Characterization of recombinant human CXCR4 in insect cells: Role of extracellular domains and N-glycosylation in ligand binding

The cDNA of the human CXCR4/fusin was isolated from a human HeLa cell cDNA library by PCR and functionally expressed in Sf9 insect cells. The recombin ant receptor was found to bind its natural ligand SDF-1 alpha with an affin ity comparable to that of the native receptor. Sequence-specific antibodies against each of the four extracellular domains were generated and used to investigate the interactions between the different domains of the receptor and the ligand. Each of the four antibodies was found to be able to inhibit ligand binding. CXCR4 was shown to be a glycoprotein. The role of N-glycos ylation of CXCR4 in ligand binding was investigated in the insect cells ove rexpressed with recombinant CXCR4. Two potential N-glycosylation sites (Asn -ll and Asn-176) were either singly or doubly mutated to a leucine residue. Both single mutant receptors exhibited a significant decrease in ligand bi nding activity and affinity. The double mutant receptor showed little bindi ng activity. Our data suggest that all of the extracellular domains are inv olved in ligand-receptor interactions and that N-glycosylation is required to maintain high-affinity ligand binding. (C) 1999Academic Press.