A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor
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A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor. / Hillig, Thore; Engelholm, Lars H; Ingvarsen, Signe; Madsen, Daniel H; Gårdsvoll, Henrik; Larsen, Jørgen K; Ploug, Michael; Danø, Keld; Kjøller, Lars; Behrendt, Niels.
In: Journal of Biological Chemistry, Vol. 283, No. 22, 30.05.2008, p. 15217-15223.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor
AU - Hillig, Thore
AU - Engelholm, Lars H
AU - Ingvarsen, Signe
AU - Madsen, Daniel H
AU - Gårdsvoll, Henrik
AU - Larsen, Jørgen K
AU - Ploug, Michael
AU - Danø, Keld
AU - Kjøller, Lars
AU - Behrendt, Niels
PY - 2008/5/30
Y1 - 2008/5/30
N2 - The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.
AB - The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.
KW - Amino Acid Substitution
KW - Cell Adhesion
KW - Cell Line
KW - Cell Movement
KW - Cell Shape
KW - Cytoskeleton
KW - Humans
KW - Integrins
KW - Peptides
KW - Protein Binding
KW - Protein Structure, Tertiary
KW - Receptors, Cell Surface
KW - Receptors, Urokinase Plasminogen Activator
KW - Urokinase-Type Plasminogen Activator
KW - Vitronectin
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1074/jbc.C700214200
DO - 10.1074/jbc.C700214200
M3 - Journal article
C2 - 18362146
VL - 283
SP - 15217
EP - 15223
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 22
ER -
ID: 14248397