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 Dana, 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 aminoterminal 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.
