Stabilizing a Flexible Interdomain Hinge Region Harboring the SMB Binding Site Drives uPAR into Its Closed Conformation

Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; Lorenzi, Valentina De; Sidénius, Nicolai; Huang, Mingdong; Ploug, Michael

doi:10.1016/j.jmb.2015.01.022

Cited by 26 publications

(33 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ligand loading/unloading loop [49] of residues 131-140 in DII domain was found to be disordered in the current structure of the unoccupied muPAR (Fig. 1C) as was the case in the three structures solved for huPAR in the absence of bound uPA [25,49,50].…”

Section: Resultsmentioning

confidence: 56%

“…Notwithstanding this proposition, two crystal structures of uPAR were actually solved without any bound uPA shielding this hydrophobic cavity from solvent exposure. Importantly, both two structures were structurally constrained either by (a) a monoclonal antibody (8B12) stabilizing the DI-DII boundary thus mimicking vitronectin binding [50] and/or (b) the presence of engineered extra disulfide bond (H47C-N259C) [69] covalently tethering DI to DIII, which structurally and functionally mimics uPA-occupied uPAR [49].…”

Section: Model Of Unoccupied Muparmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2019

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 56%

Section: Model Of Unoccupied Muparmentioning

confidence: 99%

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

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…) failed to promote the release and because an antibody (8B12) binding to the VN epitope in uPAR [25] also prevented release ( Fig 4C). The mode of action of the different compounds/ mutations utilized is depicted in Fig 4B.…”

Section: R91amentioning

confidence: 96%

“…The release of VN fragments was caused by cleavage within the RGD motif as it was impaired by the R45A substitution (Appendix Fig S3B). Furthermore, the release mediated by tc-uPA requires the following: (i) uPAR expression, as mock-transfected cells did not induce release; (ii) uPA binding to uPAR, as the release was impaired by the GFD domain of uPA; (iii) uPAR binding to VN, as cells expressing a VN binding-deficient uPAR mutant (uPAR R91A ) failed to promote the release and because an antibody (8B12) binding to the VN epitope in uPAR [25] also prevented release ( Fig 4C). The mode of action of the different compounds/ mutations utilized is depicted in Fig 4B. Similar results were observed for the treatment with sc-uPA, plasminogen and a2AP ( Fig EV4), even if the inhibition by GFD and the impairment by the uPAR 91 R?A substitution in this case were only partial.…”

Section: Upar Accelerates the Cleavage Of Matrix Vn By Upa And Plasminmentioning

confidence: 99%

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

et al. 2016

Self Cite

View full text Add to dashboard Cite

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

show abstract

“…Epitope binning of GPIHBP1 mAbs was performed with a Bi-acore3000 (GE Healthcare Life Science), as described (17).…”

Section: An Elisa To Detect Gpihbp1 In Human Plasmamentioning

confidence: 99%

Monoclonal antibodies that bind to the Ly6 domain of GPIHBP1 abolish the binding of LPL

Sleeman

Miyashita

et al. 2017

Journal of Lipid Research

Self Cite

View full text Add to dashboard Cite

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease.

show abstract

Stabilizing a Flexible Interdomain Hinge Region Harboring the SMB Binding Site Drives uPAR into Its Closed Conformation

Cited by 26 publications

References 62 publications

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

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

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

Monoclonal antibodies that bind to the Ly6 domain of GPIHBP1 abolish the binding of LPL

Contact Info

Product

Resources

About