Article Figures & Data
Figures
- Fig. 1.
Principle and functional characterization of the CXCR4-FlAsH228-CFP sensor. (A) Schematic depicting the intramolecular FRET-based sensor. Ligand-induced conformational changes in CXCR4 are monitored as changes in FRET. (B) Representative confocal images of HEK293 cells transiently expressing CXCR4-FlAsH228-CFP and FlAsH-labeled prior to the measurement. Upper panel shows CFP emission. Lower panel shows FlAsH emission. Scale bar, 10 µm. (C) Intramolecular FRET efficiency of CXCR4-FlAsH228-CFP as determined by BAL treatment. Values were calculated from the increase in the CFP fluorescence upon BAL addition. Data shows mean ± S.D. of 20 cells measured on 4 independent experimental days. A representative individual experiment is shown in Supplemental Fig. 2. (D) Gi1 activation via CXCR4 or CXCR4-FlAsH228 in response to increasing concentrations of CXCL12. Data show mean ± S.E.M. and are representative of n = 3 independent experiments conducted in quadruplicate. In this particular experiment, EC50 = 3.3 and 14.7 nM for CXCR4 and CXCR4-FlAsH228, respectively. (E) Inhibition of FSK-induced cAMP accumulation in response to increasing concentrations of CXCL12 by HEK293T cells expressing CXCR4, CXCR4-CFP, or CXCR4-FlAsH228-CFP. Data show mean ± S.E.M. and are representative of n = 4 independent experiments conducted in triplicate. In this particular experiment, EC50 = 7.3, 11.3, and 39.0 nM for CXCR4, CXCR4-CFP, and CXCR4-FlAsH228-CFP, respectively. Characterization of the CXCR4-FlAsH226-CFP and CXCR4-FlAsH229-CFP sensors is presented in Supplemental Fig. 1. RLU, relative light unit.
- Fig. 2.
The CXCR4-FlAsH228-CFP sensor reports the dynamics and kinetics of receptor activation and deactivation. (A and B) Representative traces of the FRET response from a single HEK293 cell expressing the CXCR4-FlAsH228-CFP sensor and stimulated with 30 µM CXCL12 for the indicated period of time (black line). Left panel shows corrected and normalized FRET ratio. Right panel shows corrected FlAsH (yellow) and CFP (cyan) emissions. (C) Kinetic analysis of receptor activation. The FRET change was fitted to a one-component exponential decay function to obtain the time constant τ. (D and E) On-kinetics of CXCR4 in response to CXCL12 (D) and off-kinetics of CXCR4 upon wash-out of the ligand with buffer (E). τ values from individual experiments are represented in a scatter plot. Data show median and IQR of 17 cells measured on 4 independent experimental days. a.u., arbitrary units.
- Fig. 3.
CXCL12 induces rearrangements between CXCR4 and the Gi1 protein. (A and B) Schematic depicting the settings employed to investigate the interaction between the receptor and the Gi1 protein. HEK293 cells were transfected with CXCR4-YFP and Gαi1/Gβ1/Gγ2-CFP (A) or Gαi1-CFP/Gβ1/Gγ2 (B). (C and D) Representative traces of the FRET response from a single HEK293 cell expressing CXCR4-YFP and the G protein CFP-labeled at the Gγ2 (C) or Gαi1 subunit (D) and stimulated with 30 µM CXCL12 for the indicated period of time (black line). Upper panels show corrected YFP (yellow) and CFP (cyan) emissions. Lower panels show corrected and normalized FRET ratios. (E) On-kinetics of the interaction of CXCR4 with the Gi1 protein upon CXCL12 stimulation as measured in the two settings. τ values from individual experiments are represented in a scatter plot. Data show median and IQR of 12 cells for each setting, measured on at least 3 independent experimental days. Statistical significance was tested using Mann-Whitney test. a.u.,arbitrary units; n.s., nonsignificant.
- Fig. 4.
CXCR4 resides within FRET distance from Gi proteins in the absence of agonist. (A) Schematic of acceptor photobleaching experiments. Donor and acceptor emissions were measured prior to and after photobleaching the YFP in HEK293 cells expressing the constructs Gαi1/Gβ1/Gγ2-CFP and α2A-AR-YFP or CXCR4-YFP. The latter was also measured in the presence of IT1t. (B) The change in the FCFP after YFP photobleaching from individual experiments in each condition is shown as a box plot. N = 19, 13, and 12 cells for CXCR4, CXCR4 + IT1t, and α2A-AR, respectively, measured on 3 independent experimental days. Statistical significance was tested using unpaired t test (***P ≤ 0.001; ****P ≤ 0.0001). (C–E) Representative CFP (cyan) and YFP (yellow) traces from individual experiments. The YFP photobleaching period is indicated in gray. a.u., arbitrary units.
- Fig. 5.
CXCR4 activates Gi proteins in response to CXCL12. (A) FRET-based sensors for Gi1, Gi2, Gi3, and Gq were employed to study G protein activation. A loss of FRET between the Gγ-Venus and Gα-mTurquoise2 subunits is detected upon activation. (B and C) Representative traces of the FRET response from a single HEK293 cell expressing the Gi2 sensor and CXCR4 (B) or α2A-AR (C) and stimulated with 30 μM CXCL12 or 100 µM norepinephrine, respectively (black line). Upper panels show corrected Venus (yellow) and mTurquoise2 (cyan) emissions. Lower panels show corrected and normalized FRET ratios. Supplemental Fig. 3 shows activation of Gi1 and Gi3. (D) Kinetics of Gi1, Gi2, and Gi3 protein activation via CXCR4 or α2A-AR in response to CXCL12 and norepinephrine, respectively. Table shows median and IQR. τ values from individual experiments are represented in a scatter plot with median and IQR. For CXCR4, n = 11, 22, and 11 cells for Gi1, Gi2, and Gi3 activation, respectively, measured on at least 3 independent experimental days. For α2A-AR, n = 17, 16, and 7 cells for Gi1, Gi2, and Gi3 activation, respectively, measured on at least 2 independent experimental days. Statistical significance was tested using Kruskal-Wallis test. n.s., nonsignificant. (E) CXCR4-mediated activation of Gi1, Gi2, Gi3, and Gq activation in response to increasing concentrations of CXCL12. As a control, empty plasmid was transfected instead of receptor. Data show mean ± S.E.M. and are representative of n = 5 independent experiments conducted in quadruplicate. In this particular experiment, EC50 values were 9.7, 10.8, and 17.1 nM for Gi1, Gi2, and Gi3, respectively. (F) FRET of cells expressing the CXCR4 and Gi2 sensor upon treatment with buffer, buffer supplemented with 100 µM IT1t or 100 nM CXCL12. Data are representative of three independent experiments. Data show mean ± S.D. and are normalized to buffer treatment. N = 30, 30, and 15 wells (containing 15,000 cells each) for the treatment with buffer, IT1t, and CXCL12, respectively. Statistical significance was tested using unpaired t test (**P ≤ 0.01; ****P ≤ 0.0001). a.u., arbitrary units.
- Fig. 6.
CXCR4 homodimers undergo conformational changes in response to CXCL12. (A) The rearrangement between CXCR4 protomers was investigated in HEK293 cells cotransfected with CXCR4-CFP and CXCR4-YFP, with the fluorophores fused to the C termini. (B) Representative traces of the FRET response from a single HEK293 cell expressing CXCR4-YFP and CXCR4-CFP and stimulated with 30 μM CXCL12 (black line). Upper panel shows corrected YFP (yellow) and CFP (cyan) emissions. Lower panel shows corrected and normalized FRET ratio. (C and D) On-kinetics of the rearrangement between CXCR4 protomers in response to CXCL12 (C) and off-kinetics upon wash-out of the ligand with buffer (D). τ alues from individual experiments are represented in a scatter plot with median and IQR. N = 28 and 10 cells, respectively, measured on 4 independent experimental days. a.u., arbitrary units.
- Fig. 7.
MIF induces structural rearrangements in CXCR4 but does not lead to Gi protein activation. (A, C, E, and G) Representative traces of the FRET response from a single HEK293 cell transiently expressing: CXCR4-FlAsH228-CFP sensor (A), CXCR4-YFP and Gαi1/Gβ1/Gγ2-CFP (C), CXCR4-CFP and CXCR4-YFP (E) or CXCR4 and Gi2 sensor (G), which were stimulated with 100 µM MIF and then followed by wash-out and then stimulation with 30 μM CXCL12. Upper panels show corrected acceptor (yellow) and donor (cyan) emissions. Lower panels show corrected and normalized FRET ratios. (B, D, and F) On-kinetics of receptor activation (n = 9 cells) (B), receptor/G protein interaction (n = 13 cells) (D) and rearrangement between CXCR4 protomers (n = 17 cells) (F) in response to 100 µM MIF. τ values from individual experiments are represented in a scatter plot with median and IQR. Measurements were performed on at least 2 independent experimental days. (H) Comparison of CXCR4 on-kinetics in response to CXCL12 and MIF. Data from receptor activation belong to Figs. 2D and 7B. Data from receptor/G protein interaction belong to Figs. 3E (Gγ-labeled) and 7D. Data from protomers rearrangement belong to Figs. 6C and 7F. Data from G protein activation belong to Fig. 5D (Gi2 sensor). Data are shown as a box plot in which the whiskers represent maximum and minimum values. a.u., arbitrary units.
- Fig. 8.
Summary of the findings in this manuscript regarding the CXCL12/CXCR4 axis. The upper part of the figure shows the kinetics of each step of the signaling cascade investigated using FRET. The main findings regarding the CXCL12/CXCR4 axis are: 1) Activation kinetics of CXCR4 upon CXCL12 binding are slower than other class A GPCRs; 2) Rearrangements within dimers occur faster than activation of Gi proteins; 3) This axis leads to a prolonged activation of Gi proteins; and 4) CXCR4 exhibits some degree of constitutive activity. It is tempting to speculate that the rearrangement between protomers precedes G protein activation, which might suggest that conformational changes in CXCR4 homodimers, when present, play a possible role in the signaling activation course of this receptor as depicted in model B. However, we need to emphasize that mechanistic interpretation needs to be based on measuring microscopic rate constants, and hence, model A, in which the dimer rearrangement offers an alternative pathway, is also compatible with our dataset. a.u., arbitrary units.
Additional Files
Data Supplement
- Supplemental Figures -
Supplementary Figure 1 - Characterization of the CXCR4-FlAsH226-CFP and CXCR4-FlAsH229-CFP sensors.
Supplementary Figure 2 - Determination of the intra- and intermolecular FRET efficiency of the CXCR4-FlAsH228-CFP sensor.
Supplementary Figure 3 - Activation of Gi1 and Gi3 via CXCR4 or α2A-AR upon stimulation with their respective ligands, CXCL12 and norepinephrine.
Supplementary Figure 4 - CXCL12-induced CXCR4 responses are not affected by prior stimulation of the cells with MIF.
Supplementary Figure 5 - Competition of CXCL12 with MIF for binding to CXCR4.
- Supplemental Figures -
In this issue
Early Steps in the Activation Mechanism of CXCR4
