Mimicry of the Regulatory Role of Urokinase in Lamellipodia Formation by Introduction of a Non-native Interdomain Disulfide Bond in Its Receptor

Gårdsvoll, Henrik; Kjærgaard, Magnus; Jacobsen, B.; Kriegbaum, Mette C.; Huang, Mingdong; Ploug, Michael

doi:10.1074/jbc.m111.300020

Cited by 29 publications

(70 citation statements)

References 46 publications

(85 reference statements)

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“…Importantly, we also collected SAXS data for the disulfide-constrained receptor mutant (uPAR H47C/N259C ), where domains I and III are linked by a disulfide bridge, which maintains uPAR in a conformation mimicking the uPA-bound state both functionally (29) and structurally (30). The overall SAXS parameters for all samples were in agreement with a monomeric protein of the expected size, with the exception of uPAR wt , where the apparent mass is slightly larger than expected (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…The role of uPAR in cell surface-associated proteolysis and adhesion is increasingly well described in molecular terms due to extensive biochemical studies (19,29,45,46) and several published crystal structures of ligand-bound states of the receptor (23)(24)(25)(26)(27)30). Important challenges, nevertheless, still remain to be solved.…”

Section: Discussionmentioning

confidence: 99%

“…Chemicals and Protein Preparations-Recombinant human uPAR wt and uPAR H47C/N259C were expressed as secreted proteins (residues 1-283) by Drosophila melanogaster S2 cells and affinity-purified as described (29). Recombinant uPAR DI (residues 1-92) and DIIDIII (residues 88 -283) were likewise expressed in S2 cells and affinity-purified using anti-uPAR mAbs R21 and R2, respectively (19).…”

Section: Methodsmentioning

confidence: 99%

“…We consider that the overall architectures of these two ligand binding sites on uPAR open the possibility that prior uPA binding may prime a subsequent SMB binding event by altering its composite interdomain binding site. Circumstantial cell biological (18,19,28) and biochemical (17) evidence exists to favor this scenario, including our design of a constitutively active receptor mutant (uPAR H47C/N259C ), where DI is tethered to DIII via an interdomain disulfide located distant from to the SMB binding site (29). This constrained uPAR mutant promotes lamellipodia formation on vitronectin-rich matrices in the absence of uPA (29), which suggests that a regulatory structural transition occurs at the SMB binding site when uPA binds.…”

mentioning

confidence: 99%

“…Only recently has the structure of a constitutively active receptor mutant (uPAR H47C/N259C ) (29) been solved by x-ray crystallography in the absence of bound ligands (30). As alluded to above, this structure is, nevertheless, unlikely to represent the prevalent conformation populated by unoccupied uPAR wt because the constraints we engineered into uPAR H47C/N259C render it a structural and functional surrogate for the receptor conformation selected in the uPA⅐uPAR complex (29,30).…”

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confidence: 99%

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A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

Mertens

Kjærgaard

Mysling

et al. 2012

Journal of Biological Chemistry

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Background:The urokinase receptor (uPAR) is a modular receptor containing three LU domains. Results: Ligand-free uPAR is inherently flexible with a detached N-terminal domain (DI). Conclusion: Allosteric regulation of uPAR is driven by uPA-induced compaction of the intact receptor and a concomitant stabilization of DI. Significance: This flexibility and ligand-induced allostery are expected to impact future studies on uPAR function and targeted intervention.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

Mertens

Kjærgaard

Mysling

et al. 2012

Journal of Biological Chemistry

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Crystal structure of the unoccupied murine urokinase‐type plasminogen activator receptor (uPAR) reveals a tightly packed DII–DIII unit

et al. 2019

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The urokinase‐type plasminogen activator receptor (uPAR) is a cell surface receptor that is capable of binding to a range of extracellular proteins and triggering a series of proteolytic and signaling events. Previous structural studies of uPAR with its ligands uPA and vitronectin revealed that its three domains (DI, DII, and DIII) form a large hydrophobic cavity to accommodate uPA. In the present study, the structure of unoccupied murine uPAR (muPAR) is determined. The structure of DII and DIII of muPAR is well defined and forms a compact globular unit, while DI could not be traced. Molecular dynamic simulations further confirm the rigid binding interface between DII and DIII. This study shows overall structural flexibility of uPAR in the absence of uPA.

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Re‐engineering the Immune Response to Metastatic Cancer: Antibody‐Recruiting Small Molecules Targeting the Urokinase Receptor

et al. 2016

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Developing selective strategies to treat metastatic cancers remains a significant challenge. Herein, we report the first antibody-recruiting small molecule (ARM) that is capable of recognizing the urokinase-type plasminogen activator receptor (uPAR), a uniquely overexpressed cancer cellsurface marker, and facilitating the immune-mediated destruction of cancer cells. A co-crystal Correspondence to: David A. Spiegel. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201510866. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript structure of the ARM-U2/uPAR complex was obtained, representing the first crystal structure of uPAR complexed with a non-peptide ligand. Finally, we demonstrated that ARM-U2 substantially suppresses tumor growth in vivo with no evidence of weight loss, unlike the standard-of-care agent doxorubicin. This work underscores the promise of antibody-recruiting molecules as immunotherapeutics for treating cancer. Keywordsantitumor agents; cell recognition; drug design; immunology; medicinal chemistryTumor metastasis involves the invasion of cancer cells into surrounding tissues, a process often accelerated by cell-surface proteases. One such protease, the urokinase-type plasminogen activator (uPA), breaks down extracellular matrix proteins and activates migration-inducing signal cascades upon binding to the urokinase-type plasminogen activator receptor (uPAR). [1] Both uPA and uPAR expression are substantially upregulated in invasive cancer cells com-pared to healthy cells or benign tumors. [2] In clinical settings, uPAR levels have been shown to correlate with metastatic potential and poor clinical outcomes. [1a] As such, uPAR has gained recognition as a promising target for treating metastatic cancers from diverse tissues of origin, including breast, colon, stomach, and bladder. [3,4] Antibody-recruiting molecules (ARMs) are bifunctional molecules capable of delivering endogenous antibodies to disease-causing entities, leading to their destruction and/or clearance by the immune system. Our group and others have previously developed ARMs that target various cancers and infectious agents. These novel immunotherapies have the potential both to complement protein-based agents while overcoming their challenges, such as poor oral bioavailability, high molecular weights, and immunogenicity. [5] Our group previously reported an ARM capable of targeting uPAR-expressing cancer cells for immune-mediated cell death. [5] Although effective in vitro, the reported con-struct (termed ARM-U1) contained the uPA protein at its target binding terminus (TBT), imparting many of the limitations of biologics. We therefore hypothesized that the therapeutic potential of this agent could be significantly improved upon by replacing the uPA protein with a highaffinity uPAR-binding small molecule (Figure 1 A).Herein, we report the design, synthesis, and in vitro and in vivo evaluation of a secondgeneration, low-molecular...

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Mimicry of the Regulatory Role of Urokinase in Lamellipodia Formation by Introduction of a Non-native Interdomain Disulfide Bond in Its Receptor

Cited by 29 publications

References 46 publications

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

Crystal structure of the unoccupied murine urokinase‐type plasminogen activator receptor (uPAR) reveals a tightly packed DII–DIII unit

Re‐engineering the Immune Response to Metastatic Cancer: Antibody‐Recruiting Small Molecules Targeting the Urokinase Receptor

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