DOMAIN INTERPLAY IN THE UROKINASE RECEPTOR - REQUIREMENT FOR THE 3RD DOMAIN IN HIGH-AFFINITY LIGAND-BINDING AND DEMONSTRATION OF LIGAND CONTACT SITES IN DISTINCT RECEPTOR DOMAINS

The urokinase plasminogen activator receptor (uPAR) is a membrane prot ein comprised of three extracellular domains. In order to study the im portance of this domain organization in the ligand-binding process of the receptor we subjected a recombinant, soluble uPAR (suPAR) to speci fic proteolytic cleavages leading to liberation of single domains. Tre atment of the receptor with pepsin resulted in cleavage between residu es 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 apparen t 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 chymotrypsi n. 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), D 3 has an important role in governing a high affinity in the intact rec eptor. Real-time biomolecular interaction analysis revealed that the d ecrease in affinity was caused mostly by an increased dissociation rat e of the ligand complex of D(1 + 2), Zero length cross-linking, using carbodiimide-induced, direct condensation, was used to identify region s 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 treatmen t led to cleavage between D1 and D(2 + 3), Analysis of the radiolabele d fragments revealed the expected ligand labeling of D1 but a clear la beling of D(2 + 3) was also found, indicating that this part of the mo lecule is also situated in close contact with ATF in the receptor-liga nd complex, The latter contact site may contribute to the role of mole cular regions outside D1 in high affinity binding.