C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways

Abstract

Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated G _αi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G _αi or G _αs activity. β-arrestin (βarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in βarr2 conformation. βarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by βarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of βarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.

Fig. 1.

CXCR3A and CXCR3B splice variants. Schematic representation of CXCR3A (A) and CXCR3B (B). Light pink signifies the additional 47 amino acids present on the N-terminus of CXCR3B. There is no difference in intracellular residues between these CXCR3 splice variants. (C) Sequence alignment of CXCR3A and B.

Fig. 2.

Differential G protein activation and beta-arrestin recruitment by CXCR3 splice variants. (A) cAMP signal following transient CXCR3A expression in HEK 293 cells stably expressing the cAMP activated firefly luciferase. CXCL10 and CXCL11 are full agonists in their ability to inhibit cAMP production through CXCR3A, while CXCL4 and CXCL9 are partial agonists. (B) No G_αi activity is observed after transient transfection with CXCR3B. CXCL11 recruited βarr2-YFP to both CXCR3A-Rluc (C) and CXCR3B-Rluc (D) with higher efficacy than the other endogenous ligands. CXCL11 recruited βarr1-YFP to CXCR3A-Rluc with a significantly greater efficacy than CXCL4, but not significantly greater than CXCL9 or CXCL10 (E). At CXCR3B, no ligands were observed to be significantly different in recruiting βarr1-YFP (F). Best fit calculated by a 3-parameter fit, ± S.E.M., n ≥ 3 biologic replicates per treatment group. *P < 0.05, significant effect of ligand by two-way analysis of variance; n.s denotes not significant.

Fig. 3.

CXCR3A and CXCR3B demonstrate equivalent agonist-induced phosphorylation. HEK 293 cells were transfected with plasmids encoding either no protein (Vector) or N-terminal FLAG-tagged constructs of CXCR3A or CXCR3B. After serum starvation and metabolic labeling with ³²P_i, cells were exposed to serum-free medium lacking (−) or containing (+) the CXCR3 agonist CXCL11 (100 nM) for 5 minutes (37°C), and then solubilized. CXCR3 isoforms were immunoprecipitated (IP) via their N-terminal FLAG epitope, and immune complexes were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and then subjected sequentially to autoradiography (³²P) and immunoblotting (IB) for CXCR3, as indicated. Shown are an autoradiogram and immunoblot from a single experiment, representative of three experiments performed. The arrow indicates the cell-surface, mature-glycosylated isoform of CXCR3 (which has slower electrophoretic mobility than the cotranslationally glycosylated or immature-glycosylated bands). From the ³²P signal for each CXCR3 band we subtracted the (nonspecific) signal in the cognate location of the lane from untransfected cells; the resulting specific ³²P CXCR3 signal was normalized to the cognate CXCR3 immunoblot band signal (from which nonspecific pixels in the cognate untransfected cell lane had been subtracted). In each experiment, this ratio of ³²P/CXCR3 was normalized to that obtained for CXCR3B immunoprecipitated from unstimulated cells, to obtain fold/control, plotted as individual values and corresponding means ± S.E.M. Compared with unstimulated cells: *P < 0.05 indicates a significant effect of agonist by two-way analysis of variance, with no significant effect of receptor isoform. n = 3 biologic replicates per treatment group.

Fig. 4.

CXCR3A and CXCR3B induce distinct βarr2 trafficking patterns. Confocal microscopy of HEK 293 cells transiently transfected with unlabeled CXCR3A or CXCR3B and βarr2-GFP pre-CXCL11 (left panels) and post-CXCL11 (three right panels) treatment (100 nM). Three different cells are shown post-treatment of each splice variant.

Fig. 5.

CXCR3A, but not CXCR3B, stimulation with CXCL11 causes a change in βarr2 conformation (A). Schematic of βarr2 biosensor for probing βarrestin-conformational changes following transfection with the biosensor and untagged CXCR3A or CXCR3B. HEK 293 cells were stimulated for 15 minutes with the indicated ligand (250 nM) prior to BRET measurements. (B) CXCL11 stimulation of CXCR3A led to a significant conformational change in βarrestin signified by a change in magnitude of the net BRET ratio compared with all ligands, while CXCL10 stimulation caused a significantly different signal from CXCL4. (C) Stimulation of CXCR3B resulted in no appreciable change in signal across all comparisons. *P < 0.05 by one-way analysis of variance with Tukey’s post hoc comparison between all treatment groups. n ≥ 3 biologic replicates per condition. n.s, not significant.

Fig. 6.

The internalization pattern of CXCR3 splice variants diverge. (A) Confocal images of transiently transfected untagged mVenus (left panel), myr-palm-mVenus (center panel) localized to the plasma membrane, and 2x-FYVE-mVenus (left panel) localized in an endosomal distribution. Internalization efficacy measured by BRET of myr-palm-mVenus transiently transfected cells with (B) CXCR3A-Rluc or (C) CXCR3B-Rluc CXCL11 and treated with the indicated ligand for 1 hour as a measure of total receptor internalization. 2x-FYVE-mVenus transiently transfected cells (D) CXCR3A-Rluc or (E) CXCR3B-Rluc and treated with the indicated ligand for 1 hour as a measure of receptor-endosome association. Treatment CXCL11 resulted in greater βarrestin-mediated internalization efficacy compared with all other ligands in cells transiently transfected with CXCR3A (F), but not CXCR3B (G). Aside from (G), which lacked appreciable signal with any ligand, data are normalized to CXCL11 (1 μM) stimulation and expressed as a percentage of maximal signal. *P < 0.05, significant effect of ligand by two-way analysis of variance. Scale bar is 10 μm. n ≥ 3 biologic replicates per condition.

Fig. 7.

GRK5/6 knockdown differentially attenuates βarrestin2 recruitment. (A) Illustration of CXCR3 truncation C-terminal mutants. (B) Truncation of either CXCR3A or CXCR3B reduced βarr2 recruitment as measured by BRET in response to CXCL11 (500 nM). (C) siRNA knockdown of either GRK2/3 or GRK5/6 reduced CXCL11(500 nM) induced βarr2 recruitment to CXCR3A; however, as shown in (D) only siRNA knockdown of GRK2/3, but not GRK5/6, reduced βarr2 recruitment to CXCR3B. In (B), *P < 0.05 by unpaired student’s t test between respective WT and truncation mutant; in (C) and (D), *P < 0.05 by one-way analysis of variance with Tukey’s post hoc comparison between all treatment groups. n ≥ 3 biologic replicates per condition.

Fig. 8.

Divergent ERK activation kinetics between CXCR3 splice variants. HEK 293 cells were transfected with either CXCR3A or CXCR3B and stimulated for either 5 minutes (A) or 60 minutes (B) with the indicated ligand (100 nM). The 60-minute, but not 5-minute, pERK1/2 CXCL11 signal in cells transfected with CXCR3A was significantly greater than cells transfected with CXCR3B. (C and D) Quantification of pERK/ERK signal normalized to CXCR3A vehicle treatment. Data were normalized to vehicle treatment of each respective isoform. In (C) and (D), *P < 0.05 indicates a significant effect of transfected isoform at 60 minutes by two-way analysis of variance (ANOVA). ^#P < 0.05; by one-way ANOVA and Tukey post hoc comparisons within all CXCR3A treatment groups, with * indicating a significant difference between CXCR3A CXCL11 stimulated cells compared with vehicle and CXCL4 at both 5 and 60 minutes; ^‡P < 0.05, by one-way ANOVA and Tukey’s post hoc comparisons between all CXCR3B treatment groups, with ‡ indicating a significant difference between CXCR3B CXCL11 stimulated cells compared with all treatment groups at 5 minutes. No significant differences between ligands were observed in CXCR3B transfected cells at 60 minutes; ± S.E.M., n ≥ 3 biologic replicates per condition. Western blots shown are representative of at least three separate experiments.

Fig. 9.

β-arrestin KO differentially alters pERK signaling between CXCR3 splice variants. WT or ∆ARRB1/2 cells transfected with CXCR3A (A) or CXCR3B (B) and stimulated with CXCL11 (100 nM). A significant increase in pERK1/2 signal was observed in both WT and ∆ARRB1/2 cells transfected with CXCR3A. However, only WT, but not ∆ARRB1/2, cells transfected with CXCR3B displayed a significant increase in pERK1/2 signal. The CXCR3A (C) and CXCR3B (D) phospo-ERK signal was quantified in WT and ∆ARRB1/2 cells [note the change in scale of the y-axis between (C) and (D)]. In (C) and (D), ^&P < 0.05 indicates a significant effect of cell line by two-way analysis of variance (ANOVA) in cells transfected with CXCR3B; *P < 0.05 indicates a significant difference in the 5-minute time point by two-way ANOVA followed by Bonferroni post hoc comparison (corrected for all time points) in cells transfected with CXCR3B; ^#P < 0.05 by one-way ANOVA and Dunnett’s post hoc comparison of CXCL11 stimulation to the respective isoform vehicle control at the indicated time point; ± S.E.M., n ≥ 3 biologic replicates per condition. Western blots shown are representative of at least three separate experiments.

Fig. 10.

β-arrestin overexpression differentially regulates pERK signaling between CXCR3 splice variants at 5 minutes. Either βarr1 and βarr2 or empty vector were transfected in ARRB1/2 KO cells expressing either (A) CXCR3A or (B) CXCR3B as a rescue overexpression experiment and stimulated for 5 minutes with either vehicle or CXCL11 (100 nM). (C) Rescue of β-arrestin was confirmed (nonspecific band in both lanes noted by an arrow). (D) Immunoblots were quantified by calculating pERK/total ERK signal. β-arrestin rescue resulted in decreased pERK signal in CXCR3A-expressing cells, in contrast to an increased signal in CXCR3B-expressing cells, relative to respective vehicle treatments. In (D), ^#P < 0.05 indicates a significant difference in pERK signal between pcDNA empty vector and β-arrestin rescue within isoform treatment by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc comparison, while *P < 0.05 indicates a significant difference in pERK signal between receptor isoforms treated with CXCL11 by two-way ANOVA followed by Bonferroni post hoc comparison; ± S.E.M., n ≥ 3 biologic replicates per condition. Western blots shown are representative of at least three separate experiments; IB denotes immunoblot.

Fig. 11.

CXCL11 robustly activates SRE and SRF response element signaling at CXCR3A, but not CXCR3B. HEK 293T cells were transiently transfected with either the SRE or SRF reporter and CXCR3A or CXCR3B. Prior to acquiring luminescence signal, cells were incubated for 5 hours with ligand (1 μM). CXCL11 incubation caused a significant increase in luminescence signal in both SRE-transfected (A) and SRF-transfected (C) cells. In cells transfected with CXCR3B, none of the endogenous ligands tested resulted in significant SRE (B) or SRF (D) signal, although cells still responded to the positive fetal bovine serum (FBS) control. In (E) and (F), cells were pretreated either with vehicle or with mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (20 μM). The CXCL11-induced SRE and SRF signals were sensitive to MEK inhibition (E and F); *P < 0.05. (A)–(D) One-way analysis of variance (ANOVA) and Tukey’s post hoc comparisons of treatment groups. The positive control of 10% FBS is included for reference, but not included in statistical analyses. (E) and (F) One-way ANOVA, followed by a directed Bonferroni post hoc comparison of vehicle (Veh) + CXCL11 to PD98059 + CXCL11, was conducted. n ≥ 3 biologic replicates per condition.

Fig. 12.

Summary of observed CXCR3 isoform signaling. Both CXCR3A and CXCR3B recruited β-arrestin2, became phosphorylated, and internalized in response to CXCL11. In contrast, only CXCR3A was observed to signal through G_αi since CXCR3B-transfected cells did not produce appreciable signal in either G_αi or G_αs assays. In further signaling divergence, CXCR3A and CXCR3B show distinct patterns of downstream signaling, with CXCR3A observed to display a stable class B interaction with β-arrestin2 in contrast to CXCR3B, which displayed a transient class A interaction in confocal recruitment assays. GRK2/3 siRNA knockdown attenuated βarr2 recruitment to both receptor isoforms; however, only GRK5/6 knockdown was observed to attenuate βarr2 recruitment to CXCR3A. Only CXCR3A was observed to show significant late phase (1 hour) pERK activity, and overexpression rescue of β-arrestin attenuated early phase pERK activity mediated by CXCR3A while enhanced pERK activity was mediated by CXCR3B. SRE and SRF transcriptional reporter activity was only observed downstream from CXCR3A.