalin A stimulation can be blocked by the inclusion of excess C-terminal gelatinase A but not by a truncated form of gelatinase A lacking the C-terminal domain. Scatchard analysis of the binding of 125I-progelatinase A to concanavalin A-stimulated fibroblasts has identified 950,000 gelatinase binding sites per cell with a Kd of 1.3 x 10(-8) M. Analysis of non-stimulated fibroblasts has identified 500,000 sites per cell with a Kd of 2.6 x 10(-8) M. We propose that membrane-mediated activation of progelatinase A involves binding of the proenzyme through its C-terminal domain to the cell surface and that TIMP-2 can inhibit activation by interaction with progelatinase A through the C-terminal domain, thus preventing binding of the proenzyme.
authors.affiliation: Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge, U.K.
authors.forename: R V
authors.initials: RV
authors.lastname: Ward
authors.forename: S J
authors.initials: SJ
authors.lastname: Atkinson
authors.forename: J J
authors.initials: JJ
authors.lastname: Reynolds
authors.forename: G
authors.initials: G
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D001345:Autoradiography; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology*; D018093:Gelatinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000494:pharmacology; D011869:Radioligand Assay; D019716:Tissue Inhibitor of Metalloproteinase-2
chemical_list: D004792:Enzyme Precursors; D011506:Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase