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. 2000 Jan 1;373(1):211-7.
doi: 10.1006/abbi.1999.1555.

Expression and functional characterization of mutant human CXCR4 in insect cells: role of cysteinyl and negatively charged residues in ligand binding

H Zhou  1 , H H Tai
Affiliations

Expression and functional characterization of mutant human CXCR4 in insect cells: role of cysteinyl and negatively charged residues in ligand binding

H Zhou et al. Arch Biochem Biophys. .

Abstract

Human CXCR4 was expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system. The recombinant receptor exhibited ligand binding activities with a K(d) value (3.3 nM) comparable to that of the native receptor. The role of four conserved cysteinyl residues was explored by site-directed mutagenesis. Each cysteine was individually changed to an alanine residue. All of the four mutants showed decreased ligand binding activity with increased K(d) values although comparable levels of receptor expression were observed. These results suggest that each of these four cysteinyl residues may be important for the ligand binding of the receptor. Evidence suggests that the ionic interaction may be involved in ligand binding. Point mutation of several relatively conserved acidic residues (Asp-10, Asp-262, Glu-275, and Glu-277) to an alanine residue greatly decreased the ligand binding activity and affinity. Since SDF-1alpha is a highly basic protein, these acidic residues may interact with the basic residues of SDF-1alpha by ionic pairing in addition to other molecular interactions and play an important role in ligand binding.

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