Print page Print page
Switch language
The Capital Region of Denmark - a part of Copenhagen University Hospital

Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

Research output: Contribution to journalJournal articleResearchpeer-review

  1. Tumor cell MT1-MMP is dispensable for osteosarcoma tumor growth, bone degradation and lung metastasis

    Research output: Contribution to journalJournal articleResearchpeer-review

  2. The collagen receptor uPARAP/Endo180 regulates collectins through unique structural elements in its FNII domain

    Research output: Contribution to journalJournal articleResearchpeer-review

  3. Cellular uptake of collagens and implications for immune cell regulation in disease

    Research output: Contribution to journalReviewResearchpeer-review

  4. TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding

    Research output: Contribution to journalJournal articleResearchpeer-review

  5. CCL2/MCP-1 signaling drives extracellular matrix turnover by diverse macrophage subsets

    Research output: Contribution to journalJournal articleResearchpeer-review

View graph of relations

The urokinase plasminogen activator receptor (uPAR) is a membrane protein comprised of three extracellular domains. In order to study the importance of this domain organization in the ligand-binding process of the receptor we subjected a recombinant, soluble uPAR (suPAR) to specific proteolytic cleavages leading to liberation of single domains. Treatment of the receptor with pepsin resulted in cleavage between residues 183 and 184, thus separating the third domain (D3) from the rest of the molecule, which was left as an intact fragment (D(1 + 2)). D(1 + 2) proved capable of ligand binding as shown by chemical cross-linking, but quantitative binding/competition studies showed that the apparent ligand affinity was 100- to 1000-fold lower than that of the intact suPAR. This loss of affinity was comparable with the loss found after cleavage between the first domain (D1) and D(2 + 3), using chymotrypsin. This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular interaction analysis revealed that the decrease in affinity was caused mostly by an increased dissociation rate of the ligand complex of D(1 + 2). Zero length cross-linking, using carbodiimide-induced, direct condensation, was used to identify regions within suPAR engaged in molecular ligand contact. The purified suPAR was cross-linked to the radiolabeled amino-terminal fragment (ATF) of urokinase, followed by cleavage with chymotrypsin. In accordance with the cleavage pattern found for the uncomplexed receptor, this treatment led to cleavage between D1 and D(2 + 3). Analysis of the radiolabeled fragments revealed the expected ligand labeling of D1 but a clear labeling of D(2 + 3) was also found, indicating that this part of the molecule is also situated in close contact with ATF in the receptor-ligand complex. The latter contact site may contribute to the role of molecular regions outside D1 in high affinity binding.

Original languageEnglish
JournalThe journal of biological chemistry
Issue number37
Pages (from-to)22885-94
Number of pages10
Publication statusPublished - 13 Sep 1996

    Research areas

  • Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, Cell Line, Chromatography, Gel, Chromatography, High Pressure Liquid, Cricetinae, Cricetulus, Electrophoresis, Polyacrylamide Gel, Female, Kinetics, Molecular Sequence Data, Ovary, Pepsin A, Protein Conformation, Receptors, Cell Surface, Receptors, Urokinase Plasminogen Activator, Structure-Activity Relationship, Urokinase-Type Plasminogen Activator

ID: 46435177