Spatial regulation and activity modulation of plasmin by high affinity binding to the G domain of the alpha(3) subunit of laminin-5

Cells in complex tissues contact extracellular matrix that interacts with i ntegrin receptors to influence gene expression, proliferation, apoptosis, a dhesion, and motility, During development, tissue remodeling, and tumorigen esis, matrix components are modified by enzymatic digestion with subsequent effects on integrin binding and signaling. We are interested in understand ing 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 acti vity 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 alpha (3) subunit, with e quilibrium dissociation constants of 50 nM for plasminogen and 80 nM for tP A, No high affinity binding to the G(2), G(3), and G(4) subdomains was obse rved. As a result of binding to the G(1) subdomain, the catalytic efficienc y of tPA-catalyzed plasminogen activation is enhanced 32-fold, leading to i ncreased matrix-associated plasmin that is positioned favorably for cleavag e 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), T hus, physical constraints dictated by interaction of proteinase and matrix macromolecule control not only enzymatic activity but may regulate substrat e targeting of proteinases.