Identification of CXCR4 Domains That Support Coreceptor and Chemokine Receptor Functions

Abstract

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, is capable of inhibiting human immunodeficiency virus type 1 (HIV-1) utilization of CXCR4 as a coreceptor, and has been implicated in delaying disease progression to AIDS in vivo. Because of the importance of this chemokine-chemokine receptor pair to both development and disease, we investigated the molecular basis of the interaction between CXCR4 and its ligands SDF-1 and HIV-1 envelope. Using CXCR4 chimeras and mutants, we determined that SDF-1 requires the CXCR4 amino terminus for binding and activates downstream signaling pathways by interacting with the second extracellular loop of CXCR4. SDF-1-mediated activation of CXCR4 required the Asp-Arg-Tyr motif in the second intracellular loop of CXCR4, was pertussis toxin sensitive, and did not require the distal C-terminal tail of CXCR4. Several CXCR4 mutants that were not capable of binding SDF-1 or signaling still supported HIV-1 infection, indicating that the ability of CXCR4 to function as a coreceptor is independent of its ability to signal. Direct binding studies using the X4 gp120s HXB, BH8, and MN demonstrated the ability of HIV-1 gp120 to bind directly and specifically to the chemokine receptor CXCR4 in a CD4-dependent manner, using a conformationally complex structure on CXCR4. Several CXCR4 variants that did not support binding of soluble gp120 could still function as viral coreceptors, indicating that detectable binding of monomeric gp120 is not always predictive of coreceptor function.

Mutations at the CXCR4 interaction sites for AMD3100 influence anti‐CXCR4 antibody binding and HIV‐1 entry

“…Such diversity among HIV strains in their structural requirements for functional interaction with CXCR4 was previously reported by Brelot et al [29,31]. Also, Doranz et al found that amino acid residues in all four extracellular regions of CXCR4 contribute to coreceptor activity, implying that the viral envelope interacts with a conformationally complex structure of the receptor protein [32]. Yet, AMD3100 is potently and consistently active in the nanomolar range against every CXCR4using HIV-1 strain tested so far, including those viruses that for their infectivity do not depend on the presence of aspartic acid residues at positions 171 and/or 262 of the coreceptor (e.g.…”

Cloning and characterization of SDF‐1γ, a novel SDF‐1 chemokine transcript with developmentally regulated expression in the nervous system

“…The ®rst 17 AA, which are identical in all isoforms, are required for the binding of the SDF-1 proteins to CXCR4 (Loetscher et al, 1998;Doranz et al, 1999). At the other end, in the carboxy-terminal region of SDF-1b, a pair of basic AA (lys 89, arg 90) provides a motif for proteolytic cleavage by a membrane-bound protease of the trans-Golgi apparatus and secretory vesicles, that would result in a pentapeptide (lys 89 ± met 93) and a shortened core protein.…”

Characterization of aXenopus laevis CXC chemokine receptor 4: implications for hemato-poietic cell development in the vertebrate embryo

“…1) raises the possibility that the remaining 19 residues are, either directly or indirectly, involved in specifically recognizing SDF-1 and/ or mediating CXCR4 activation by SDF-1. Functional analysis of hCXCR4/hCXCR2-chimera and hCXCR4 mutants showed that the amino-terminal-most portion of hCXCR4 up to the residue corresponding to Pro 31 of xCXCR4 is important for high-affinity SDF-1 binding, but is neither necessary nor sufficient for receptor activation by high concentrations of SDF-1 [37]. Results obtained very recently by NMR solution structure analysis of a complex between the CXC chemokine IL-8 and an amino-terminal hCXCR1 peptide revealed that nine amino-terminal residues of hCXCR1, corresponding to Gly 23 -Pro 31 of xCXCR4 are likely to directly interact with, and thus constitute part of, the receptor recognition site for IL-8 [38].…”

“…Importantly, four of these residues, Gly 23 , Asp 24 , Glu 30 , and Pro 31 of xCXCR4, are absolutely conserved between X. laevis and mammalian CXCR4, suggesting an important role of these residues in the binding of SDF-1 to CXCR4. On the other hand, residues present within the second and the third extracellular loop appear to be important for SDF-1-mediated activation of CXCR4 [37]. For example, replacement of the Glu 179 -Ala-Asp-Asp motif present within the second extracellular loop of hCXCR4 by Gln-Ala-Ala-Asn blocked activation of CXCR4 by SDF-1.…”