Immunologic Detection of the Cellular Receptor for Urokinase Plasminogen Activator

Mizukami, Ikuko F.; Garni-Wagner, B. A.; DeAngelo, Laura; Liebert, Monica; Flint, Andrew; Lawrence, David A.; Cohen, Robert; Todd, Robert F.

doi:10.1006/clin.1994.1057

Cited by 31 publications

(20 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The peptides used were colorless, odorless, and Ͼ95% pure as determined by reverse phase high pressure liquid chromatography and mass spectrometry. A monoclonal antibody against uPAR (3B10FC) was generously provided by Dr. Robert F. Todd III from the University of Michigan (Ann Arbor, MI) (16). Monoclonal anti-uPAR antibodies E180 and E33 were the kind gifts of Dr. A. Mazar (Attenuon, San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

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

Mahdi

Shariat‐Madar

Kuo

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (su-PAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC 50 of 60, 110, and 8 nM, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His 477 -Gly 496 ) and a lower affinity site within the heavy chain (Cys 333 -Lys 345 ). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Recent investigations indicate that high molecular mass kininogen (HK)1 binds to endothelial cell membranes through an interaction with a multiprotein receptor complex comprising at least cytokeratin 1, gC1qR, and the urokinase plasminogen activator receptor (uPAR) (1-5). The three proteins co-localize on the endothelial cell membrane (6). The same three proteins form a receptor complex for factor XII (7), but binding of factor XII in vivo is likely limited both by the low plasma concentration of free Zn 2ϩ , which is below the requirement for FXII binding and by the much higher plasma concentration of HK (7). Binding of HK to this multiprotein receptor complex predominates, localizing prekallikrein (PK) to the cell surface. The plasma concentration of PK and the ambient free Zn 2ϩ concentration in plasma also prevent FXI from binding to HK under conditions where platelets or other cells are not activated (8). PK bound to HK on endothelial cells is proteolyzed by membrane-expressed prolylcarboxypeptidase to form plasma kallikrein (9, 10). This multiprotein receptor complex thereby regulates the assembly and activation of the plasma kallikrein/ kinin system.The requirements for HK binding to each component of this receptor complex and the effects of other biologically relevant ligands, e.g. urokinase, on this binding have not been well delineated. It is known that both the heavy and light chains of HK interact with a region of cytokeratin 1 coded by exon 1 (2). Antibody to this region completely inhibits HK binding to cytokeratin 1 as well as to the receptor complex (6). Likewise, some antibodies to gC1qR and uPAR completely block HK binding to the proteins individually as well as when they are part of the complex expressed on endothelial cells (6, 8). However, it is unclear whether each component of the cellular complex recognizes discrete or overlapping portions of HK and whether each molecule of HK binds to more than one component of the complex at the same time. To begin to address these issues, we sought to identify the regions in HK and uPAR required for binding. The results indicate the existence of multiple potential sites of interaction between this ligand and receptor and provide insight into the assembly of HK on its multireceptor co...

show abstract

Section: Methodsmentioning

confidence: 99%

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

Mahdi

Shariat‐Madar

Kuo

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The differentiated U937 cell line was used as a model of CD87-expressing monocyte/macrophage-type cells (3,43,52). The proteolytic activity of purified EL and CG on membrane CD87 was first explored by FACS analysis, using the already mentioned anti-D1 mAb 3931 and anti-D2 mAb 3932 as well as mAb 3936, which recognizes a still unknown conformational epitope (41,48). Exposure of cells to EL for 15 min (Fig.…”

Section: El Cleaves Membrane Cd87 On Monocytic Cells Preferentially Wmentioning

confidence: 99%

“…6). It must be noted that the conformational, yet unknown epitope recognized by mAb 3936, which interferes with uPA binding and has been extensively used in immunohistochemistry studies (41,48), appears to be unrelated to expression of domain D1.…”

Section: Binding Of Upa To Monocytic Cells Is Drastically Reduced By mentioning

confidence: 99%

Proteolytic Regulation of the Urokinase Receptor/CD87 on Monocytic Cells by Neutrophil Elastase and Cathepsin G

Beaufort

Leduc

Rousselle

et al. 2004

The Journal of Immunology

View full text Add to dashboard Cite

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.

show abstract

“…The epitopes and function blocking effects of these mAbs have recently been reviewed (5). Anti-uPAR mAb 3B10 and the generation of its F(abЈ) 2 coupled to tetramethylrhodamine isothiocyanate (TRITC) were previously described (17,29,30). Anti-CD11a mAb TS1/22 was provided by Dr. Springer and G43-25B was purchased from BD PharMingen (San Diego, CA).…”

Section: Abs and Other Reagentsmentioning

confidence: 99%

Function of the Lectin Domain of Mac-1/Complement Receptor Type 3 (CD11b/CD18) in Regulating Neutrophil Adhesion

Xia

Borland

Huang

et al. 2002

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by β-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcγRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) β2 integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by β-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by β-glucan. A single CD11b lectin site for β-glucan and uPAR was suggested because the binding of either β-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of 125I-labeled β-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of 125I-labeled β-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.

show abstract

Immunologic Detection of the Cellular Receptor for Urokinase Plasminogen Activator

Cited by 31 publications

References 0 publications

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

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

Proteolytic Regulation of the Urokinase Receptor/CD87 on Monocytic Cells by Neutrophil Elastase and Cathepsin G

Function of the Lectin Domain of Mac-1/Complement Receptor Type 3 (CD11b/CD18) in Regulating Neutrophil Adhesion

Contact Info

Product

Resources

About