Characterization of the Functional Epitope on the Urokinase Receptor

Abstract

The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655-1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface-exposed side chains in this interaction. Mutations causing ⌬⌬G > 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross-linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR⅐uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction.The urokinase-type plasminogen activator receptor (uPAR) 2 is a glycosylphosphatidylinositol-anchored membrane glycoprotein (1) that has a primary role in focalizing plasminogen activation at the cell surface through its specific high affinity interaction with the urokinase-type plasminogen activator (uPA). Besides facilitating the generation of plasmin activity in the vicinity of uPAR-expressing cells, which is directly or indirectly involved in remodeling of the extracellular matrix (2, 3), the uPAR/pro-uPA interaction also assists in regulating other aspects of cell adhesion and migration. Among these molecular processes is the direct interaction with matrix-deposited vitronectin (4); the modulation of integrin function, in particular ␣ M ␤ 2 , ␣ 5 ␤ 1 , and ␣ 3 ␤ 1 (5-8); and the activation of the chemotactic FPRL1 receptor (9). As an increased expression level of uPAR is often found in the invasive areas of various human cancers and correlates with poor prognosis (10), the uPAR/uPA interaction and uPA catalytic activity are considered relevant molecular targets for drug development (11-13). Intervention strategies developed for targeting the uPAR/uPA interaction with a view to cancer therapy include recombinant fusion proteins containing the receptor-binding module of uPA (14,...

Conformational Regulation of Urokinase Receptor Function

“…In contrast, Tyr 57 is located at the bottom of the hydrophobic uPA binding cavity and is completely shielded from solvent in the corresponding uPAR-ATF complex (16,40). The affinity of uPAR Y57A for uPA is accordingly decreased by ϳ7-fold compared with both uPAR wt and uPAR W32A (34,39), whereas vitronectin binding is unaffected.…”

“…2). This may at first sight appear counterintuitive, bearing in mind that the K D for the interaction between pro-uPA and uPAR Y57A is 3.5 nM compared with only 0.5 nM for uPAR W32A (34). This paradox is, however, easily reconciled by taking into account that the uPA-uPAR Y57A complexes exhibit a vitronectin binding similar to that of uPAR wt complexes, whereas uPAuPAR W32A complexes display an impaired vitronectin binding due to the mutation of a hotspot residue (Trp 32 ) for this interaction (18).…”

“…Purified Protein Preparations-Soluble forms of recombinant human uPAR (residues 1-283) were expressed by stably transfected Drosophila melanogaster S2 cells (33), and a library of Ͼ300 purified uPAR mutants carrying single-site substitutions was prepared and characterized as described (34). Recombinant human pro-uPA S356A (residues 1-411) without catalytic activity due to the active-site mutation, pro-uPA ⌬GFD (residues 45-411), and the N-terminal fragment (ATF) of uPA (residues 1-143) were all expressed by D. melanogaster S2 cells and affinity-purified using the immobilized anti-uPA monoclonal antibody, clone-6 (34).…”

“…Epitope Mapping of Anti-uPAR mAbs-To identify the functional epitopes for various uPAR-specific mAbs, we analyzed their binding kinetics toward a large number of purified uPAR mutants carrying defined single-site alanine substitutions (34). In some cases we analyzed the interaction at 5°C to minimize the risk of irreversibly denaturing the less stable mAbs during the repeat cycles of regeneration.…”

“…S1. The kinetic rate constants, k on and k off , were subsequently derived from these real-time interaction analyses by fitting the association and dissociation phases to a bimolecular interaction model using the BIAevaluation 4.1 software (Biacore) as described in detail previously (34). For low affinity interactions, K D was also estimated by fitting binding isotherms at equilibrium.…”