Regulation of Urokinase/Urokinase Receptor Interaction by Heparin-like Glycosaminoglycans

Pucci, Marco; Fibbi, Gabriella; Magnelli, Lucia; Rosso, Mario Del

doi:10.1074/jbc.m005993200

Cited by 12 publications

(9 citation statements)

References 67 publications

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“…Cells were then sorted for the Green Fluorescent Protein (GFP) marker and selected with 1 µg/mL puromycin for 2-3 weeks (Sigma-Aldrich, Saint Louis, MO, USA), which is singularly characterized using Western Blotting, qPCR and PCR on the full-length mRNA. For the uPAR rescue expression experiment, cells were stably transfected using an Okayama-Berg vector containing uPAR cDNA, and they were selected with G418 as resistance marker (0.5 mg/mL) as previously reported [23].…”

Section: Transfection and Plasmidmentioning

confidence: 99%

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

Biagioni

Laurenzana

Chillà

et al. 2020

Cells

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Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB.

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Section: Transfection and Plasmidmentioning

confidence: 99%

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

Biagioni

Laurenzana

Chillà

et al. 2020

Cells

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“…This impact was readily apparent with a more prolonged dissociation phase than has been explored previously 9 and with suPAR oriented on the sensor surface in a homogeneous manner using a monoclonal nonblocking antibody that simulated the cellular context, allowing less direct influence by the dextran surface. 59 Second, the amino-terminal fragment (ATF) of scuPA, which contains the GFD linked to the kringle, inhibits binding of full-length scuPA to suPAR more effectively than does the GFD fragment alone. Third, whereas the apparent K d 's of ATF and full-length scuPA calculated from the initial "on-rate" an "offrates" are nearly identical, 28 ATF but not GFD is able to displace full-length scuPA from suPAR upon incubation.…”

Section: Discussionmentioning

confidence: 99%

The kringle stabilizes urokinase binding to the urokinase receptor

Bdeir

Kuo

Sachais

et al. 2003

Blood

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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...

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“…To compare the interactions of PAPP-A with the unknown, natural heparan sulfate-substituted receptor and the interactions with isolated heparin, we analyzed the PAPP-A/PAPP-A2 chimeras by affinity chromatography on heparin-Sepharose ( Fig. 2), a method commonly used to determine the relative affinities of proteins for heparan sulfate-like GAGs [31][32][33][34][35][36]. Recombinant wild-type PAPP-A, contained in culture medium, eluted from the column at 0.68 M NaCl.…”

Section: Heparin and Cell Surface Binding Of Papp-a Is Mediated Primamentioning

confidence: 99%

Cell surface adhesion of pregnancy‐associated plasma protein‐A is mediated by four clusters of basic residues located in its third and fourth CCP module

Weyer

Overgaard

Laursen

et al. 2004

European Journal of Biochemistry

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The metalloproteinase pregnancy‐associated plasma protein‐A (PAPP‐A) cleaves a subset of insulin‐like growth factor binding proteins (IGFBP), which inhibit the activities of insulin‐like growth factor (IGF). Through this proteolytic activity, PAPP‐A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP‐A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP‐A to the cell surface. Based on site‐directed mutagenesis, we here delineate the PAPP‐A GAG‐binding site in the C‐terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP‐A with heparin. In a model of PAPP‐A CCP3‐CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP‐A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP‐A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP‐A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP‐A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP‐A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP‐A.

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Regulation of Urokinase/Urokinase Receptor Interaction by Heparin-like Glycosaminoglycans

Cited by 12 publications

References 67 publications

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

The kringle stabilizes urokinase binding to the urokinase receptor

Cell surface adhesion of pregnancy‐associated plasma protein‐A is mediated by four clusters of basic residues located in its third and fourth CCP module

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