N-LINKED GLYCOSYLATION OF THE LIGAND-BINDING DOMAIN OF THE HUMAN UROKINASE RECEPTOR CONTRIBUTES TO THE AFFINITY FOR ITS LIGAND

Variations in glycosylation exist among urokinase plasminogen activato r receptors (u-PARs) from different cell types. We have studied the fu nctional role of N-linked carbohydrate within the ligand-binding domai n of u-PAR. Treatment with glycosidases demonstrated that all the N-li nked carbohydrates on u-PAR are complex-type oligosaccharides. Substit ution of a single Asn (Asn52) to Gln by means of site-directed mutagen esis led to an active receptor mutant with a ligand-binding domain dev oid of carbohydrate. The cellular distribution, the glycosyl-phosphati dylinositol anchoring, and the conformational stability after solubili zation were unaffected by this single substitution. However, ligand bi nding analysis demonstrated a 4-5-fold decrease in affinity as compare d with the wild type receptor. Two different strategies were used in o rder to obtain a u-PAR type completely devoid of N-linked carbohydrate s. 1) Tunicamycin treatment of wild type u-PAR-expressing cells. 2) Mu tation of all glycosylation sites (Hu-PAR(N5-mut)). In neither case, u nglycosylated receptors with ligand binding activity were identified. However, immunofluorescence studies demonstrated that the-Hu-PAR(N5-mu t) was retained inside the cells in the endoplasmic reticulum. The sam e result was found for Hu-PAR(N4-mut), where only the glycosylation si tes outside the binding domain were mutated. These results demonstrate that some extent of glycosylation of u-PAR is necessary for cellular transport and for molecular maturation events leading to ligand bindin g activity. Glycosylation of the binding domain per se affects only th e affinity of the receptor. The positive modulation of the Asn52 carbo hydrate side chain on ligand affinity suggests that the u-PAR glycosyl ation variants observed in various cell types may have different funct ional roles.