Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Apr;168(7):1662-74.
doi: 10.1111/bph.12064.

Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes

A O Watts  1 , M M H van Lipzig, W C Jaeger, R M Seeber, M van Zwam, J Vinet, M M C van der Lee, M Siderius, G J R Zaman, H W G M Boddeke, M J Smit, K D G Pfleger, R Leurs, H F Vischer
Affiliations

Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes

A O Watts et al. Br J Pharmacol. 2013 Apr.

Abstract

Background and purpose: The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.

Experimental approach: CXCR3-CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity.

Key results: We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific β-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation.

Conclusions and implications: This study indicates that heteromeric CXCR3-CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.

PubMed Disclaimer

Figures

Figure 1
Figure 1
CXCR3 and CXCR4 form heteromers. HEK293T cells were transfected with HA-CXCR3 and/or FLAG-CXCR4 (500 ng/106 cells). Cells expressing HA-CXCR3 were collected and mixed (1:1) with cells expressing FLAG-CXCR4 (i.e. mix), whereas cells co-expressing HA-CXCR3 and FLAG-CXCR4 were mixed (1:1) with cells transfected with the empty vector pcDEF3 (i.e. co). (A) For co-immunoprecipitation experiments, cells were solubilized and lysates were immunoprecipitated with anti-HA beads, and both lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-HA (top) or anti-FLAG antibodies (bottom). Immunoblots shown are from a representative experiment performed three times. (B) For trFRET analysis, cell surface expressed HA-CXCR3 and FLAG-CXCR4 were labelled with Eu3+-conjugated anti-HA and XL665-conjugated anti-Flag antibodies, respectively, and trFRET was determined by measuring emission at 665 nm 100 μs after excitation of Eu3+ at 337 nm. Specific trFRET between GPCR heteromers is given by the trFRETco/trFRETmix ratio. Pooled data from five independent experiments are shown. ***Cotransfected cells emitted a significantly higher FRET signal in comparison to the mix control (P < 0.0001). WB, Western blot.
Figure 2
Figure 2
CXCR3–CXCR4 heteromers are present on the cell surface. (A) HEK293T cells were cotransfected with CXCR3–CFP and either CXCR3-Venus (left panels), CXCR4-Venus (middle panels), or GABAB2-Venus (right panels). CXCR3–CFP fluorescence images are shown in the upper panels, receptor-Venus fluorescence images are shown in the middle panels, whereas sensitized emission FRET is shown in the bottom panels. (B) Quantification of FRET efficiency from the sensitized emission FRET images.
Figure 3
Figure 3
Hetero- and homomerization of CXCR3 and CXCR4. HEK293T cells were transiently cotransfected with a constant amount (150 ng/106 cells) of CXCR4-Rluc (A and B) or CXCR3-Rluc (C and D) DNA and increasing amounts of CXCR3-EYFP (B and D) or CXCR4-EYFP (A and C) (0–2200 ng/106 cells). Saturation curves were obtained by measuring BRET ratio as function of acceptor/donor ratio (i.e. EYFP/Rluc). Data were obtained from at least three independent experiments each performed in triplicate. Curves were fitted using nonlinear regression, assuming a single binding site.
Figure 4
Figure 4
Evidence for the CXCR3-CXCR4 heteromer recruiting β-arrestin2 using the GPCR-HIT assay. eBRET kinetic profiles (Pfleger et al.,2006a) were generated with live HEK293FT cells co-expressing CXCR3-Rluc8 and β-arrestin2-Venus (A), CXCR4-Rluc8 and β-arrestin2-Venus (B) or CXCR4-Rluc8, β-arrestin2-Venus and CXCR3 (C). These cells were treated with 100 nM CXCL11, CXCL12 or both. Data are mean ± SEM of three independent experiments.
Figure 5
Figure 5
CXCR3 and CXCR4 heteromers display negative ligand binding cooperativity for endogenous and low molecular weight agonists. Membranes for [125I]-CXCL10 and [125I]-CXCL12 binding experiments were prepared from HEK293T cells transfected with 500 ng/106 cells CXCR3 DNA (A), 125 ng/106 cells CXCR4 DNA (C) or cotransfected with CXCR3 and CXCR4 DNA (B and D). Competition binding experiments were performed with approximately 50 pM of [125I]-CXCL10 (A and B) and [125I]-CXCL12 (C and D) and increasing concentrations of the CXCR3 chemokine CXCL10, small CXCR3 agonist VUF10661 and the CXCR4 chemokine CXCL12. Graphs shown are representative of three or more independent experiments performed in triplicate.
Figure 6
Figure 6
Low molecular weight antagonists of CXCR3 and CXCR4 do not have negative binding cooperativity with endogenous agonists. Membranes for [125I]-CXCL10 and [125I]-CXCL12 binding experiments were prepared from HEK293T cells transfected with 500 ng/106 cells CXCR3 DNA (A), 125 ng/106 cells CXCR4 DNA (C) or cotransfected with CXCR3 and CXCR4 DNA (B and D). Competition binding experiments were performed with approximately 50 pM of [125I]-CXCL10 (A and B) and [125I]-CXCL12 (C and D) and increasing concentrations of the CXCR3 chemokine antagonists VUF10085 and TAK-779 and the CXCR4 antagonist AMD3100. Graphs shown are representative of three or more independent experiments performed in triplicate.
Figure 7
Figure 7
Heteromerization of CXCR3 and CXCR4 increases the dissociation rate of CXCL12. [125I]-CXCL12 dissociation half-life was determined in HEK293T membranes expressing CXCR4 (A) alone or (B) in combination with CXCR3, in the absence (asterisk with dotted line) and presence of the CXCR3 endogenous agonist CXCL10 (open circles), the small CXCR3 agonist VUF10661 (open squares), and the CXCR4 chemokine CXCL12 (closed circles). Representative graphs of three or more independent experiments performed in triplicate are shown.
Figure 8
Figure 8
Chemokines display no negative binding cooperativity on intact cells. Binding of approximately 50 pM [125I]-CXCL10 (A) or [125I]-CXCL12 (B), in the absence or presence of 100 nM unlabelled CXCL10 or CXCL12, was measured on intact HEK293T cells transiently transfected with 500 ng CXCR3 DNA/106 cells (open bars), 125 ng CXCR4 DNA/106 cells (hatched bars), or cotransfected with CXCR3 and CXCR4 DNA (closed bars). Graphs show mean ± SEM of two or more independent experiments performed in triplicate.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, Iizawa Y, et al. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A. 1999;96:5698–5703. - PMC - PubMed
    1. Babcock GJ, Farzan M, Sodroski J. Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor. J Biol Chem. 2003;278:3378–3385. - PubMed
    1. Birdsall NJ. Class A GPCR heterodimers: evidence from binding studies. Trends Pharmacol Sci. 2010;31:499–508. - PubMed
    1. Chabre M, Deterre P, Antonny B. The apparent cooperativity of some GPCRs does not necessarily imply dimerization. Trends Pharmacol Sci. 2009;30:182–187. - PubMed
    1. Christopoulos A, Lanzafame A, Ziegler A, Mitchelson F. Kinetic studies of co-operativity at atrial muscarinic M2 receptors with an ‘infinite dilution’ procedure. Biochem Pharmacol. 1997;53:795–800. - PubMed

Publication types