Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28
© The Author(s). 2016
Received: 27 June 2016
Accepted: 2 December 2016
Published: 12 December 2016
Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency.
We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection.
We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.
KeywordsViral G protein-coupled receptor US28 Chemokine receptor CXCR4 Constitutive activity Bioluminescence resonance energy transfer Bioluminescence complementation Signaling crosstalk
Plain English Summary
Some herpesviruses like human cytomegalovirus encode viral G protein-coupled receptors. These membrane receptors facilitate dissemination of the virus and often prevent immune surveillance. As we demonstrate in this work, the G protein-coupled receptor US28 of HCMV is able to severely disturb the signaling and surface expression of the human chemokine receptor CXCR4 and thus limit the immune response.
Certain herpesviruses like human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are known to encode viral G protein-coupled receptors (vGPCRs) [1, 2]. These vGPCRs had most probably been hijacked from the human genome as they resemble human GPCRs in structure and function. HCMV-encoded vGPCRs have previously been shown to interact with the signaling machinery of the host cell in a remarkably efficient manner . This reprogramming of cell signaling by vGPCRs is often aimed at facilitating dissemination of the virus, preventing immune surveillance and establishing life-long latency . HCMV encodes four vGPCRs, US27, US28, UL33 and UL78, among which US28 is the best-characterized. US28 plays a crucial role in the viral life cycle by promoting viral spread  and by activating the immediate early HCMV promoter , which is necessary for the transactivation of other viral genes. US28, which is constitutively active, can also bind to a wide range of chemokines , possibly acting as a “chemokine sink” to reduce immune responses at the site of inflammation . Alternatively, the constitutive or chemokine-induced signaling activities of US28 may modulate intracellular signaling pathways consequently promoting virus replication. In addition, US28 was reported to act as a HIV coreceptor in certain cell types  and has been associated with pathogenic processes leading to atherosclerosis .
Host responses to viral infections involve complex interactions between chemokines and other cytokines that provide key communication signals resulting in the effective development of innate and adaptive immunity. Thus, innate immune responses are critical in limiting viral spread and averting virus-induced disease. The human chemokine receptor CXCR4 is a promising target for manipulation by vGPCRs as it is expressed on cells, which are infected by HCMV or display reservoirs of latency . CXCR4 is specific for stromal cell-derived factor-1α (SDF-1α or CXCL12) and is highly expressed on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow niche as well as on differentiated circulating blood cells . It serves as a coreceptor for the cell entry of HIV , highly contributes to trafficking and homeostasis of human immune cells, stem cell homing in tissue regeneration , but also tumorigenesis and progression of various types of cancer [14–16]. CXCR4 is prone to function in various homo- and heteromeric complexes to deploy its differential effects as revealed by various crystal structures and additional methods [17–19]. Importantly, CXCR4 has been associated with vGPCR-mediated manipulation of the chemokine receptor homeostasis. The Epstein-Barr virus - encoded vGPCR BILF1 was found to attenuate CXCL12-induced CXCR4 signaling by scavenging Gαi-proteins and impairing CXCL12 binding to CXCR4. Interestingly, the G protein-coupling deficient mutant BILF1-K3.50A affected CXCL12 - induced signaling less effectively, indicating that BILF1 - mediated CXCR4 inhibition is a consequence of its constitutive activity . Additionally, it was reported that the HCMV - encoded vGPCRs UL33 and UL78 modulate CXCR4 signaling, surface expression as well as its HIV coreceptor activity . In these reports, the observed manipulations of CXCR4 signaling and surface expression were mainly attributed to a direct physical contact or heteromerization of CXCR4 with the viral GPCRs BILF1, UL33 and UL78.
As GPCRs can physically affect each other’s signaling by forming heteromeric complexes , we thoroughly investigated the possibility of physical interaction or heteromerization of the vGPCR US28 with the human chemokine receptor CXCR4. Indeed, US28 seems to employ a subtler but nevertheless very effective way to influence CXCR4 signaling. Our data support the assumption that the observed attenuation of the CXCR4 surface expression and signaling in the presence of US28 is partly attributed to the high constitutive activity of US28. We believe that the G protein-dependent constitutive signaling of US28 leads to indirect signaling crosstalk via shared intracellular signaling networks, which results in disturbed chemokine receptor signaling and reduced surface expression.
US28 abates chemokine-induced G protein-mediated signaling of CXCR4
Influence of US28 coexpression on efficacy and potency of CXCL12-induced cAMP concentrations
Emax (mean ± SEM)
pEC50 (mean ± SEM)
CXCR4 + mock (control)
100 ± 4
9.85 ± 0.09
CXCR4 + US28
32 ± 11 (***)
9.54 ± 0.91 (n.s.)
CXCR4 + US28Δ300
35 ± 18 (***)
8.96 ± 1.10 (n.s.)
CXCR4 + US28DQY
73 ± 7 (n.s.)
10.02 ± 0.22 (n.s.)
CXCR4 + US28Δ300/DQY
76 ± 8 (n.s.)
10.34 ± 0.26 (n.s.)
US28 affects interactions between Gi proteins and CXCR4
We also determined the effect of US28 expression on the G protein-dependent signaling of CXCR4 as early as on the level of Gαi1 protein recruitment. Therefore, we again used a BRET-based method. Gαi1-91mVenus and unlabeled Gβ1 and Gγ2 were coexpressed with CXCR4 carrying a Renilla reniformis luciferase 8 (Rluc8) at its C-terminus . The agonist-induced recruitment of the Gαi1 subunit to the chemokine receptors was monitored in presence and absence of US28wt or US28 mutants. In CXCR4-expressing HEK293T cells, stimulation with 100 nM CXCL12 resulted in a significant increase in ligand-promoted BRET signal (ΔBRET). In US28wt or US28Δ300 coexpressing cells Gαi1 recruitment to CXCR4 was significantly reduced in efficacy, whereas the coexpression of US28DQY or US28Δ300/DQY did not significantly suppress the agonist-induced Gαi1 protein recruitment to CXCR4 (Fig. 1b). As evident from Additional file 1: Figure S1b, the maximal Gαi1 recruitment to CXCR4 in presence of US28 does not increase with an increasing pool of Gαi1 proteins, indicating that the recruitment of Gαi1 proteins is not influenced by a limited pool of Gαi1 proteins.
US28 restrains surface expression and CXCL12-mediated β-arrestin 2 recruitment to CXCR4
Radioligand-displacement studies to detect changes in CXCR4 and US28 surface expression
% of max. bound radioligand
(mean ± SEM)
(mean ± SEM)
CXCR4 + mock (control)
100 ± 2
8.13 ± 0.07
CXCR4 + US28wt
74 ± 5 (***)
8.27 ± 0.26 (n.s.)
US28wt + mock (control)
99 ± 3
8.82 ± 0.08
US28wt + CXCR4
70 ± 10 (*)
8.71 ± 0.43 (n.s.)
To validate our data obtained in the BRET-based β-arrestin 2 recruitment assay, we employed a bioluminescence complementation (BiLC)-based system developed by Ozawa et al. [37, 38]. They optimized the complementation of split luciferase fragments from click beetle (Brazilian pyrearinus termitilluminans) to provide a BiLC-system with high sensitivity and low signal-to-noise ratio. As shown in Fig. 3b, the data obtained from BiLC-based β-arrestin 2 recruitment are comparable to the data from the BRET-based approach. Additionally, we verified that the attenuation of β-arrestin 2 recruitment to CXCR4 in the presence of US28wt is not caused by a limited pool of β-arrestin 2 available for the interaction. As shown in Additional file 3: Figure S3, the reduction of β-arrestin 2 recruitment to CXCR4 in presence of US28wt is not influenced by an increasing pool of β-arrestin 2.
CXCR4 does not heteromerize with US28
Among GPCR researchers there is an ongoing debate about the existence and importance of GPCR dimers in vivo. However, several reports showed the organization of class A GPCRs in homodimeric, oligomeric or even heteromeric complexes [21, 39–41]. Methods such as pull-down assays, protein crystallography, BRET, fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP) and single molecule imaging provide tools to track GPCR dimerization in living cells [42–45]. Also bitopic ligands are important tools to analyze and manipulate receptor dimerization .
BRETmax and BRET50 values (mean ± SEM) from BRET donor saturation curves to detect receptor dimerization
(mean ± SEM)
(mean ± SEM)
2.75 ± 0.042
0.11 ± 0.007
0.54 ± 0.031
0.24 ± 0.036
0.53 ± 0.023
0.33 ± 0.031
Overall, BiLC and BRET results demonstrated that US28 most likely does not form heterodimers with CXCR4. Considering that also images from confocal laser scanning microscopy showed that CXCR4 and US28 only weakly colocalize and that this colocalization is restricted to intracellular vesicles, we showed that US28 does not influence CXCR4 signaling by heteromerization. The localization of US28 in endosomes implies that the effect of US28 on CXCR4 is ligand-independent.
US28 is involved in downregulation of CXCR4 in HCMV-infected HUVEC
In this study we show that expression of the cytomegaloviral chemokine receptor US28 leads to downregulation of CXCR4 surface expression and agonist-induced signaling in HEK293T cells. These findings are in accordance with the observation that in primary HUVEC, infected with the endotheliotropic TB40E strain of HCMV, CXCR4 is significantly downregulated from the surface of infected cells. In contrast, using a TB40E strain lacking the US28 gene we detected that CXCR4 downregulation is strongly impaired, in particular at late times after infection indicating an important role of US28 for CXCR4 modulation during the course of HCMV infection.
We assessed the consequences of US28 expression for the responsiveness of CXCR4 and could narrow down the underlying mechanism to structural motifs of US28. Our data demonstrate that the presence of US28 in CXCR4-expressing cells leads to a dampening of CXCL12-induced Gαi protein-dependent signaling. This inhibition could be observed as early as on the level of Gαi protein recruitment as well as on the level of the secondary messenger cAMP. Additionally, US28 seems to antagonize the ligand-induced Gαi1 recruitment of CXCR4 by its constitutive G protein activation. The G protein-uncoupled mutants US28DQY and US28Δ300/DQY had no significant effect on agonist-induced second messenger formation and Gαi protein recruitment of CXCR4, clearly showing that mainly the DRY motif, enabling the high constitutive activity of US28, is responsible for dampening of the G protein-dependent signaling of CXCR4, while the C-terminal domain does not seem to play a role.
The constitutive signaling activity of US28 attracted attention before and is suspected to represent one of the mechanisms employed by vGPCRs to disturb the host immune homeostasis [28, 51]. US28 was reported to constitutively activate phospholipase C-β (PLC-β) and NFκB via Gq/11-dependent pathways [23, 52]. Infection with US28R129A mutant virus failed to induce PLC-β signaling, which also shows the clinical relevance of constitutive G protein activation by US28 . Moreover, US28-mediated constitutive G protein activation is also involved in tumor formation and progression . The C-terminally truncated forms of US28, lacking important serine and threonine residues, are expressed to a higher degree on the cell surface than US28wt and therefore show even higher G protein-dependent constitutive activity than wild type US28 . This also explains the effect of the US28Δ300 mutant on Gαi protein-dependent signaling of CXCR4 being comparable to US28wt.
Interestingly, in presence of US28wt, US28Δ300 and US28DQY the CXCL12-induced β-arrestin 2 recruitment to CXCR4 was abrogated. Only the concomitant expression of US28Δ300/DQY led to partial recovery of the initial agonist-induced β-arrestin 2 recruitment to CXCR4. This indicates that the constitutive G protein activation as well as the C-terminal phosphorylation sites of US28 are involved in US28-promoted abrogation of β-arrestin 2 recruitment to CXCR4, but are not the main determinants. Recently, it was shown that activation of ERK1/2 leads to a β-arrestin 2-dependent reduction of constitutive GPCR cell surface expression and consequently blunted G protein and β-arrestin signaling . US28 was reported to activate ERK1/2 in an agonist-dependent manner, engaging the G proteins Gαi1 and Gα16, in response to RANTES/CCL5 . As described before, also an agonist-independent downstream activation of ERK1/2 can be observed in US28-expressing HEK293T cells . With use of the MEK1/2 inhibitor PD184352 and the ERK1/2 inhibitor FR180204 we intended to suppress US28-mediated ERK1/2 activation and thus reduction of CXCR4 surface expression. However, this experiment did not reveal significant changes in steady-state surface expression levels (data not shown). Consequently, in case of US28, the weak constitutive activation of ERK1/2 via the Gq/11 pathway does not act as the main determinant leading to the radical dampening of CXCR4 signaling. US28 itself was shown to employ multiple routes for internalization including dynamin-dependent pathways. However, US28 trafficking is not dependent on β-arrestin, as in β-arrestin deficient cells endocytosis and subcellular localization of US28 was unaltered . Nevertheless, the presence of US28 in HEK293T cells was shown to cause a redistribution of β-arrestin 2 from the plasma membrane to intracellular vesicles in absence of ligand stimulation . In contrast to the G protein-uncoupled mutant US28R129A, a GRK phosphorylation site-deficient mutant of US28, US28S1-12A , showed the same effect on subcellular β-arrestin 2 localization. This indicates that the DRY motif, conserved in TM3 of US28 and responsible for constitutive G protein activation, is sufficient to cause a redistribution of β-arrestin 2 to intracellular vesicles, which reduces its availability to interact with other receptors. Still, the signaling-deficient US28DQY was not sufficient to prevent abrogation of agonist-induced β-arrestin 2 recruitment to CXCR4 in our hands. Therefore, the mechanism of US28-mediated abrogation of agonist-induced β-arrestin 2 recruitment to CXCR4 remains to be unraveled.
As heteromerization is one of the mechanisms that enables receptors to influence and disturb each other’s signaling we also thoroughly investigated the possibility of CXCR4/US28 heteromerization. However, our data indicate that the observed dampening of CXCR4 responsiveness by US28 cannot be explained by receptor heteromerization. Data from BiLC and BRET saturation experiments suggest a weak, most probably non-significant interaction between CXCR4 and US28. Furthermore, qualitative analyses of images from colocalization studies using confocal laser scanning microscopy show that CXCR4 does not colocalize with US28 on the cell surface and intracellular colocalization was confined to single vesicles.
We hypothesize that the attenuation of G protein- and β-arrestin 2-dependent signaling of CXCR4 is related to a reduced density of CXCR4 at the cell surface as we found the surface expression of CXCR4 to be downregulated for up to 50% in the presence of US28wt in HEK293T cells. We observed that coexpression of the signaling-impaired mutants US28Δ300 and US28DQY still significantly reduced CXCR4 surface expression. Only when the double mutant US28Δ300/DQY was coexpressed, CXCR4 surface expression was restored. This indicates that only when the constitutive G protein signaling and the recycling-machinery of US28 are impaired at the same time CXCR4 surface expression is not attenuated. However, we could also show that not only in HCMV strain TB40E-infected monocytes , but also in infected HUVEC CXCR4 surface expression is significantly attenuated, which underlines the relevance of our study. We observed that TB40E/IE2eYFP- and TB40E/IE2eYFP-delUS28-infected HUVEC show strong downregulation of CXCR4 surface expression at 24 hpi. At late time points of infection (96 hpi) CXCR4 surface expression was significantly downregulated in TB40E/IE2eYFP-infected HUVEC, whereas in TB40E/IE2eYFP-delUS28-infected HUVEC, CXCR4 surface expression was restored to mock level. This indicates that US28 a critical factor involved in attenuation of CXCR4 surface expression in particular at late time points of infection, which also correlates with the late expression kinetic of US28 . The observed downregulation of CXCR4 at early time points of infection is most probably attributed to other factors. However, downregulation of chemokine receptors in infected monocytes eventually impaired immune response to viral infection as shown by Frascaroli et al. . HCMV - infected monocytes failed to recruit lymphocytes, monocytes and neutrophils as a result of downregulated CCR1, CCR2, CCR5 and CXCR4 levels at the cell surface. Endothelial cells (EC) are described to play a role in the dissemination of HCMV throughout the body . Interestingly, during acute disease EC can detach from the blood vessel and enter the blood stream . In contrast to detection of HCMV infected EC during acute infection in immunocompromised patients, their role during latency is controversial. There are reports demonstrating HCMV DNA in vessel walls of major arteries of sero-positive individuals , whereas others classify EC as unlikely sites of HCMV latency in vivo . Downregulation of chemokine receptors from the surface of EC might facilitate detachment of EC from the blood vessel and entry into the blood stream, thereby facilitating viral dissemination. In accordance with reports about US28 being directly involved in facilitation of viral spread [60, 65], we propose that US28 might also indirectly promote viral dissemination by downregulation of chemokine receptors from the surface of infected cells.
In summary, our data support the assumption that the observed attenuation of CXCR4 surface expression and signaling in presence of US28 is mainly caused by the high constitutive activity of US28. By use of well-characterized mutants of US28, we could attribute the reduction of G protein-dependent signaling and surface expression of CXCR4 to an activity relying partially on the DRY motif and C-terminus of US28. We propose that the constitutive signaling of US28 leads to indirect signaling crosstalk via shared intracellular signaling networks. This eventually results in disturbed chemokine receptor signaling and reduced constitutive surface expression, which is also reflected in HCMV-infected primary HUVEC.
Cell culture and transfection
Human Embryonic Kidney 293 (HEK293) T cells were cultured in DMEM/F-12 supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1% penicillin-streptomycin, 2 mM L-glutamine and incubated at 37 °C/5% CO2. Transient transfections were performed using linear polyethylenimine 25 kDa (PEI) (Polysciences, Inc.) or TransIT-293 transfection reagent (Mirus corporation) as transfection reagent at a transfection reagent/DNA ratio of 3:1. COS-7 cells were grown in Dulbecco’s modified Eagle’s medium 1885 supplemented with 10% FBS, 2 mM glutamine, 180 units/ml penicillin and 45 μg/ml streptomycin at 37 °C/10% CO2. Primary human foreskin fibroblasts (HFFs) were prepared from human foreskin tissue  and cultured in Eagle’s minimal essential medium supplemented with 7.5% FBS, 1% L-glutamine and gentamicin at 37 °C/5% CO2. Primary HUVEC (a kind gift from M. Mach, Erlangen, Germany) were isolated from single blood veins from human umbilical cord tissue and cultured in Endothelial Growth Medium supplemented with 5% FBS, hydrocortisone, human Fibroblast Growth Factor B (hFGF-B), Vascular Endothelial Growth Factor (VEGF), human insulin-like growth-factor-I (R3-IGF-1), ascorbic acid, human epidermal growth factor (hEGF) and GA-1000 (Gentamicin, Amphotericin B) at 37 °C/5% CO2.
Infection experiments were performed with the recombinant viruses TB40E/IE2-eYFP  and TB40E/IE2eYFP-delUS28. Titration of the viral stocks was performed by IE1p72 fluorescence . Briefly, HFFs (8 × 104 cells) in 0.5 ml medium were seeded into 24-well plates and infected the next day with 300 μl of various dilutions (1:5 to 1:55) of viral supernatant. At 2 hpi 500 μl of fresh culture medium were added. At 36 hpi, cells were fixed with 4% PFA and stained with monoclonal antibody p63-27, which is directed against IE1p72 . Subsequently, the number of IE1-positive cells was determined in duplicate wells and was used to calculate viral titers in IE1 protein-forming units (IE1U) per ml. For infection, 2 × 105 HUVEC, between passage two and seven, were seeded per well in 6-well plates. The day after, culture medium was replaced by 2 ml of infectious cell culture supernatant of TB40E/IE2-eYFP or TB40E/IE2-eYFP-delUS28 and the plates were centrifuged for 30 min at 1900 × g. After 3 h of incubation, the supernatant was substituted with fresh culture medium.
Generation of the recombinant virus TB40E/IE2eYFP-delUS28
For generation of the recombinant virus TB40E/IE2eYFP-delUS28 the coding region of US28 was removed from the already described HCMV TB40E/IE2eYFP  by BAC (bacterial artificial chromosome) mutagenesis according to Datsenko & Wanner . E.coli strain DH10B, which had beforehand been transformed with TB40E/IE2eYFP BAC DNA and pKD46 (Red recombinase expression plasmid with a temperature sensitive, L-arabinose inducible promoter) , were grown in LB medium supplemented with chloramphenicol, ampicillin and 0.2% L-arabinose at 30 °C. In order to accomplish homologous recombination E. coli cells were transformed with PCR fragments, generated by amplification of an FRT-kanamycin-FRT cassette from plasmid pKD13  using primers that are homologous to the adjacent regions of the US28 gene. DpnI was added to digest template DNA and the amplicon was purified from an agarose gel. Positive transformants were identified using agar plates containing chloramphenicol and kanamycin at 37 °C and additionally checked for the clearance of the Red recombinase plasmid pKD46 by use of agar plates containing ampicillin. Subsequently, chloramphenicol/kanamycin-resistant, but ampicillin-sensitive clones were transformed with pcP20 in order to enable elimination of the kanamycin cassette. pcP20 encodes for a FLP recombinase expression plasmid, which is chloramphenicol/ampicillin-resistant and shows temperature-sensitive replication and thermal induction of FLP recombinase expression . Chloramphenicol/ampicillin-resistant mutants were selected at 30 °C and then purified for pCP20 at 43 °C. Finally, chloramphenicol-resistant but ampicillin/kanamycin-sensitive transformants were selected at 37 °C. BAC DNA was isolated from bacteria and the obtained BACs were verified by distinct PCR reactions and subsequent sequencing as well as restriction fragment length polymorphism analysis (RFLP) as described previously . In order to reconstitute infectious particles, HFFs were transfected with the obtained BAC DNA using X-tremeGENE transfection reagent (Roche, Mannheim, Germany). Cells were incubated until the appearance of distinct cytopathic changes. Cell culture supernatant containing infectious particles was harvested, centrifuged to remove cellular debris and stored at −80 °C until use.
Fluorescence-Activated Cell Sorting (FACS) analysis
For FACS analysis of TB40E/IE2eYFP or TB40E/IE2eYFP-delUS28 infected cells HUVECs were harvested at indicated time points post-infection using Accutase Solution for 5–10 min at 37 °C. Cells were washed once with PBS, followed by FBS-containing buffer (2% FBS and 2 mM EDTA in PBS). Next, cells were stained with anti-CXCR4-APC or anti-IgG2ab-APC antibodies in FBS-containing buffer for 1 h at 4 °C. Finally, cells were washed with FBS-containing buffer and fixed with 2% PFA. Samples were analyzed with the BD LSR II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the results were evaluated with FCS Express V3 (De Novo Software, Los Angeles, CA, USA).
The cDNA encoding hCXCR4 was purchased from the UMR cDNA Resource Center (University of Missouri-Rolla, USA). The cDNA encoding US28wt and US27wt receptor from TB40E strain of HCMV were used. BiLC: Rluc8 plasmids: Rluc8 cDNA was provided by Jonathan A. Javitch, Columbia University, USA. The used plasmids were designed in accordance to the previously described D2sR constructs . The cDNAs encoding full-length Rluc8 or fragments for the Rluc8N (residues 1–229) or Rluc8C (residues 230–311) were fused to the C-terminus of the respective receptors by a 24 aa linker in pcDNA5/FRT (Invitrogen). Emerald luciferase (Eluc) and firefly luciferase (Fluc) split plasmids: The used plasmids were designed in accordance to the described plasmids . Fragments of Eluc, ElucN (residues 1–415) or ElucC (residues 394–542), were C-terminally linked to the respective receptors by a 20 aa linker sequence (4 × SGGGG). Fragments of Fluc, FlucN (residues 1–416) and FlucC (residues 416–550), were C-terminally linked to the respective receptors by a 4 aa linker (SGGG). PCR products were subcloned into pcDNA3.1(+) or pcDNA4/V5-His(B). BRET sensors: CXCR4 was C-terminally fused to the YFP derivative mVenus. G α i1-91mVenus was a gift from Jonathan A. Javitch, Columbia University, USA. The Gβ1 and Gγ2 subunits as well as RlucII-β-arrestin 2 were kindly provided by Michel Bouvier, University of Montreal, Canada. The CAMYEL biosensor was purchased from ATCC, USA. ELISA: CXCR4 cDNA was tagged by N-terminally inserting a FLAG-tag (DYKDDDAAAD) immediately before the start codon and cloned in pcDNA3.1. The truncated version of US28wt, US28Δ300, was constructed by inserting a STOP-codon after residue Gln-300. The DRY-lock mutant of US28, US28DQY, was constructed by mutating the Arg in position 129 of the DRY-motif to Gln as previously described [30, 31]. The double mutant US28Δ300/DQY was constructed by inserting a STOP-codon after residue Gln-300 of the US28DQY mutant. US28wt, US28Δ300, US28DQY, US28Δ300/DQY and NTS1 were N-terminally fused to an HA-tag (YPYDVPDYA) in pcDNA3.1(+). The identity of all plasmids was confirmed by sequencing (LGC Genomics).
Reagents, antibodies and radioligands
CXCL12 was purchased from PeproTech. Anti-HA, anti-FLAG antibody and secondary peroxidase-conjugated anti-IgG antibody for ELISA as well as Forskolin and Accutase Solution were purchased from Sigma-Aldrich. The anti-human CD184(CXCR4)-APC (clone 12G5) as well as the isotype control anti-mouse IgG2ab-APC were purchased from Miltenyi Biotec. Coelenterazin-h as well as BrightGlo substrate were purchased from Promega. Cell culture reagents for HEK293T, HFF and COS-7 cells were purchased from Gibco/Thermo Fisher Scientific. Medium growth factors for culturing HUVEC was purchased from Lonza. The radiolabelled tracer of CX3CL1 was made by applying the oxidative iodination technique to CX3CL1, which incorporates 125I at the meta-position of tyrosine residue side chains, and the tracer was characterized and purified by RP-HPLC . The 12G5 tracer was instead produced by Bolton-Hunter labelling, which incorporates 125I at the amino terminus of the protein.
BiLC to assess receptor dimerization
HEK293T cells were transiently transfected in 96-well plates with a pair of receptors fused to the C-terminal (Cluc) and N-terminal (Nluc) split of Rluc8, Fluc or Eluc, respectively using TransIT-293 transfection reagent, while the DNA ratio of receptor-Cluc:receptor-Nluc was 1:1. Luminescence was measured 24 h after transfection using the microplate reader Clariostar (BMG Labtech, no emission filter, 2 s recording), following the addition of 100 μl BrightGlo Substrate (Promega) and 5 min incubation at RT.
BiLC to assess β-arrestin 2 recruitment
HEK293T cells were transiently transfected with ElucN-β-arrestin 2, CXCR4-ElucC and US28, US28 mutants or empty vector (mock) using PEI, while the DNA ratio was 2:1:1. At 48 hours post transfection (hpt), culture medium was replaced by HBSS supplemented with 0.1% BSA. After 30 min incubation at 37 °C/5% CO2, cells were stimulated with 100 nM CXCL12 or vehicle (HBSS-0.1%BSA). At 10 min post ligand addition, luminescence was measured using the microplate reader Clariostar (BMG Labtech, no emission filter, 2 s recording), following the addition of 100 μl BrightGlo Substrate and 5 min incubation.
Bioluminescence resonance energy transfer measurements
In this study, BRET480-YFP also termed BRET1 was used for all the following described BRET-based assays. For BRET1, one of the proteins is fused to Rluc or brighter forms of Rluc (RlucII/Rluc8) and the other protein is fused to mVenus. Rluc and mVenus serve as energy donors and acceptors, respectively. We used Coelenterazin-h (Promega) as a substrate for the luciferase, which generates light with a maximal emission peak at 480 nm. The emission spectrum of Rluc overlaps with the excitation spectrum of mVenus, which leads to energy transfer and excitation of mVenus, if the two proteins are about less than 10 nm apart from each other. For use of Rluc8 and mVenus a Förster distance (R 0) of 5.55 nm is described . R 0 describes the intermolecular separation of donor and acceptor which allows 50% of the maximal energy transfer. BRET values were collected 5 min after addition of Coelenterazin-h at a final concentration of 5 μM with the microplate reader ClarioStar (BMG Labtech) equipped with the BRET480-YFP filter set (475 ± 30 nm and 535 ± 30 nm). BRET ratio was determined as the ratio of the emitted light by acceptor (filter: filter: 535 ± 30 nm) over donor (475 ± 30 nm).
BRET titration curves to assess receptor dimerization
For BRET titration experiments a constant amount of the receptor-Rluc8 plasmid (energy donor) was cotransfected with increasing amounts of the receptor-mVenus plasmid (energy acceptor) using PEI. At 2 d post transfection, culture medium was replaced by HBSS complemented with 0.1% BSA and cells were incubated for 30 min at 37 °C/5% CO2 before measurement of BRET. To determine the specific BRET signal (net BRET), the BRET signal detected in cells expressing the energy donor only was subtracted from the BRET signal obtained from cells expressing the acceptor and donor. The net BRET values were plotted as a function of the expression level of the acceptor over the expression of the donor for each individual transfection. The expression level of the acceptor was determined by measuring mVenus fluorescence (ex: 497 ± 15 nm, em: 535 ± 30 nm) and the expression level of the donor was determined as emitted light by the donor (filter 475 ± 30 nm).
BRET-based measurements of Gi protein activation
HEK293T cells were cotransfected with a beforehand optimized DNA ratio of CXCR4-Rluc8, Gαi1-mVenus, Gβ1, Gγ2 and US28wt, US28 mutants or empty vector (mock), whereas the DNA-ratio of CXCR4-Rluc8 to US28, US28 mutant or empty vector was 1:1. Cells were transfected using PEI and seeded in 96-well plates at a density of 25,000 cells per well and incubated for 48 h. For the assay, culture medium was replaced by HBSS complemented with 0.1% BSA and cells were incubated for 30 min at 37 °C/5% CO2. Cells were treated with 100nM CXCL12 or vehicle (HBSS-0.1% BSA) and BRET was measured 2 min later. To determine the ligand-promoted BRET signal (ΔBRET), BRET signal detected in vehicle-treated cells was subtracted from BRET signal detected in stimulated cells for each transfection.
BRET-based measurements of β-arrestin 2 recruitment
HEK293T cells were cotransfected with RlucII-β-arrestin 2, CXCR4-Rluc8 and US28, US28 mutants or empty vector (mock) whereas the CXCR4:US28 DNA ratio was 1:1. Cells were transfected using PEI and seeded in 96-well plates at a density of 25,000 cells per well and incubated for 48 h. For the assay, culture medium was replaced by HBSS complemented with 0.1% BSA and cells were incubated for 30 min at 37 °C/5% CO2. BRET was measured 5 min after stimulation with endogenous ligands. To determine the ligand-promoted BRET signal (ΔBRET), BRET signal detected in vehicle-treated cells was subtracted from BRET signal detected in stimulated cells for each transfection.
BRET-based cAMP assay (CAMYEL-sensor)
HEK293T cells were cotransfected with chemokine receptor cDNA and US28wt, US28Δ300 or US28DQY and CAMYEL biosensor at a DNA ratio of 1:1:2 using PEI and seeded into half-area 96-well plates at a density of 1,5 × 104 cells per well. At 48 h after transfection, the culture medium was removed and replaced by HBSS complemented with 0.1% BSA and incubated for 30 min at 37 °C/5%CO2. BRET values were collected 15 min after simultaneous treatment with indicated concentrations of CXCL12 and a final concentration of 10 μM Forskolin.
Confocal laser scanning microscopy
The day before transfection, HEK293T cells were seeded in 6-well plates at a density of 2 × 105 cells/well. Cells were transfected with C-terminally eGFP-tagged CXCR4 and C-terminally mCherry-tagged US28 or empty vector (mock) using TransIT293(MIRUS). Cells were transferred to Poly-L-Lysine coated glass coverslips 24 h after transfection. At 2 days after transfection, cells were washed with PBS and fixed with 4% paraformaldehyde (RotiHistofix,Carl Roth) for 10 min. After washing three times with PBS, the glass coverslips were mounted on microscope slides using Dako Fluorescent Mounting Medium and investigated using a Leica SP5II confocal microscope (Software LAS AF v188.8.131.5223) equipped with Leica hybrid detectors. Excitation energy and gain were set to the same level to make all data set-ups comparable in intensity. Microscopy/Image analysis was performed with support from the Optical Imaging Center Erlangen (OICE). Post image processing (adjusting brightness and contrast) was performed for a better visualization.
Enzyme linked immunosorbent assay (ELISA)
HEK293T cells were transiently cotransfected with Flag-tagged chemokine receptors and HA-tagged US28 wildtype or mutants or empty vector (mock) at a DNA ratio of 1:1. 24 h after transfection, cells were seeded in Poly-D-Lysine-coated 48-well plates. At 48 hours post transfection the cells were fixed with 4% PFA for 10 min at RT. Cells were permeabilized or not for 5 min in PBS/0.1% TritonX-100 at RT. In separate wells, cells were stained with monoclonal anti-Flag or anti-HA antibody produced in mouse followed by an anti-mouse, IgG-peroxidase conjugated antibody. Absorbance at 492 nm was measured 10 min after incubation in substrate buffer containing 6 mM o-phenylenediamine using the microplate reader ClarioStar (BMG LabTech).
Data and statistical analysis
All graphs were generated and analyzed using PRISM 6.0 (GraphPad Software, San Diego, CA). Curves were fitted using least square nonlinear regressions assuming a one site hyperbola where Kd corresponds to BRET50 and Bmax corresponds to BRETmax or linear regression (BRET saturation experiments) or sigmoidal fit (dose-response curves), in which the logIC50 and Hill coefficient were free parameters. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test if more than two values were compared with the control or Student’s t test if two values were compared.
Radioligand competition binding assay
Two days before the assays, the calcium phosphate precipitation method was used to transiently transfect cells with pcDNA3.1(+) vectors expressing either CXCR4 or US28, and on the next day, the transfected cells were seeded to 96-well plates. For the competition binding assays, the cells were washed in HEPES buffer (50 mM) supplemented with BSA (5 g/l) and chilled at 5 °C. Unlabeled ligands were added to the cells 5 min before adding the tracer, which was administered at levels leading to 10% tracer binding. Following an incubation period of 3 h at 4 °C, the cells were washed in HEPES buffer with BSA (5 g/l) and NaCl (29.22 g/l) to remove any unbound tracer, and gamma radiation of the remaining tracer was measured.
Bacterial artificial chromosome
Bioluminescence resonance energy transfer
Epstein Barr virus
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Fetal bovine serum
Fluorescence recovery after photobleaching
Fluorescence resonance energy transfer
Human immunodeficiency virus
Hematopoietic stem and progenitor cells
Human umbilical vein endothelial cells
- IC50 :
half maximal inhibitory concentration
Kaposi's sarcoma-associated herpesvirus
Monocyte chemotactic protein-1
multiplicity of infection
Neurotensin receptor type 1
Regulated on activation, normal T-cell expressed and secreted
Renilla reniformis luciferase 8
Stromal cell-derived factor-1α
viral G protein-coupled receptors
We thank Michel Bouvier (Université de Montréal, Canada) for providing us with the RlucII-β-arrestin 2 and Gβ1 and Gγ2 construct, Jonathan A. Javich (Columbia University, USA) for G protein-mVenus and -luciferase constructs, Michael Mach (Institute for Clinical and Molecular Virology, Erlangen, Germany) for providing us with primary HUVEC and Nevin A. Lambert (Augusta University, USA) for inspiring discussions about dimerization of GPCRs. N.T., T.F. and T.S. were financially supported by Collaborative Research Center 796 (SFB796) of German Research Foundation. N.T. participates in the European COST Action CM1207 (GLISTEN: GPCRLigand Interactions, Structures, and Transmembrane Signaling: a European Research Network).
This research was financially supported by Collaborative Research Center 796 (SFB796) of German Research Foundation (N.T., Project A6).
TF, AR, OL and ACS planned and conducted the experiments. TF, OL, MMR, TS, TO and NT made substantial contributions to conception and design, and analysis and interpretation of the data. All authors participated in drafting of the article. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethical approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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