TY - JOUR T1 - Mutation of Asp<sup>171</sup> and Asp<sup>262</sup> of the Chemokine Receptor CXCR4 Impairs Its Coreceptor Function for Human Immunodeficiency Virus-1 Entry and Abrogates the Antagonistic Activity of AMD3100 JF - Molecular Pharmacology JO - Mol Pharmacol SP - 164 LP - 173 DO - 10.1124/mol.60.1.164 VL - 60 IS - 1 AU - Sigrid Hatse AU - Katrien Princen AU - Lars-Ole Gerlach AU - Gary Bridger AU - Geoffrey Henson AU - Erik De Clercq AU - Thue W. Schwartz AU - Dominique Schols Y1 - 2001/07/01 UR - http://molpharm.aspetjournals.org/content/60/1/164.abstract N2 - The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunodeficiency virus (HIV)-1 and HIV-2, which use CXCR4 as coreceptor for host cell entry. Here, we investigated the interaction of AMD3100 with CXCR4 at the molecular level by mutational analysis. We established a set of stably transfected U87.CD4 cell lines expressing different mutant forms of CXCR4 (i.e., CXCR4[WT], CXCR4[D171N], CXCR4[D262N], CXCR4[D171N,D262N], and CXCR4[H281A]), to compare the activity of the compound against mutated versus wild-type CXCR4. We found that the antagonistic action of AMD3100 against CXCR4—as assessed by the inhibitory effects of the compound on stromal cell-derived factor (SDF-1) binding to its receptor and on SDF-1-induced intracellular calcium signaling, and by displacement of the CXCR4-specific antibody, clone 12G5—was greatly reduced by substitution of Asp171 and/or Asp262 by neutral asparagine residue(s). Both aspartates, but most particularly Asp262, also proved essential for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His281 by a neutral alanine potentiated the antagonistic and antiviral effects of the compound in the different assay systems. Importantly, compared with the wild-type receptor, CXCR4[D262N] was much less effective, whereas CXCR4[D171N,D262N] completely failed as a coreceptor for infection by HIV-1 NL4.3. Thus, the negatively charged aspartate residues at positions 171 and 262, located in transmembrane domains 4 and 6 of the 7-transmembrane receptor, respectively, may represent crucial sites for electrostatic interaction of the positive charges of the bicyclams, as well as for the highly basic V3 loop of the gp120 envelope protein of certain HIV-1 strains. HIVhuman immunodeficiency virusSDF-1stromal cell-derived factor-1PBSphosphate-buffered salineFBSfetal bovine serumPEphycoerythrinmAbmonoclonal antibodyRT-PCRreverse transcription polymerase chain reactionLTRlong terminal repeatTMtransmembrane domainAgantigenGAPDHglyceraldehyde 3-phosphate dehydrogenaseWTwild-type ER -