The kringle stabilizes urokinase binding to the urokinase receptor

Abstract

The structural basis of the interaction between single-chain urokinase-type plasminogen activator (scuPA) and its receptor (uPAR) is incompletely defined. Several observations indicated the kringle facilitates the binding of uPA to uPAR. A scuPA variant lacking the kringle (⌬K-scuPA) bound to soluble uPAR (suPAR) with the similar "on-rate" but with a faster "off-rate" than wild-type (WT)-scuPA. Binding of ⌬K-scuPA, but not WT-scuPA, to suPAR was comparably inhibited by its growth factor domain (GFD) and amino-terminal fragment (ATF).ATF and WT-scuPA, but not GFD, scuPA lacking the GFD (⌬GFD-scuPA), or ⌬K-scuPA reconstituted the isolated domains of uPAR. ATF completely inhibited the enzymatic activity of WT-scuPAsuPAR unlike comparable concentrations of GFD. Variants containing mutations that alter the charge, length, or flexibility of linker sequence (residues 43-49) between the GFD and the kringle displayed a lower affinity for uPAR, were unable to reconstitute uPAR domains, and their binding to uPAR was inhibited by GFD in the same manner as ⌬K-scuPA. IntroductionUrokinase-type plasminogen activator (uPA) has been implicated in diverse physiologic and pathophysiologic processes that involve cell adhesion, fibrinolysis, and signal transduction. 1-3 Several of these processes are mediated by the catalytic activity of the molecule, 4 while others involve the participation of the uPA receptor (uPAR) 1,[5][6][7][8] or other cell surface molecules. 8,[9][10][11] uPA is expressed as a single-chain molecule (scuPA) composed of an N-terminal fragment (ATF; amino acids 1-135) and a protease domain (amino acids 136-411), also known as low molecular weight uPA (LMW-uPA). The amino-terminal fragment (ATF) is itself composed of 2 independent domains, the amino-terminal growth factor-like domain (GFD; amino acids 1-43), which is known to bind to the uPA receptor, and a single kringle (K; amino acids 47-135), the function of which is uncertain. [12][13][14][15][16][17] scuPA expresses plasminogen activator activity when converted to a 2-chain molecule (tcuPA) by plasmin 18 or as a single-chain molecule when bound to its receptor. [19][20][21][22] The mature urokinase receptor (uPAR) is a 283-amino acid glycolipid-anchored protein composed of 3 partial repeat domains. 5,6,23 Cooperation among the 3 domains is required to optimize scuPA binding and activation. [24][25][26] Binding of scuPA is inhibited by peptides that cross-link residues Arg53 and Leu66 in domain I and His251 in domain III of uPAR, respectively, 27,28 or by mutations in a region of domain II, which has been implicated in interdomain cooperativity. 26 There is compelling evidence to indicate that scuPA binds to uPAR through its GFD. [28][29][30][31][32][33][34] However, the potential involvement of other sites in uPA in receptor binding has not been studied in detail.Evidence favoring the existence of such auxiliary sites comes from studies in which uPA-induced, uPAR-mediated adhesion to vitronectin (Vn) and other integrin ligands has been examined using purif...

Species-specific urokinase receptor ligands reduce glioma growth and increase survival primarily by an antiangiogenesis mechanism

“…48,49 More recently, it has been suggested that the uPA kringle stabilizes uPA binding to its receptor mediated by the amino-terminal growthfactor-like domain. 50 Our data demonstrate that PEG 1-48 inhibits, like ATF, the binding of uPA to its receptor, although it is missing the kringle domain. The use of recombinant uPA1-48 rather than proteolytically derived uPA4-43 may account for this difference.…”

Mapping the Interaction between High Molecular Mass Kininogen and the Urokinase Plasminogen Activator Receptor

“…Preparation of Wild Type and Mutant suPAR-Soluble urokinase plasminogen activator receptor (suPAR), isolated domains of suPAR (1-3), a fragment containing recombinant soluble suPAR domains 1 and 2 and 3 (S-D1, S-D2D3) and scuPA were prepared and expressed using the Drosophila Expression System (Invitrogen, Carlsbad, CA) according to manufacturer's instructions, as described previously (17,18). Isolated domains 1,2, and 3 of suPAR were prepared from wild type protein by sequential digestion with chymotrypsin and pepsin as previously reported (19).…”

Structural Basis of Interaction between Urokinase-type Plasminogen Activator and its Receptor

“…To further reduce receptor heterogeneity resulting from differences in N-linked oligosaccharide structures, four of the five N-glycosylation sites of the suPAR WT were mutated (N162Q, N172Q, N200Q, and N233Q), and the resulting mutant was denoted suPAR 2345 (reported earlier in ref. 12). Both suPAR constructs were expressed in Drosophila Schneider's S2 cells and purified to homogeneity by combination of immunoaffinity chromatography and size-exclusion chromatography (data not shown).…”