Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF-1α: implications for development of selective CXCR4 antagonists
Introduction
Chemokines are structurally and functionally related proteins that orchestrate immunological and inflammatory processes, such as leukocyte chemotaxis, adhesion, hematopoiesis and angiogenesis [1]. Stromal cell derived factor-1 (SDF-1), is a C-X-C chemokine [2], [3] that interacts with its specific receptor, CXCR4 [4], [5]. Both SDF-1 and CXCR4 are constitutively expressed in a variety of tissues and cell types [2], [3], [4], [6], and cause efficacious chemoattraction of T and B-lymphocytes, monocytes, CD34+ hematopoietic progenitors and endothelial cells [7], [8], [9], [10]. Furthermore, SDF-1 is involved in angiogenesis and induces the formation of capillary vessels in mice [11]. In contrast to other chemokines, both SDF-1 and CXCR4 are remarkably conserved with >90% identity across diverse species [12], implying a fundamental biological role during development. It is noteworthy that murine knockouts of SDF-1 and CXCR4 genes display a similar embryologically lethal phenotype, characterized by deficient B-lympho and myelopoiesis, abnormal neuronal and cardiac development, and defects in vasculogenesis [13], [14], [15]. CXCR4 is also a co-receptor for the entry of T-lymphocyte tropic strains of HIV-1, and SDF-1 can inhibit the fusion and replication of HIV-1 in CD4+ and CXCR4+ cells [5], [16]. This involvement of CXCR4 and SDF-1 in HIV life-cycle has generated widespread interest in the comprehensive biochemical and pharmacological characterization of this receptor.
In common with other chemokine receptors, CXCR4 belongs to the superfamily of seven transmembrane G-protein coupled receptors that couple to, and signal via heterotrimeric guanine nucleotide-binding proteins (G-proteins). The binding kinetics of SDF-1 is complex and not completely characterized, with reports of CXCR4-independent binding in cell lines like CHO-K1 and ECV-304 [17]. Furthermore, based on three-dimensional structural analysis of SDF-1 a two-site model for their interaction has been proposed recently [18]. In this study, we have attempted to delineate the pharmacological properties of the CXCR4 receptor in two human leukemia cell lines, HL-60 cells [19], used extensively as an in vitro model for myeloid differentiation; and Jurkat cells [20] with a T-lymphocytic origin. Our data obtained from [125I]-SDF-1α radioligand and [35S]-GTPγS binding studies, along with the discordant nature of inhibition mediated by CXCR4 specific antagonist AMD3100 in binding versus functional assays, are best explained by invoking a model based on multiple site ligand-receptor interaction. These data will lead to a further understanding of the molecular interactions between SDF-1 and CXCR4, and will be useful for the future design of potent SDF-1 agonists and antagonists.
Section snippets
Materials and cell culture
[125I]-SDF-1α (2200 Ci/mmol) and [35S]-GTPγS (1250 Ci/mmol) were obtained from NEN/DuPont. SDF-1α and anti-CXCR4 antibody (clone 12G5) were from R&D Systems (Minneapolis, MN). AMD3100 [21] was synthesized at GlaxoSmithKline, King of Prussia, PA. Human HL-60 and Jurkat T-cells were from ATCC (Rockville, MD), and cultured in RPMI-1640 containing 10% FBS.
Radioligand binding assays
Cells were split 1:2 in culture medium, 24 h prior to performing binding assays. Cells were washed twice in PBS, and cell pellets were
Results and discussion
SDF-1α induces a particularly efficacious and dose dependent chemotactic response in both HL-60 (EC50=33.9 nM) and Jurkat-T (EC50=1 nM) cell lines; and upto 60% of the input cells migrate after 4 h with 100 nM SDF-1α (data not shown). In addition, SDF-1α also causes a dose dependent mobilization of intracellular calcium in both HL-60 cells and the Jurkat T-cells. Moreover, SDF-1α-mediated migration is chemotactic, and not merely chemokinesis as revealed by checkerboard analysis. These results
Acknowledgements
We wish to acknowledge Dr Juan Luengo for synthesis of AMD3100. We also thank Dr John White for critical reading of this manuscript
References (32)
- et al.
Genomics
(1995) - et al.
J. Biol. Chem.
(1998) - et al.
Am. J. Pathol.
(1999) - et al.
J. Biol. Chem.
(1999) Blood
(1987)- et al.
J. Biol. Chem.
(1997) - et al.
Antivir. Res.
(1997) - et al.
J. Neuroimmunol.
(2000) - et al.
Neurosci. Lett.
(1997) - et al.
Annu. Rev. Immunol.
(1997)
Proc. Natl. Acad. Sci. USA
Science
Genomics
Nature
J. Exp. Med.
J. Exp. Med.
Cited by (78)
Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies
2020, Cell Chemical BiologyDesign, synthesis, and biological characterization of a new class of symmetrical polyamine-based small molecule CXCR4 antagonists
2020, European Journal of Medicinal ChemistryCXCR4-targeting nanobodies differentially inhibit CXCR4 function and HIV entry
2018, Biochemical PharmacologyCitation Excerpt :Chemokine receptors, including CXCR4, bind different ligand types with a variety of binding modes [37]. In the case of the endogenous chemokine ligand CXCL12, the interaction involves multiple epitopes: the N-terminus and ECLs of CXCR4 interact with the globular core of CXCL12 (Chemokine Recognition Site 1, CRS1) while the N-terminus of CXCL12 interacts within the CXCR4 TM bundle (Chemokine Recognition Site 2, CRS2) [48,49]. CXCR4-targeting small molecules and peptidergic ligands usually bind to an epitope in CRS2 that partially overlaps with the CXCL12 binding site [37].
The chemical diversity and structure-based evolution of non-peptide CXCR4 antagonists with diverse therapeutic potential
2018, European Journal of Medicinal ChemistryDisulfide Trapping for Modeling and Structure Determination of Receptor: Chemokine Complexes
2016, Methods in EnzymologyStructure-based studies of chemokine receptors
2013, Current Opinion in Structural Biology