Spatial Regulation and Activity Modulation of Plasmin by High Affinity Binding to the G domain of the α3 Subunit of Laminin-5

Abstract

Cells in complex tissues contact extracellular matrix that interacts with integrin receptors to influence gene expression, proliferation, apoptosis, adhesion, and motility. During development, tissue remodeling, and tumorigenesis, matrix components are modified by enzymatic digestion with subsequent effects on integrin binding and signaling. We are interested in understanding the mechanisms by which broad spectrum proteinases such as plasmin are targeted to their extracellular matrix protein substrates. We have utilized plasmin-mediated cleavage of the epithelial basement membrane glycoprotein laminin-5 as a model to evaluate molecular events that direct plasmin activity to specific structural domains. We report that plasminogen and tissue plasminogen activator (tPA) exhibit high affinity, specific binding to the G 1 subdomain of the N terminus of the laminin-5 ␣ 3 subunit, with equilibrium dissociation constants of 50 nM for plasminogen and 80 nM for tPA. No high affinity binding to the G 2 , G 3 , and G 4 subdomains was observed. As a result of binding to the G 1 subdomain, the catalytic efficiency of tPA-catalyzed plasminogen activation is enhanced 32-fold, leading to increased matrix-associated plasmin that is positioned favorably for cleavage within the G 4 subdomain as we have reported previously (Goldfinger, L. E., Stack, M. S., and Jones, J. C. R. (1998) J. Cell Biol. 141, 255-265). Thus, physical constraints dictated by interaction of proteinase and matrix macromolecule control not only enzymatic activity but may regulate substrate targeting of proteinases.Cells in epithelial tissues are in contact with an array of extracellular matrix (ECM) 1 molecules. Through interaction with cell surface receptors, ECM proteins have a profound influence on gene expression as well as proliferation, adhesion, and motility (see for example, Refs. 1-4). During development, tissue remodeling, and tumorigenesis, protein components of the ECM are often modified by enzymatic digestion (4 -9). Proteolyzed ECM components may bind different cell surface receptors than their intact counterparts and thereby trigger alternative signaling events (10). We have utilized plasminmediated cleavage of the epithelial basement membrane glycoprotein laminin-5 as a model to evaluate molecular events that direct plasmin activity to specific structural domains.Laminin-5 secreted by epithelial cells is a heterotrimeric protein with an ␣ 3 ␤ 3 ␥ 2 subunit composition and promotes epithelial cell migration (11,12). However, we have previously demonstrated that proteolytic processing of the 190-kDa ␣ 3 subunit within the C-terminal globular (G) domain renders laminin-5 competent to induce formation of hemidesmosomes, cell matrix anchorage structures formed in part via binding of laminin-5 to the ␣ 6 ␤ 4 integrin ( Fig. 1) (13, 14). As a consequence of hemidesmosome formation, cellular motility is significantly reduced. In cultured cells, this functional and structural modification of the laminin-5 ␣ 3 subunit occurs as a result of limited plasmin...

The tissue plasminogen activator-plasmin system participates in the rewarding effect of morphine by regulating dopamine release

“…However, it is known that the tPAplasmin system degrades several extracellular matrix proteins (42), including laminin (43). Laminin in the synaptic cleft localizes calcium channels to the sites of active zones (44) and induces a small but significant increase in calcium levels in ciliary ganglion neurons when applied in soluble form to the culture medium (45).…”

Involvement of Tissue Plasminogen Activator-Plasmin System in Depolarization-Evoked Dopamine Release in the Nucleus Accumbens of Mice

“…Second, it was reported that tPA and plasmin bind to laminin in vitro (Goldfinger et al, 2000), and plasmin degrades several extracellular matrix components such as laminin (Nakagami et al, 2000). Laminin in the synaptic cleft causes calcium channels to localize to the active zones (Sunderland et al, 2000) and induces a small but significant increase in the level of calcium in ciliary ganglion neurons when added in soluble form to the culture medium (Bixby et al, 1994).…”

Effects of urokinase-type plasminogen activator in the acquisition, expression and reinstatement of cocaine-induced conditioned-place preference

“…Urokinase plaminogen activator is an extracellular serine protease, which is expressed in most brain regions in the CNS [1,[43][44][45] and exerts its main enzymatic actions by converting inactive precursor plasminogen to the active protease plasmin [46][47][48] and by degrading certain components in extracellular matrix, such as laminin [47,[49][50][51][52][53]. The role of uPA for extracellular matrix degradation and dendritic spine dynamics has been well established [54,55].…”