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 recombinant receptor was found to bind its natural ligand SDF-1alpha with an affinity 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-glycosylation of CXCR4 in ligand binding was investigated in the insect cells overexpressed with recombinant CXCR4. Two potential N-glycosylation sites (Asn-11 and Asn-176) were either singly or doubly mutated to a leucine residue. Both single mutant receptors exhibited a significant decrease in ligand binding activity and affinity. The double mutant receptor showed little binding activity. Our data suggest that all of the extracellular domains are involved in ligand-receptor interactions and that N-glycosylation is required to maintain high-affinity ligand binding.
Zhou
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Division of Pharmaceutical Sciences, College of Pharmacy, Lexington, Kentucky, 40536-0082, USA.
Tai
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D000818:Animals; D000918:Antibody Specificity; D016367:Baculoviridae; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000378:metabolism*; D018076:DNA, Complementary; D015723:Gene Library; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D019718:Receptors, CXCR4 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D018076:DNA, Complementary; D008024:Ligands; D010446:Peptide Fragments; D019718:Receptors, CXCR4; D011994:Recombinant Proteins