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. 2011 Sep 23;286(38):33466-77.
doi: 10.1074/jbc.M111.233742. Epub 2011 Jul 13.

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Vikas Saini  1 , Daniel M Staren, Joshua J Ziarek, Zayd N Nashaat, Edward M Campbell, Brian F Volkman, Adriano Marchese, Matthias Majetschak
Affiliations

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Vikas Saini et al. J Biol Chem. .

Abstract

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.

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Figures

FIGURE 1.
FIGURE 1.
CXCR4, but not CXCR7 overexpression increases ubiquitin receptor binding. A, HA-tagged open reading frame cDNA clones of CXCR4 and CXCR7 were transfected into HEK293 cells followed by immunoblotting of whole cell lysates with anti-HA and anti-GAPDH. B, quantification of HA expression by flow cytometry after transfection as in A. Thick lines, cells labeled with mouse anti-HA/anti-mouse Alexa Fluor 488 goat IgG. Thin lines, control; cells labeled with rabbit IgG/anti-rabbit FITC goat IgG. Gray, unstained cells. Red, cells transfected with HA-tagged CXCR4. Blue, cells transfected with HA-tagged CXCR7. Black, cells transfected with empty plasmid. C, FITC-ubiquitin binding (1 min, 4 °C) after transfection as in A. ●, CXCR4. □, CXCR7. ○, empty vector. ●, nonspecific binding (NSB)-CXCR4. □, NSB-CXCR7. ●, NSB-empty vector; n = 4. D, quantification of CXCR4 expression by flow cytometry after transfection as in A. Thick lines, cells labeled with anti-human CXCR4 FITC-conjugated IgG. Thin lines, control, cells labeled with FITC-conjugated IgG2A. Gray, unstained cells. Red, cells transfected with HA-tagged CXCR4. Blue, cells transfected with HA-tagged CXCR7. Black, cells transfected with empty plasmid.
FIGURE 2.
FIGURE 2.
Phosphorylation of mitogen-activated protein kinases after stimulation with SDF-1α and ubiquitin. THP-1 cells were stimulated with 0 or 1 μm ubiquitin or SDF-1α for 10 min at 37 °C. Whole cell lysates were probed for protein kinase phosphorylations utilizing a proteome array. A, proteome array membranes showing the spot densities in untreated (ctrl.), ubiquitin-treated and SDF-1α-treated cells. The numbers on the array membrane correspond to the spot positions for phosphorylated ERK1 (1), ERK2 (2), RSK1 (3), Akt1 (4), Akt2 (5), and Akt pan (6). B, densitometric quantification of the spot densities after treatment as in A, n = 4. Spot densities are given as normalized pixel densities (1 = unstimulated cells, dashed line). The bars (white, SDF-1α treatment; gray, ubiquitin treatment) extend from the minimum to the maximum, the horizontal line shows the mean.
FIGURE 3.
FIGURE 3.
CXCR4-induced protein kinase phosphorylation. Western blot analyses of MAPK phosphorylation after stimulation of THP-1 cells with ubiquitin and SDF-1α. A, phospho-ERK1/2. B, phospho-Akt. C, phospho-RSK1. Left panels, cells were pretreated with or without AMD3100 and stimulated with 1 μm ubiquitin or SDF-1α for 10 min at 37 °C. Center panels, time course of MAPK phosphorylations after stimulation of cells with 1 μm ubiquitin or SDF-1α. Right panels, quantification of the chemiluminescence signals after cell stimulation as in B. White bars, SDF-1α stimulation. Gray bars, ubiquitin stimulation, n = 5–10. *, p < 0.05 versus unstimulated cells.
FIGURE 4.
FIGURE 4.
CXCR4-mediated chemotaxis. A, dose-dependent migration of THP-1 cells toward a ubiquitin (●) and SDF-1α (■) gradient; n = 7. *, p < 0.05 versus cells in the presence of PBS in the lower compartment. B, migration of THP-1 cells in the presence or absence of ubiquitin, SDF-1α, or AMD3100 (AMD, 10 μm) in the upper (top) and lower (bottom) compartment, as indicated in the graph (n = 4). Ubiquitin and SDF-1α were used at concentrations (two experiments with 1 nm, two experiments with 10 nm) that showed maximal chemotactic activity, as determined in A. *, p < 0.05 versus cells in the presence of PBS in the upper and lower compartment. #, p < 0.05 versus cells in the presence of PBS in the upper compartment and ubiquitin in the lower compartment. ‡, p < 0.05 versus cells in the presence of PBS in the upper compartment and SDF-1α in the lower compartment.
FIGURE 5.
FIGURE 5.
Ubiquitin functions as a CXCR4 agonist in P4.R5 MAGI cells. A, FITC-ubiquitin binding (1 min, 4 °C). ●, FITC-ubiquitin; □, nonspecific binding. Dashed line, specific binding curve (=total FITC-ubiquitin binding − nonspecific binding); n = 6. B, competition binding (1 min, 4 °C) curve for unlabeled ubiquitin (n = 6, ●) and AMD3100 (n = 5, ●) with 1.16 μm FITC-ubiquitin. FITC-ubiquitin binding is expressed as % of the fluorescence signal measured in the absence of unlabeled ubiquitin (=100%). C, top, ubiquitin (left panels) and SDF-1α (right panels) induced Ca2+ flux. Bottom, cells were pretreated with AMD3100 (10 μm); n = 3. Arrows indicate the time point when ubiquitin or SDF-1α was added (●, 1.16 μm; □, 116 nm; ○, 16 nm; ■, 1.6 nm). D, AMD3100 (10 μm) abolishes ubiquitin and SDF-1α (116 nm) induced reduction of cAMP levels in forskolin-stimulated cells; n = 4. Data are expressed as % of untreated cells (=100%). *, p < 0.05 versus untreated cells.
FIGURE 6.
FIGURE 6.
Ubiquitin does not affect HIV-1 infection. Effects of CXCR4 ligands on X4 tropic HIV-1 R9 (A) and R5 tropic HIV-1 R9BaL (B) infection in P4.R5 MAGI cells (n = 3). One h before infection with 0.5 multiplicity of infection of virus, P4.R5 MAGI cells were treated with the indicated concentrations of CXCR4 ligands. Thirty-six hours postinfection cells were assayed for β-galactosidase expression. Experiments with lower multiplicity of infection (0.25 and 0.125) and X4 and R5 tropic pseudotyped virions showed identical results (not shown). *, p < 0.05 versus cells cultured in the absence of CXCR4 ligands.
FIGURE 7.
FIGURE 7.
The ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain. A, ubiquitin does not bind CXCR4-(1–38). 15N-1H heteronuclear single quantum coherence of 250 μm [U-15N]CXCR4-(1–38) in the absence (black) and presence (gray) of 325 μm SDF-1α (left) or ubiquitin (right); the chemical shift of CXCR4-(1–38) residues change in the presence of SDF-1α, whereas they are unperturbed by ubiquitin. B, combined 1H/15N shift perturbations of SDF1α (top) and ubiquitin (bottom) plotted as a function of the CXCR4-(1–38) residue. Tyr-7 and Thr-8 were not present at the end of titration with SDF-1α due to line broadening. Shift changes for Pro-27 were not measured because it does not contain an amide proton. C, FITC-ubiquitin binding (1.16 μm) to THP-1 cells after labeling of cells with anti-CXCR4-(1–14), anti-CXCR4-(176–293), or anti-IgG. Gray bars (left y axis), RFU from ubiquitin binding assays. Open bars (right y axis), mean RFU from FACS analyses. Ub, ubiquitin, 30 μm. Data are expressed as % of the RFU after incubation with FITC-ubiquitin alone (=100%); n = 3. *, p < 0.05 versus cells incubated with FITC-ubiquitin alone. D, THP-1 cells were coincubated with each of the CXCR4 ligands (116 nm for ubiquitin (light gray bars) and SDF-1α (dark gray bars), 10 μm for AMD3100 (open bars)) and anti-CXCR4-(1–14) or anti-CXCR4-(176–293) at 4 °C. Antibody binding was detected by FACS and mean RFU (% of max) were quantified; n = 3. Data are expressed as % of the RFU after incubation with antibody alone. *, p < 0.05 versus cells after incubation with antibody alone. E, cAMP levels in forskolin (5 μm)-treated THP-1 cells 15 min after ubiquitin or SDF-1α (116 nm) stimulation in the presence or absence of anti-CXCR4-(1–14), anti-CXCR4-(176–293), or anti-IgG, n = 3. Data are expressed as % of untreated cells (=100%). White bars, cells were incubated with antibodies alone. Light gray bars, coincubations with antibodies and ubiquitin. Dark gray bars, coincubations with antibodies and SDF-1α. *, p < 0.05 versus untreated cells.
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