Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/08/10/mol.116.105502.DC1

1521-0111/90/4/483–495$25.00 http://dx.doi.org/10.1124/mol.116.105502 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:483–495, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

CXC 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways s

Yamina A. Berchiche1 and Thomas P. Sakmar Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York (Y.A.B.; T.P.S.); and Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, Huddinge, Sweden (T.P.S) Received June 3, 2016; accepted August 9, 2016 Downloaded from

ABSTRACT The G -coupled receptor (GPCR) C-X-C chemokine CXCL10 displayed full agonistic activity for Gai activation and receptor 3 (CXCR3) is a potential drug target that mediates extracellular signal regulated kinase (ERK) 1/2 phosphorylation signaling involved in cancer metastasis and inflammatory dis- and partial agonist activity for b-arrestin recruitment, CXCL9 eases. The CXCR3 primary transcript has three potential alter- triggered only modest ERK1/2 phosphorylation. CXCL11 in- native splice variants and cell-type specific expression results duced CXCR3B-mediated b-arrestin recruitment and little ERK molpharm.aspetjournals.org in receptor variants that are believed to have different func- phosphorylation. CXCR3Alt signaling was limited to modest tional characteristics. However, the molecular pharmacology ligand-induced receptor internalization and ERK1/2 phosphory- of ligand binding to CXCR3 alternative splice variants and their lation in response to CXCL11, CXCL10, and CXCL9. downstream signaling pathways remain poorly explored. To These results show that CXCR3 splice variants activate different better understand the role of the functional consequences of signaling pathways and that CXCR3 variant function is not alternative splicing of CXCR3, we measured signaling in re- redundant, suggesting a mechanism for tissue specific biased sponse to four different chemokine ligands (CXCL4, CXCL9, agonism. Our data show an additional layer of complexity for CXCL10, and CXCL11) with agonist activity at CXCR3. Both signaling that might be exploited to target CXCL10 and CXCL11 activated splice variant CXCR3A. Whereas specific CXCR3 splice variants. at ASPET Journals on September 28, 2021

Introduction is the expression of alternative splice variants, which remains poorly explored (Wise, 2012). Because many chemokine re- Chemokine receptors are members of the G protein-coupled ceptors and chemokines are coexpressed by the same cell receptor (GPCR) family that bind peptidic chemotactic cyto- types or are present in the same cellular environment during kines also known as chemokines. One hallmark of chemo- inflammation, it is challenging to assess differences in the kine receptors is that they tend to have multiple endogenous function of chemokine receptors and their alternative splice agonist ligands. Conversely, some chemokines can activate variants in response to different ligands in in vivo settings. multiple separate chemokine receptor subtypes. Accumulat- Inflammatory chemokine receptor CXCR3 is of increasing ing in vitro and in vivo evidence suggests that this promiscuity clinical interest because it controls leukocyte and is results in a diversity of specific chemokine receptor signaling, involved in inflammatory disorders such as as rather than mere functional redundancy (Schall and Proud- foot, 2011). Furthermore, chemokine receptor signaling is also well as in cancer metastasis (Hancock et al., 2000; Murphy controlled by chemokine binding to proteoglycans (Brady and et al., 2000; Groom and Luster, 2011a,b; Li et al., 2015; Lleo Limbird, 2002; Proudfoot et al., 2003; Groom and Luster, et al., 2015; Zhu et al., 2015). CXCR3 is expressed at the surface 2011b; Zweemer et al., 2014), posttranslational modifications, of a plethora of cells, including monocytes, lymphocytes, natural and chemokine dimerization (Ludeman and Stone, 2014). killer, and endothelial cells (García-López et al., 2001). CXCR3 Another potential mechanism controlling receptor function displays classic receptor-ligand promiscuity and binds four chemokine ligands: CXCL4, CXCL9, CXCL10, and CXCL11 This work was supported by a Fonds de la Recherche en Santé du Québec (Loetscher et al., 1996; Cole et al., 1998; Mueller et al., 2008). fellowship, Québec, Canada (Y.A.B.). CXCR3 couples to Gai/o heterotrimeric G , which are 1Current affiliation: B cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, US pertussis toxin (PTX)-sensitive, and can also activate signaling National Institutes of Health, Bethesda, MD. pathways including MAPK, intracellular calcium flux, actin dx.doi.org/10.1124/mol.116.105502. s This article has supplemental material available at molpharm. polymerization, and chemotaxis in response to chemokines aspetjournals.org. (Loetscher et al., 1996; Smit et al., 2003; Kouroumalis et al.,

ABBREVIATIONS: BRET, bioluminescence resonance energy transfer; BSA, bovine serum albumin; CXCL, CXC chemokine ligand; CXCR, CXC chemokine receptor; ERK, extracellular signal regulated kinase; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; HEK, human embryonic kidney; PBMC, peripheral blood mononuclear cell; PBS, phosphate buffered saline; PTX, pertussis toxin; RLuc, Renilla Reniformis luciferase; RT, room temperature; TM, transmembrane.

483 484 Berchiche and Sakmar

2005; Thompson et al., 2007). The CXCR3 receptor displays from Life Technologies (Carlsbad, CA). bovine serum albumin three alternative splice variants. After the identification of the (BSA) fraction V, fatty acid free was from EMD Millipore (Bedford, canonical receptor CXCR3A (Loetscher et al., 1996), two MA), and 96-well white microplates with clear bottom and 384-well naturally occurring alternative splice variants, CXCR3B and black microplates with clear bottom plates were from Corning CXCR3Alt, were also identified (Lasagni et al., 2003; Ehlert (Woburn, MA). Plasmids. The CXCR3A (Uniprot identifier P49682-1) plasmid et al., 2004). Compared with CXCR3A, CXCR3B has an addi- was purchased from the Missouri S&T cDNA Resource Center (Rolla, tional 51 amino acids at its N-terminal tail, whereas CXCR3Alt MO: www.cdna.org). The cDNA of splice variants CXCR3B (Uniprot has a major truncation of intracellular loop three and trans- identifier P49682-2) and CXCR3Alt (Uniprot identifier P49682-3) membrane (TM) helices six and seven, which results in a ma- were synthesized by Bio Basic (Markham, Canada) and subcloned ture protein with possibly only five TM helices and a short into pcDNA3.11 using with BstEII and XbaI restriction sites. The cytoplasmic C-terminal tail (Supplemental Figs. 1 and 2). cAMP EPA2 sensor was a gift from Michel Bouvier (Université de The expression patterns and function of CXCR3 splice Montréal, Canada). CXCR3A-GFP10, CXCR3B-GFP10, and CXCR3Alt- variants in normal and disease states remain largely unex- GFP10 were constructed by ligating the coding sequence of CXCR3 plored. Tissue specific expression of CXCR3 alternative var- isoforms to GFP10, amplified by PCR from the cAMP EPAC reporter iants has been mainly quantified using reverse-transcription using XhoI and XbaI, or NotI and XhoI, or NotI and XbaI, respec- tively. The amino acid sequence of the linker region between the polymerase chain reaction, and variant-specific antibodies

terminal receptor residue and the fluorophore was described earlier Downloaded from that might allow measurement of expression levels remain (Percherancier et al., 2005; Berchiche et al., 2011). b-Arrestin1-Rluc3 unavailable. Nevertheless, in prostate cancer specimens and b-arrestin2-Rluc3 sensors were constructed by C-terminally CXCR3A mRNA levels are upregulated, whereas CXCR3B fusing the coding sequence of b-arrestin to Rluc3, amplified from the mRNA levels are downregulated (Wu et al., 2012). These cAMP EPAC reporter including the linker region, using HindIII and differences receptor variant expression levels are reported XbaI unique restriction sites (Leduc et al., 2009). All sequences were to alter the migration and invasion capabilities of prostate verified by direct sequencing. cancer cells (Wu et al., 2012). In addition, mRNA levels of Cell Culture and Transfection. HEK293T cells (passage num- molpharm.aspetjournals.org CXCR3A are decreased, whereas CXCR3Alt mRNA levels ber 5 to 15, ATCC, Manassas, VA) were maintained in Dulbecco’s ’ are increased in CD31 peripheral blood lymphocytes in modified Eagle s medium Glutamax, 1% penicillin-streptomycin, and 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA). patients suffering from Crohn’s disease, suggesting a variant ’ Transient transfection was performed in six-well plates using the specific role of CXCR3Alt with Crohn s disease (Manousou polyethylenimine method as described previously (Percherancier et al., et al., 2008). 2005). Transient high-throughput in-plate transfections were performed We set out to determine whether CXCR3 alternative splice in 384-well plates using Lipofectamine 2000 according to manufacturer’s variants activated different signaling pathways in response to instructions with some modifications. Briefly, cells were trypsinized, their chemokine ligands. We dissected signaling profiles counted, and mixed with the DNA-Lipofectamine 2000 complex then across multiple pharmacological assays when CXCR3 directly plated in 0.01% poly-D-lysine coated 384-well plates at a density at ASPET Journals on September 28, 2021 variants were individually expressed in an human embry- of 20,000 cells/well. The total amount of transfected DNA was kept onic kidney (HEK) 293T expression system. We carefully constant at 2 mg/well for six-well plates and 30 ng/well for 384-well plates 1 quantitated receptor expression levels and measured by adding empty vector pcDNA3.1 . Transfected DNA amounts were chemokine-induced Gaiactivity,b-arrestin recruitment, adjusted to obtain comparable cell surface expression among the unfused and GFP10- fused CXCR3 splice variants in all experiments. receptor internalization, and ERK1/2 phosphorylation to Flow Cytometry. HEK293T cells transfected in six-well plates demonstrate that the nature of the activated signaling path- with CXCR3 alternative variants were detached in ice-cold phosphate- ways depends on both the alternative receptor variant and the buffered saline. Human peripheral blood mononuclear cells (PBMC) chemokine tested. For example, nearly all ligands induced obtained by leukapheresis (Gift from Prof. Michel Nussenzweig, The CXCR3 internalization, whereas selected ligands induced Rockefeller University) and purified T cells (Gift from Dr. Helen Su, b-arrestin recruitment in a PTX-insensitive manner, support- National Institute of Health/National Institute of Allergy and In- ing a possible Gai-independent mechanism of b-arrestin re- fectious Diseases, NIH/NIAID) obtained using a Pan isolation cruitment to CXCR3A and CXCR3B. Moreover, our results (Miltenyi Biotec, San Diego, CA) were stimulated with 10 mg/ml strongly suggest that b-arrestin2 is recruited to both CXCR3A phytohemagglutinin and cultured for 10 days in RPMI containing 10% and CXCR3B in a ligand-independent manner. In summary, we fetal bovine serum and 50 IU/ml interleukin-2. Cells were labeled with monoclonal 1C6 phycoerythrin-conjugated antibody (BD Biosciences, show that CXCR3 splice variants have different signaling San Jose, CA) that recognizes all three CXCR3 alternative variants for profiles in response to different ligands, which could lead to 30 minutes at 4°C in BRET buffer [phosphate buffered saline (PBS) different pathophysiological roles. Our data support the concept containing 0.5 mM MgCl2 and 0.1% BSA]. Cells were then washed of biased agonism in CXCR3 variant-mediated signaling and three times in ice-cold PBS. Cell surface expression was quantified by suggest that individual CXCR3 variants might prove to be flow cytometry using the Accuri C6 flow cytometer (BD Biosciences). viable drug targets. We also determined receptor surface expression of transfected CXCR3 variants in HEK293T and the total CXCR3 quantities in both activated PBMC and T cells using the Quanti-BRITE standardization Materials and Methods beads (BD Biosciences). Receptor quantities typically reached around 1.3 to 2.5 Â 104 antibody-binding sites per cell for both HEK293T Materials. Recombinant chemokines were from PeproTech, Inc. transfected CXCR3 alternative variants and endogenous CXCR3 (Rocky Hill, NJ). Coelenterazine 400A for BRET2 experiments was expressed in activated PBMCs and purified T cells. from Biotium (Hayward, CA). Forskolin, pertussis toxin (PTX), and BRET Measurements. HEK293T cells transfected in six-well poly-D-lysine were from Sigma (St. Louis, MO), and the anti-CXCR3 plates were seeded in 0.01% poly-D-lysine coated 96-well, white mAb (clone 1C6) directly coupled to phycoerythrin was from R&D microplates with clear bottom 24 hours after transfection at a density Systems (Minneapolis, MN). Dulbecco’s modified Eagle’s medium of 90,000 cells/well. Forty-eight hours posttransfection, media was Glutamax, 1% penicillin-streptomycin, and Lipofectamine 2000 were replaced with BRET buffer. Coelenterazine 400A was added at a final Differential Activation of CXC Chemokine Receptor 3 Splice Variants 485 concentration of 5 mM followed by a 5 minutes incubation at room post t test when appropriate. When indicated, differences of top or temperature (RT). Luminescence and fluorescence readings were bottom values were also determined using sigmoidal dose response collected using the Synergy NEO plate reader from Biotek (Winooski, simultaneous curve fitting. VT) and Gen5 software. BRET2 readings between Rluc3 and GFP10 were collected by sequential integration of the signals detected in the 365 to 435 nm (Rluc3) and 505 to 525 nm (GFP10) windows. BRET2 Results ratios were calculated as described previously (Leduc et al., 2009; a Berchiche et al., 2011). All BRET experiments were performed while G i Activity of CXCR3 Is Limited to Specific Alter- cells remained attached to the 96-well plates. native Splice Variants. We evaluated the ability of CXCR3 Adenylyl Cyclase Activity. cAMP was determined by using the alternative splice variants to activate Gai signaling in re- Rluc3-EPAC-GFP10, a BRET2 cAMP sensor, as described previously sponse to chemokines (Fig. 1). Receptor cDNA quantities were (Leduc et al., 2009). Briefly, cells cotransfected with 1.0 mg CXCR3A adjusted to obtain comparable cell surface expression for all or 1.5 mg CXCR3B or 1.0 mg CXCR3Alt and 0.03 mg Rluc3-EPAC- CXCR3 variants, which were kept constant across all assays GFP10 reporter were seeded into poly-D-lysine coated 96-well plates (Supplemental Fig. 3). HEK293T cells coexpressing the EPAC 24 hours after transfection. Coelenterazine 400A was added to the cells cAMP biosensor previously described (Leduc et al., 2009) with followed by a 5-minute incubation at RT. Cells were then stimulated CXCR3 alternative variants were stimulated with increasing with ligand in the presence of 5 mM of forskolin at RT for 3 minutes. For concentrations of chemokines in the presence of forskolin in experiments involving PTX, 100 ng/ml of PTX was added to the media Downloaded from and cells were treated with PTX for 16 hours before stimulation with live cells and the inhibition of forskolin induced cAMP pro- a forskolin and chemokines. duction as a consequence of G i activation was measured. Arrestin Recruitment. b-Arrestin recruitment was measured Chemokine receptor CXCR3A inhibited cAMP production by BRET2 by cotransfection of 1.0 mg of CXCR3A-GFP10 or 1.5 mg when stimulated with CXCL10 and CXCL11, and both chemo- of CXCR3B-GFP10 or 0.5 mgCXCR3Alt-GFP10with0.05mgof kines had similar efficacies and potencies (Fig. 1A, Table 1). b-arrestin2-Rluc3 or b-arrestin1-Rluc3. Transfected cells were seeded These results are in line with previous reports (Scholten et al., into poly-D-lysine coated 96-well plates. We first determined the opti- 2012). Yet, chemokines CXCL9 and CXCL4 failed to trigger molpharm.aspetjournals.org mal donor (RLuc3) quantity and then performed acceptor (GFP10) significant Gai activity even at chemokine concentrations as titrations experiments with optimal donor quantity to determine high as 100 nM. Furthermore, CXCR3B-mediated Gai activa- BRET ratios of each -GFP10 and -Rluc3 fusion pair as described max tion was only detected after stimulation with 100 nM of previously (Berchiche et al., 2007; Kalatskaya et al., 2009; Bonneterre et al., 2016). For dose-response experiments, cells expressing the -GFP10 CXCL11 but not at lower concentrations or in the presence 2 of the other chemokine ligands (Fig. 1B). In addition, we and -Rluc3 fusion proteins at BRET max ratios were stimulated for 5 minutes at 37°C with increasing concentrations of the indicated assessed the ability of chemokines to trigger Gas activation ligand before the addition of the substrate, unless specified other- via CXCR3B using the EPAC biosensor in the absence of wise. The values were corrected to net BRET by subtracting the back- forskolin (Leduc et al., 2009). As opposed to previously re- 2 ground BRET signal detected when the -Rluc3 construct was ported Gas activity in human microvascular endothelial cell at ASPET Journals on September 28, 2021 expressed alone. line-1 (HMEC-1) stably expressing CXCR3B (Lasagni et al., Endocytosis. HEK293T transfected with 1.0 mg CXCR3A or 2003), we found that in our assays it failed to activate Gas m m 1.5 g CXCR3B or 1.0 g CXCR3Alt were incubated with 100 nM of upon treatment with its chemokine ligands (data not shown). chemokines at 37°C with gentle agitation, followed by immediate Chemokine receptor variant CXCR3Alt failed to induce incubation on ice to stop the reaction. Surface bound chemokines were significant Gai activation in response to all chemokines tested removed with acid washing (50 mM glycine, pH 2.7, 150 mM NaCl) and cells subsequently washed twice with ice-cold PBS. Cells were then (Fig. 1C), although the truncated receptor was expressed labeled with anti-CXCR3 1C6 phycoerythrin-conjugated antibody for at the cell surface as measured by flow cytometry. Results 30 minutes on ice following manufacturer’s instructions. Antibody in- obtained with CXCR3Alt were similar to our results obtained cubation was followed by two washes with ice-cold PBS. Cell surface when cells were cotransfected with the EPAC biosensor and receptor expression was quantified by flow cytometry using the Accuri empty vector pcDNA3.11 (Fig. 1D). C6 flow cytometer (BD Biosciences). Surface expression of CXCR3 after CXCR3 Splice Variants Differentially Recruit ligand incubation at 37°C was expressed as percentage of CXCR3 b-Arrestins. We measured b-arrestin recruitment to CXCR3 expression compared with a sample drawn before addition of the ligands in live cells using a BRET proximity assay, which was exten- and kept on ice before the acid wash and antibody staining. sively used to study receptor interaction with arrestins, ligand- Erk1/2 Phosphorylation. Chemokine-induced MAPK pathway induced receptor/arrestin conformational changes, as well as activation was determined by InCell Western assay (Li-COR). Briefly, cells were transfected in a high-throughput in-plate transfection receptor dimerization (Hamdan et al., 2005; Kalatskaya et al., manner with 10 ng of CXCR3A, 15 ng of CXCR3B, or 10 ng of 2009; Berchiche et al., 2011). We performed acceptor/donor CXCR3Alt. After 24 hours, the media was removed and replaced with titration experiments by coexpressing increasing quantities of 1Â Hank’s Balanced Salt Solution-20 mM HEPES pH 7.4 supple- CXCR3-GFP10 acceptors with fixed quantities of b-arrestin1- mented with 0.2% BSA (HBSS-H 0.2% BSA). Transfected cells were Rluc3 or b-arrestin2-Rluc3 donors (Fig. 2). Surprisingly, we then stimulated with 100 nM of chemokines at 37°C for different measured a significant and saturating basal BRET signal in the periods of time (2, 5, 7, 10, and 15 minutes) or in the presence of absence of ligands for CXCR3A with both b-arrestins and increasing concentration of ligand. ERK activity was stopped by fixing CXCR3B with b -arrestin2, which further increased after stim- cells with a solution of PBS containing 3.7% formaldehyde for ulation with 100 nM CXCL11 (Fig. 2, A–C). As for CXCR3B/ 20 minutes at RT. Detection of phosphor-ERK1/2 and total ERK2 b-arrestin1, acceptor/donor titration in the absence of ligand proteins were performed as described previously (Wong, 2004). Plates were scanned using the Li-COR Odyssey infrared reader. resulted in a bystander curve, most likely due to random collision Data Analysis. Data were analyzed using Prism 6.0 software of coexpressed BRET pairs (Bonneterre et al., 2016). However, (GraphPad Software, San Diego, CA). Statistical significance of the stimulation with CXCL11 resulted in a saturating curve sup- differences between the various conditions was determined using porting ligand-induced b-arrestin1 recruitment to CXCR3B one-way analysis of variance with Tukey’spostt test or Bonferroni’s (Fig. 2D). This suggests a preference of CXCR3B for b-arrestin2 486 Berchiche and Sakmar Downloaded from molpharm.aspetjournals.org

Fig. 1. CXCR3 alternative splice variants display different Gai activities in response to their chemokines. Receptor splice variants CXCR3A (A), CXCR3B (B), CXCR3Alt (C), or empty vector pcDNA3.1+ (D) were cotransfected with the cAMP Rluc3-EPAC-GFP10 sensor in HEK293T cells. Cells were exposed to 5 mM forskolin with increasing concentrations of the indicated ligands at room temperature for 5 minutes before readings. Results are expressed as the % of inhibition of forskolin-induced cAMP production. Data are reported as mean values of 4–6 independent experiments performed in triplicate 6 S.E.M.

in the absence of ligand. Curve fitting of the data obtained acceptor and donor concentrations determined at saturation with at ASPET Journals on September 28, 2021 from the titration experiments allowed us to determine both optimal sensitivity, which we used to assess ligand-induced BRETmax and BRET50 values in the absence and presence of CXCR3/b-arrestin interaction. Furthermore, BRET50 corre- 100 nM CXCL11 (Table 2). BRETmax corresponds to the best sponds to the acceptor/donor ratio giving 50% of the maximal

TABLE 1 Summary of fitted curve parameters for results shown in Figs. 1 (Gai activity), 5 (b-arrestin recruitment), and 8 (ERK1/2 phosphorylation).

pEC50 values are given and maximal responses are shown in absolute (b-arrestin recruitment) and relative (ERK1/2 phosphorylation and Gai activity) units. Errors are presented as 6 S.E.M.

CXCR3A CXCR3B

CXCL11 CXCL10 CXCL11 cAMP inhibition N446 IC50 (nM) 0.7 0.5 N.D. pIC50 6 S.E.M. 29.5 6 0.3 29.4 6 0.2 N.D. Emax 6 S.E.M. 89 6 11 70 6 7 N.D. b-arrestin2 N 33 3 EC50 (nM) 8 71 61 pEC50 6 S.E.M. 28.2 6 0.1 27.3 6 0.2 27.2 6 0.1 2 a BRET max 6 S.E.M. 0.11 6 0.01 0.07 6 0.01 0.026 6 0.001 b-arrestin1 N 33 3 EC50 (nM) 16 32 33 pEC50 6 S.E.M. 27.8 6 0.1 27.5 6 0.1 27.6 6 0.2 2 a a BRET max 6 S.E.M. 0.06 6 0.01 0.025 6 0.003 0.01 6 0.001 ERK1/2 phosphorylation N 44 6 EC50 (nM) 2 3 N.D. pEC50 6 S.E.M. 28.9 6 0.1 28.5 6 0.1 N.D. Emax 6 S.E.M. 368 6 32 293 6 32 N.D.

aTheoretical value, experimental curves did not reach saturation. N.D., not detectable. Differential Activation of CXC Chemokine Receptor 3 Splice Variants 487 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 28, 2021

Fig. 2. CXCR3 variants constitutively interact with select b-arrestins. Acceptor/donor titration curves of CXCR3 splice variants fused with GFP10 and b-arrestin-Rluc3 were obtained after transfection of HEK293T cells with increasing quantities of CXCR3-GFP10 fusion with 0.05 mg b-arrestin-Rluc3 fusions as indicated: A, CXCR3A/b-arrestin2; B, CXCR3A/b-arrestin1; C, CXCR3B/b-arrestin2; D, CXCR3B/b-arrestin1; E, CXCR3Alt/b-arrestin2; F, CXCR3Alt/b-arrestin1. Measurements were carried out at room temperature in the absence or presence of 100 nM CXCL11. Data were plotted and fitted 2 to a one-site binding hyperbola equation. BRET50 and BRETmax values were calculated for each BRET couple. Results obtained from at least three independent experiments, carried out in triplicate 6 S.E.M are shown. signal and reflects the propensity of BRET partners to -independent association, indicating that CXCR3Alt does not interact. interact with b-arrestins (Fig. 2, E and F). Stimulation of CXCR3A/b-arrestin1, CXCR3A/b-arrestin2, as Moreover, we assessed the specificity of the interactions we well as CXCR3B/b-arrestin2 pairs with 100 nM CXCL11 resulted measured to ascertain whether select CXCR3 variants in- in an increased BRETmax (Fig.2,A–C, and Table 2) compared teract with b-arrestins in the absence of ligand. We cotrans- with basal conditions. This increase is a direct consequence of fected cells expressing the BRET2 b-arrestin donor and changes in the distance and/or orientation between the -Rluc CXCR3 acceptor fusions with the corresponding FLAG- donor and -GFP10 acceptor fusions. This strongly suggests that b-arrestin and measured its impact on the basal BRET CXCL11 induces conformational changes in these receptor/ signal. We observed a transfected FLAG-b-arrestin quantity- b-arrestin preformed complexes. In addition, CXCL11 stimulation dependent decrease of the basal BRET signal for CXCR3A also decreased BRET50 values, shifting the saturation curve to the and both b-arrestins as well as CXCR3B and b-arrestin2, left for these receptor/arrestin pairs, which indicates a higher whereas CXCR3B and b-arrestin1 remained unaffected (Fig. propensity of CXCR3A and B variants to interact with b-arrestins 3). A decrease in a dose-dependent manner of the basal BRET in the presence than in the absence of CXCL11 (Table 2). signal indicates a competition between the donor fused In contrast, titration of the CXCR3Alt-GFP10 acceptor b-arrestin and the corresponding FLAG-tagged b-arrestin, with both b-arrestins donors showed no ligand-dependent or which in turn supports a specific ligand-independent 488 Berchiche and Sakmar

TABLE 2

Fitted curve parameters of CXCR3 (acceptor)/b-arrestin (donor) titration curves. BRET50 and BRET maximal values are given in absolute units Errors are presented as 6 S.E.M.

Receptor Variant

CXCR3A-GFP10 CXCR3B-GFP10

CXCL11 Basal CXCL11 (100 nM) Basal (100 nM) b-Arrestin2-Rluc3 N 4433 BRET50 6 S.E.M 24 6 556 1 9.5 6 0.4 7.9 6 0.3 BRETmax 6 S.E.M 0.08 6 0.001 0.13 6 0.01 0.024 6 0.002 0.036 6 0.001 b-Arrestin1-Rluc3 N 4433 BRET50 6 S.E.M 47 6 12 2 6 1ND96 3 BRETMax 6 S.E.M 0.034 6 0.002 0.07 6 0.01 ND 0.023 6 0.001

N.D., not detectable. Downloaded from interaction between select CXCR3 alternative variants and indicate a receptor preference for specific b-arrestins, which is b-arrestin. selectively influenced by chemokines. Next, we quantified ligand-mediated b-arrestin recruitment Also, CXCL11 increased b-arrestin2 recruitment to to CXCR3 at BRETmax by performing kinetic experiments. CXCR3B with the signal reaching a plateau within the first These were carried out at RT to fully capture the details of 5 minutes of stimulation (Fig. 4B). CXCL10, CXCL9, and molpharm.aspetjournals.org recruitment kinetics after chemokine stimulation (100 nM). CXCL4, a chemokine suggested to be a selective high affinity CXCR3A treatment with CXCL11 or CXCL10 resulted in a ligand of CXCR3B (Lasagni et al., 2003), failed to trigger rapid b-arrestin2 recruitment reaching a plateau in 5 to b-arrestin2 recruitment to CXCR3B. Similar results were 7 minutes (Fig. 4A). Similar results were obtained with obtained with b-arrestin1 (Supplemental Fig. 4B). As for b-arrestin1 (Supplemental Fig. 4). A small but statistically CXCR3Alt, all chemokines failed to induce both b-arrestin1 significant elevation in b-arrestin2 recruitment to CXCR3A and b-arrestin2 recruitment to this C-terminally truncated after stimulation with CXCL9 or CXCL4 was also observed alternative variant (Fig. 4C, Supplemental Fig. 4C). (Fig. 4A, Supplemental Fig. 5). In contrast, CXCL9 or CXCL4 To catalog the pharmacology of CXCR3 variants in response failed to induce b-arrestin1 recruitment to CXCR3A (Supple- to their chemokines, we measured the effect of increasing at ASPET Journals on September 28, 2021 mental Fig. 4A). Our data show that CXCR3A and CXCR3B concentrations of ligands on b-arrestin recruitment to acceptor fusions are functional and are able to recruit both CXCR3A and CXCR3B (Table 1). Our results obtained with b-arrestins in response to ligands. In addition, these results CXCL11 and CXCL10 are consistent with previously reported

Fig. 3. Constitutive b-arrestin interaction is receptor variant specific. HEK293T cells transfected with the in- dicated receptor variant/b-arrestin at BRETmax,intheab- sence (control) or presence of increasing levels of C-terminally FLAG-tagged b-arrestin. A, CXCR3A/b-arrestin2; B, CXCR3A/b- arrestin1; C, CXCR3B/b-arrestin2; D, CXCR3B/b-arrestin1. The fold increase shown on the x-axis represents the ratio of micrograms of b-arrestin-Rluc3 to b-arrestin-FLAG trans- fected in cells coexpressing CXCR3 variant GFP10 fusion. Statistical significance of the differences between the control, in the absence of FLAG-tagged b-arrestin and increasing levels of coexpressed C-terminally FLAG-tagged b-arrestin: *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 [one-way analysis of variance (ANOVA), Bonferroni’smultiplecompar- ison test]. Results obtained for three independent experi- ments carried out in triplicate 6 S.E.M are shown. Differential Activation of CXC Chemokine Receptor 3 Splice Variants 489 Downloaded from

Fig. 4. Receptor variants selectively recruit b-arrestin2 in response to 100 nM of chemokines. HEK293T cells transiently coexpressing the indicated CXCR3 variant GFP10 fusion with b-arrestin2-Rluc3 at BRETmax were incubated with 100 nM of the indicated ligands at room molpharm.aspetjournals.org temperature. A, CXCR3A/b-arrestin2; B, CXCR3B/ b-arrestin2; C, CXCR3Alt/b-arrestin2. BRET was mea- sured immediately after ligand addition. Data are re- ported as mean values of four independent experiments performed in duplicate 6 S.E.M. at ASPET Journals on September 28, 2021

observations obtained with a BRET1 arrestin proximity assay terminus of CXCR3Alt are sufficient to perturb its ability to (Scholten et al., 2012). Indeed, CXCL11 acts as a full agonist recruit b-arrestins. with a potency of 8 nM, whereas CXCL10 acts as a partial Chemokine Triggered b-Arrestin Recruitment to agonist with a potency of 70 nM (Fig. 5A) on the b-arrestin2 CXCR3 is PTX Insensitive. To determine whether ligand- recruitment to CXCR3A. A similar order of potencies was induced b-arrestin recruitment to CXCR3A and CXCR3B observed with b-arrestin1 (Table 1, Supplemental Fig. 6A). As are linked to Gai signaling, we tested their sensitivity to for CXCR3B, CXCL11 caused arrestin recruitment with Gai/o-inactivating PTX treatment (Fig. 6). Ligand-mediated potencies of 61 and 33 nM for b-arrestin2 and b-arrestin1, b-arrestin2 (Fig. 6, A-C) and b-arrestin1 (Supplemental Fig. 7) respectively (Fig. 5B, Table 1, Supplemental Fig. 6B). These recruitment to CXCR3A and CXCR3B were resistant to PTX results suggest that the longer N terminus of CXCR3B is treatment. As expected, PTX treatment inhibited ligand- sufficient to change the receptor’s ability to respond to chemo- induced Gai activity mediated by CXCR3A (Fig. 6A). This kines and recruit b-arrestins. Moreover, these data suggest result suggests that b-arrestin recruitment to CXCR3A occurs that although the N-terminal tail of CXCR3Alt is identical independently from Gai signaling in response to CXCL11 and to that of CXCR3A, not surprisingly differences in the C CXCL10. PTX treatment also impaired CXCL11 triggered 490 Berchiche and Sakmar

Fig. 5. CXCR3A and CXCR3B recruit b-arrestin2 in the presence of increasing concentrations of chemokine. HEK293T cells transiently coexpressing A, CXCR3A-GFP10 or B, CXCR3B-GFP10 with b-arrestin2-Rluc3 at BRETmax were incubated with increasing concentrations of the indicated ligands for 5 minutes at 37°C. BRET was measured 5 minutes after ligand addition. Data are reported as the mean values of three independent experiments Downloaded from performed in triplicate 6 S.E.M (see Table 1 for curve fitting parameters).

CXCR3B Gai activity, even if it was only detected with 100 nM with the other chemokines tested for this receptor variant, yet of chemokine (Fig. 6C) comparable to CXCR3A. our results with CXCL11 remain comparable to previously Chemokines Induce Internalization of All CXCR3 reported observations (Scholten et al., 2012). Moreover, nearly Splice Variants. We stimulated HEK293T cells expressing 40% of CXCR3A receptors internalized within the first molpharm.aspetjournals.org CXCR3 variants with chemokine ligands (100 nM) for various 10 minutes of stimulation with CXCL10 and CXCL4, com- time periods at 37°C to assess ligand-induced receptor in- pared with CXCL9, which required 30 minutes of stimulation ternalization. Excess chemokine was removed by acid wash to reach 40% internalization. Similarly, nearly 50% of CXCR3B and remaining surface receptors were quantified by flow receptors internalized during the first 10 minutes after incubation cytometry. Stimulation with chemokines induced internaliza- with CXCL10, CXCL9, and CXCL4. tion of all receptor variants (Fig. 7). Chemokine CXCL11 Yet CXCL11 only induced moderate receptor internaliza- induced CXCR3A internalization was modest in comparison tion in the same fashion as CXCL11-induced CXCR3A at ASPET Journals on September 28, 2021

Fig. 6. PTX treatment only affects chemokine induced Gai activity of CXCR3A and CXCR3B. HEK293T cells transfected with CXCR3 and the EPAC cAMP biosensor or CXCR3-GFP10 fusion with b-arrestin2-Rluc3 were incubated with 100 ng/ml PTX for 16 hours at 37°C. Gai activity of CXCR3A (A) and CXCR3B (C) are expressed as the % of forskolin-induced cAMP production in the presence of 100 nM of the indicated chemokine in the absence or presence of PTX. The forskolin induced cAMP production measured in the absence of chemokine and PTX was set to 100% (white bars). b-Arrestin2 recruitment to CXCR3A (B) and CXCR3B (D) induced with 100 nM of the indicated ligands in the absence or presence of PTX. Results represent data obtained from three to five independent experiments performed in triplicate 6 S.E.M. Differential Activation of CXC Chemokine Receptor 3 Splice Variants 491 Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 28, 2021

Fig. 7. Chemokines induce CXCR3 splice variant’s internalization. HEK293T cells expressing CXCR3A; CXCRB, and CXCR3Alt were incubated with 100 nM of each chemokine at 37°C. Aliquots were removed on ice at the indicated times after ligand addition. Surface-bound ligand was removed by acid washing, and remaining surface CXCR3 was measured by flow cytometry. Data are mean of three to four independent experiments 6 S.E.M. internalization. Interestingly, CXCL11, CXCL9, and CXCL4 significant response starting as early as 2 minutes after induced a robust and rapid CXCR3Alt internalization, ligand addition (294 6 37% of basal) and lasting 10 minutes whereas CXCL10 had little to no effect. Surprisingly, in our (247 6 21% of basal), with a peak phosphorylation mea- assay CXCL4 (100 nM), a CXCR3B ligand, induced robust sured at 5 minutes (402 6 37% of basal). In comparison with receptor internalization of all three CXCR3 variants. CXCL4 CXCL11, CXCL10-mediated ERK1/2 phosphorylation through chemokine is reported to bind to CXCR3A and trigger CXCR3A was shorter and more modest. Its signal was de- chemotaxis at high chemokine concentrations of 500 to 750 nM tected between 2 (250 6 60% of basal) and 5 minutes (199 6 (Mueller et al., 2008; Korniejewska et al., 2011). Our results 25% of basal) after stimulation. Chemokine CXCL9 also indicate that all ligands (100 nM) are able to bind CXCR3 triggered a moderate elevation of ERK1/2 phosphorylation variants expressed at the cell surface and thus trigger their starting at 5 minutes (225 6 24% of basal), which declined function after 10 minutes (217 6 26% of basal). In contrast, CXCR3B CXCR3 Splice Variants Display Different ERK1/2 activation by chemokines showed a different ERK1/2 phos- Phosphorylation Profiles. We quantitatively measured phorylation profile than for CXCR3A (Fig. 8B). Indeed, ligand-induced ERK1/2 phosphorylation in HEK293T cells CXCL11 triggered a modest ERK1/2 phosphorylation starting expressing CXCR3 variants. Transfected cells were stimu- at 5 minutes (203 6 18% of basal) and lasted 10 minutes lated with chemokines (100 nM) at 37°C for various time (170 6 13% of basal). Moreover, CXCL9 also induced a short periods (Fig. 8, A, C, and D). ERK1/2 phosphorylation was ERK1/2 phosphorylation, peaking at 10 minutes (192 6 22% quantified using an InCell Western approach. This method of basal) and a short and weak, yet statistically significant, was previously used to measure ERK1/2 phosphorylation of signal at 5 minutes (159 6 22% of basal) after stimulation cannabinoid receptor CB1, a member of the GPCR family with CXCL4. Surprisingly, cells expressing CXCR3Alt variant coupled to Gai/o heterotrimeric G proteins (Daigle et al., also responded to ligands and we measured ERK1/2 phosphor- 2008). Three out of the four chemokines tested induced ylation after stimulation with CXCL11, CXCL10, and CXCL9 CXCR3A-mediated ERK1/2 phosphorylation (Fig. 8A). In cells (Fig. 8D). Treatment with CXCL11 increased ERK1/2 expressing CXCR3A, CXCL11 induced the longest and most phosphorylation between 5 to 10 minutes, with a peak 492 Berchiche and Sakmar Downloaded from molpharm.aspetjournals.org

Fig. 8. CXCR3 variants induced ERK1/2 phosphorylation with different intensities after stimulation with chemokines. HEK293T cells transiently expressing CXCR3 variants were incubated at 37°C with the indicated ligands CXCR3A (A), CXCR3B (C), CXCR3Alt (D) with 100 nM of chemokines at the indicated times or CXCR3A (B) with increasing concentrations of CXCL11 and CXCL10 at maximum stimulation time of 5 and 2 minutes, respectively. Data are reported as mean values of at least four independent experiments performed in duplicate 6 S.E.M. Statistical significance of the differences between stimulated and control condition: *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 (one-way ANOVA, Bonferroni’s multiple comparison test). at ASPET Journals on September 28, 2021 phosphorylation detected after 7 minutes (195 6 16% of different CXCR3 splice variants induce quantitatively distinc- basal). We also quantified modest ERK1/2 phosphorylation tive signaling responses after ligand stimulation and that from 5 to 15 minutes after treatment with CXCL10. In receptor signaling efficacy depends on the splice variant/ contrast, CXCL9 triggered ERK1/2 phosphorylation at a chemokine pair assessed. Receptor variant/chemokine pairs single time point of 10 minutes (155 6 14% of basal). and the corresponding pathways activated are summarized in Compared with CXCR3A, chemokines induced weaker Table 3. The ability of ligands to activate different intracellu- ERK1/2 phosphorylation through CXCR3B and CXCR3Alt. lar signaling pathways via the same receptor to different However, these changes were statistically significant com- extents is a widely observed phenomenon and has been pared with cells transfected with the receptor and treated with documented for many GPCRs, including chemokine receptors. assay buffer only. Furthermore, to ensure that the signals This characteristic has been given several names including were indeed the result of CXCR3 alternative variants, all biased agonism, stimulus trafficking, and functional selectiv- experiments were performed in parallel with cells transfected ity. Biased agonism of GPCRs supports the existence of with empty vector pcDNA3.11. These cells failed to respond to multiple active receptor conformations that ligands may chemokines (data not shown), indicating that the responses differentially stabilize, leading to the activation of specific measured for CXCR3 were ligand induced and receptor- signaling pathways (Kenakin, 2007, 2009). The nonredundant variant specific. We also collected pharmacological data for signaling responses we report for CXCR3 alternative splice CXCR3A by taking advantage of favorable dynamic range and variants could be explained if chemokines stabilized distinct the quantitative nature of the ERK1/2 phosphorylation InCell receptor splice variant conformations leading to the activation Western assay. We measured ERK1/2 phosphorylation in cells of different intracellular signaling pathways. transfected with CXCR3A in response to increasing concen- Indeed, CXCR3A-mediated Gai activity was only detectable trations of CXCL11 and CXCL10 (Fig. 8B). Our results in response to CXCL11 and CXCL10, which are in line with indicate that both CXCL11 and CXCL10 induce ERK1/2 the Gai activity previously reported for this canonical receptor phosphorylation with similar efficacy and potency and are (Scholten et al., 2012). Interestingly, CXCL11 also induced full agonists for this pathway (Table 1). CXCR3B-mediated Gai activity, yet only at a saturating concentration of 100 nM. Sulfation of Tyr27 and Tyr29 in Discussion the N-terminal region of CXCR3A is essential for chemokine binding and thus receptor function (Colvin et al., 2006). The The goal of this study was to explore the specific intracel- longer N terminus of CXCR3B contains two additional lular signaling pathways activated by CXCR3 splice variants potential sulfation sites (Tyr6 and Tyr40). These Tyr residues in response to their chemokine ligands. We report that could also be sulfated or influence the sulfation of the Tyr Differential Activation of CXC Chemokine Receptor 3 Splice Variants 493

TABLE 3 Summary of CXCR3 variant activated signaling pathways following stimulation with chemokines

CXCR3A CXCR3B CXCR3Alt Gai activation CXCL11, CXCL10 CXCL11a N.D. b-arrestin2 recruitment CXCL11, CXCL10 CXCL11 N.D. CXCL9*, CXCL4* b-arrestin1 recruitment CXCL11, CXCL10 CXCL11 N.D. Internalization CXCL11, CXCL10, CXCL9, CXCL4 CXCL11, CXCL10, CXCL9, CXCL4 CXCL11, CXCL9, CXCL4 ERK1/2 phosphorylation CXCL11, CXCL10, CXCL9* CXCL11, CXCL9, CXCL4* CXCL11, CXCL10* CXCL9*

aOnly measureable at 100 nM chemokine. *Weak but statistically significant response. N.D., not detectable. residues common to both variants, and therefore cotransla- CXCL11-induced internalization. Likewise, CXCL11 and tional Tyr sulfation might explain the Gai activity measured CXCL10 provoked receptor internalization in a b-arrestin- for CXCR3B. independent fashion when CXCR3A was expressed in .2 As expected, Gai activation of CXCR3A was inhibited by murine cells (Meiser et al., 2008). Furthermore, b-arrestins Downloaded from PTX treatment. Surprisingly, CXCR3B-induced Gai activa- are well recognized for their role as scaffold proteins for tion was also inhibited by PTX treatment, suggesting that this downstream signaling phosphorylation cascades (DeWire Gai activity is specific. Relatedly, stimulation of CXCR3A with et al., 2007). ERK1/2 phosphorylation induced by CXCR3 CXCL11 or CXCR10 and of CXCR3B with CXCL11 further splice variants does not correlate either with ligand-induced increased basal b-arrestin recruitment to the receptor and b-arrestin recruitment. This indicates that b-arrestin may was PTX insensitive. Taken together, our results indicate that not be required for ERK1/2 phosphorylation provoked by molpharm.aspetjournals.org ligand-mediated Gai signaling and b-arrestin recruitment specific receptor variant/ligand pairs such as CXCR3B/CXCL9 occur independently from each other. Our findings also and CXCR3Alt/CXCL10. Nevertheless, b-arrestins may suggest that chemokine receptor variant/ligand pairs selec- play a role in ERK1/2 phosphorylation provoked by the tively determine the receptor/b-arrestin conformation as well CXCR3A/CXCL11 pair. Similarly, only CXCR3A/CXCL11 and as the efficacy of Gai activity. Similar to the ligand-initiated CXCR3A/CXCL10 pairs provoke Gai activity that correlates b-arrestin recruitment and Gai signaling, we report that the with their ERK1/2 phosphorylation profile, supporting the CXCR3-mediated ERK1/2 phosphorylation intensity and sig- possibility that ERK1/2 phosphorylation may be a conse- nal duration measured depend on the splice variant/chemo- quence of Gai activation. kine pair assessed. For example, CXCL11-mediated ERK1/2 Many GPCRs with highly truncated alternative splice at ASPET Journals on September 28, 2021 phosphorylation through CXCR3A was stronger and lasted variants lacking some TM helices are reported in the litera- longer than the one mediated through both CXCR3B and ture (Wise, 2012). Alternative splice variant CXCR3Alt is a CXCR3Alt. predicted five TM domain receptor with a short C-terminal Ligand-independent b-arrestin recruitment was previ- end (Ehlert et al., 2004). CXCR3Alt lacks the third intracel- ously reported for chemokine receptor CCR1 (Gilliland et al., lular loop important for G-protein interaction and subse- 2013), atypical chemokine receptor ACKR2/D6 (McCulloch quent activation of intracellular components (Thelen and et al., 2008), as well as a chimeric receptor composed of Thelen, 2008). Stimulation of this splice variant resulted in ACKR3/CXCR7 with its C-terminal tail swapped with CXCR4 weak but statistically significant ERK1/2 phosphorylation (Gravel et al., 2010). C-terminal tails of both D6 and CCR1 and chemokine-induced receptor internalization. Neverthe- show basal phosphorylation, which plays an important role in less, CXCR3Alt failed to induce Gai activation and b-arrestin their ligand independent activity. Our data are the first to recruitment in the absence or presence of chemokines. Com- indicate that ligand-independent b-arrestin recruitment can pared with CXCR3A and CXCR3B, the C terminus of CXCR3Alt also differentially occur for the different CXCR3 alternative lacks the Ser and Thr phosphorylation sites typically phos- splice variants. Such constitutive b-arrestin recruitment to phorylated by GRKs and required for b-arrestin recruitment. CXCR3A and CXCR3B might also arise from low levels of This fundamental difference of CXCR3Alt and its limited basal receptor phosphorylation. Indeed, the C-terminal tail of signaling further supports the idea that CXCR3 splice vari- CXCR3A and CXCR3B contains nine Ser residues and one Thr ants play different roles in fine tuning chemokine-induced residue that might allow for some basal level of Ser/Thr signaling responses. phosphorylation, favoring the interaction with b-arrestins. It may be speculated that CXCR3Alt splice variant behaves An earlier report of a low level of phosphorylation measured as an atypical chemokine receptor. Its role could be to for transfected CXCR3 further supports this hypothesis scavenge chemokines and contribute to the establishment of (Colvin et al., 2004). a chemokine gradient, similarly to chemokine receptor Receptor internalization may occur in both b-arrestin- CXCR7, which does not activate Gai heterotrimeric G protein, dependent and -independent fashion. Here, we demonstrate yet still recruits b-arrestin and induces ERK1/2 phosphoryla- that the internalization profiles of all three CXCR3 alterna- tion and internalization in response to CXCL12 (Boldajipour tive splice variants fail to correlate with ligand-induced et al., 2008; Kalatskaya et al., 2009; Levoye et al., 2009). b-arrestin recruitment, supporting a b-arrestin-independent Another explanation for our results could be that the role of internalization mechanism. Similarly, Rajagopal et al. (2013) CXCR3Alt is to modulate the function of a chemokine receptor recently showed that CXCL11-induced b-arrestin recruitment such as CXCR3A, because these variants can be coexpressed to the canonical CXCR3 receptor does not correlate with in cells (Aksoy et al., 2006) and because truncated GPCRs 494 Berchiche and Sakmar could act as dominant negative of CXCR3A (Wise, 2012). The Authorship Contributions later possibility is further supported by the atypical and Participated in research design: Berchiche and Sakmar. predicted five TM arrangement of CXCR3Alt. An additional Conducted experiments: Berchiche. possibility could be that activation of CXCR3Alt leads to other Performed data analysis: Berchiche. functional consequences we did not assess. For example, Wrote or contributed to the writing of the manuscript: Berchiche and Sakmar. coupling to Ga12/13 (Kouroumalis et al., 2005) or Gai proteins isoforms i2 and i3 (Thompson et al., 2007) are also reported for CXCR3. References We cannot rule out that our observations are cell-type Aksoy MO, Yang Y, Ji R, Reddy PJ, Shahabuddin S, Litvin J, Rogers TJ, and Kelsen SG (2006) CXCR3 surface expression in human airway epithelial cells: cell cycle specific, yet CXCR3 natural ligands behave as perfect biased dependence and effect on cell proliferation. Am J Physiol Lung Cell Mol Physiol ligands (Kenakin and Christopoulos, 2013), which activate 290:L909–L918. Berchiche YA, Chow KY, Lagane B, Leduc M, Percherancier Y, Fujii N, Tamamura only specific signaling pathways but not others. 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V A S P E A T P A R A L C C L N T H A D L C P A C C N Q G N E S Q L G Y L L N D D W V F R R Q N V F S F G E D H2-N F E Q S E S V S D P M S M H S N L A G H Q L R V D S E N A L H L S Y G F V I V A D C D E V D Y D S G D Extracellular R V K L R V V

A F A V A F T L A L P L W G A I V K F L A V S A L P R V Y S T L L F H L T S N I P D Q L Y G L V L L L V L P L F L L N T G F Y F A A G W Y CXCR3A L G L A L L V L L D T L C M H A G L P F A C C A L C F L G N A V L W G L G A L V A N P L L V L V V V V A L H A C L A L L A V F L L Y I C V V L S F T M A V A S L T L Y L F V Cytosol R D D T C R G Y R T R V M V R Q A COOH T S Y R A K Q P A L S H F L S L L A R I R G S G N P L L E R S S I P R R Q R Y A V G R M N R S H V R W P D A L Q V I L Y A R V A S P T L Y G M C S E T G G T Q L E A T P V S R L G S S G G P A A R A L C C L N L L W T A A L P T H A D L C P A C C L R S E L A F S N Q G N E S Q L Y G R Q E S V L L N D D W F R N R G S K P Q G E D V F S F F E P K T F V H2-N Q S E S S D M G S I P P S H S N L A G H M Y Q S P Q L D S E N R E K T D S A L H S Y G F V I V L R K S Q A D C D V D Y D S G D Extracellular R V K L R V V

A F A V A F T L A L P L W G A I V K F L A V S A L P R V Y S T L L F H L T S N I P D Q L Y G L V L L L V L P L F L L N T G CXCR3B F Y F A A G W Y L G L A L L V L L D T L C M H A G L P F A C C A L C F L G N A V L W G L G A L V A N P L L V L V V V V A L H A C L A L L A V F L L Y I C V V L S F T M A V A S L T L Y L F V Cytosol R D D T C R G Y R T R V M V R Q A COOH T S Y R A K Q P A L S H F L S L L A R I R G S G N P L L E R S S I P R R Q R Y A V A S P V G R M N R S H V E A T P R W P D A L Q A R M C S E A L C C T Q L Y L G V D L C P N A T S R L G S S L N E S Q R H L L W T C L N D D W V F E L R S E Q V F S F G D Q H2-N Q S E S V S H Y M H S N L A G N V D S E N H A L F L S Y G F A D C E V D Y D S P Extracellular R V K L Q A A V G F L W A I F S S P L G A F A L P S G Y S T L L F D S N I L P L L L G C F L L V G C N F F A L Y A L L G L T L C CXCR3Alt L A D S C G A L C G N V L W G G A L A A W L L V A V A H A C A A V L L L F C A P I S T T S L L T F L RE Cytosol R D D T E T R R T R V G P A T S Y R S P C A L S L A R R E Q N P G A S P G I P H R I V G H R L R R L G A A R COOH T Q L Y P P P A H L I G I S P Supplemental Figure 1. Schematic representation of CXCR3 alternative splice variants. Residues in blue are unique to CXCR3B, in orange to CXCL3Alt, residues in red are conserved motifs and residues in magenta are common to CXCR3A and CXCR3Alt.The predicted positionsof the TM domains common for all three variants is according to www..org data base. The position of TM V of CXCR3Alt was predicted by an algorithm based analysis of TM domains (http://www.ch.embnet.org/software/TMPRED_form.html). Authors: Yamina A. Berchiche & Thomas P. Sakmar Title: CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways Journal title: Molecular Pharmacology

P49682 hCXCR3A 1 ------M VLE VSDHQVLND 13 CXCR3B N-terminus P49682-2 hCXCR3B 1 MELRKYGPGRLAGTVIGGAAQSKSQTKSDSITKEFLPGLYTAPSSPF P PSQ VSDHQVLND 60 P49682-3 hCXCR3Alt 1 ------M VLE VSDHQVLND 13 CXCR3A and CXCR3Alt specific TM I Conserved motifs P49682 hCXCR3A 14 AEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDR AFLPALYSLLFLLGLLGNGA 73 P49682-2 hCXCR3B 61 AEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDR AFLPALYSLLFLLGLLGNGA 120 CXCR3Alt specific P49682-3 hCXCR3Alt 14 AEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDR AFLPALYSLLFLLGLLGNGA 73 Transmembrane (TM) domain TM II TM III P49682 hCXCR3A 74 VAAVLLS RRTALSSTD TFLLHLAVADTLLVLTLPLWA VDAAVQWVFGSG LCK VAGALFNI 133 P49682-2 hCXCR3B 121 VAAVLLS RRTALSSTD TFLLHLAVADTLLVLTLPLWA VDAAVQWVFGSG LCK VAGALFNI 180 P49682-3 hCXCR3Alt 74 VAAVLLS RRTALSSTD TFLLHLAVADTLLVLTLPLWA VDAAVQWVFGSG LCK VAGALFNI 133

TM IV P49682 hCXCR3A 134 NFYAGALLLACISF DRY LNIVHATQLYRRGPPARVT LTCLAVWGLCLLFALPDFIF LSAH 193 P49682-2 hCXCR3B 181 NFYAGALLLACISF DRY LNIVHATQLYRRGPPARVT LTCLAVWGLCLLFALPDFIF LSAH 240 P49682-3 hCXCR3Alt 134 NFYAGALLLACISF DRY LNIVHATQLYRRGPPARVT LTCLAVWGLCLLFALPDFIF LSAH 193 Y TM V P49682 hCXCR3A 194 HDERLNATHCQYNFPQVGR TA L RVL QLV AGF LL PLLVMAY CYAHILAVL LVSRGQRRLRA 253 P49682-2 hCXCR3B 241 HDERLNATHCQYNFPQVGR TA L RVL QL VAGF LL PLLVMAY CYAHILAVL LVSRGQRRLRA 300 P49682-3 hCXCR3Alt 194 HDERLNATHCQYNFP QGSSSGSGCGCCSCAWAA PTR------EGSRGSHRLPA 240

TM VI TM VII P49682 hCXCR3A 254 MR LVVVVVVAFALCWTPYHLVVL VDILMDL GALARNCGRESRVDV AKSVTSGLGYMHCCL 313 P49682-2 hCXCR3B 301 MR LVVVVVVAFALCWTPYHLVVL VDILMDL GALARNCGRESRVDV AKSVTSGLGYMHCCL 360 P49682-3 hCXCR3Alt 241 G I H------PGL------RPQRPPTRACE AG IRAP L------264

P49682 hCXCR3A 314 NPLLY AFV GVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL 368 P49682-2 hCXCR3B 361 NPLLY AFV GVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL 415 P49682-3 hCXCR3Alt 265 SP I 267

Supplemental Figure 2. Amino acid sequence alignment of CXCR3 alternative splice variants. Identically to Supplemental Figure 1, the predicted positionsof the TM domains common for all three variants is based on www.uniprot.org data base. The position of TM V of CXCR3Alt was predicted by an algorithm based analysis of TM domains (http://www.ch.embnet.org/software/TMPRED_form.html). ± S.E.M. surface expressionwasmeasuredbyflowcytometry. Dataaremeanofninetotenindepdendentexperiments as acontrol.24hoursaftertransfectioncellswerestainedwithanti-CXCR3PE(clone1C6)antibodyand cell with 1.0μgofCXCR3A,1.5CXCR3B,CXCR3Altand2emptyvectorpcDNA3.1+serving Supplemental Figure 3.Cellsurfaceexpression ofCXCR3splicevariants.HEK293T cells weretransfected Journal title:Molecular Pharmacology Pathways Title: CXCChemokineReceptor 3 Alternative Splice Variants Selectively Activate Different Signaling Authors: Yamina A. Berchiche & Thomas P. Sakmar

CXCR3 surface expression (Mean Fluorescence PE) 2 4 6 8 0 0 0 0 0 C C C C ont X X X CR3 CR3 CR3 r o l B A A

l t Authors: Yamina A. Berchiche & Thomas P. Sakmar Title: CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways Journal title: Molecular Pharmacology

A CXCR3A/β-arrestin1 100nM CXCL11 0.08 100nM CXCL10 100nM CXCL9 100nM CXCL4 Control 0.06 T2 E 0.04 Ne t B R 0.02

0.00 0 5 10 15 20 25 Time (minutes)

B CXCR3B/β-arrestin1 0.020 100nM CXCL11 100nM CXCL10 100nM CXCL9 100nM CXCL4 Control 0.015 T2 E 0.010 Ne t B R

0.005

0.000 0 5 10 15 20 25 Time (minutes)

C CXCR3Alt/β-arrestin1 0.08 100nM CXCL11 100nM CXCL10 100nM CXCL9 100nM CXCL4 0.06 Control T2 E 0.04 Ne t B R

0.02

0 5 10 15 20 25 Time (minutes)

Supplemental Figure 4. Selective β-arrestin1 recruitement to CXCR3 variants in response to 100nM of chemokine. HEK293T cells transiently co-expressing the indicated CXCR3 variant GFP10 fusion with β-arrestin1 -Rluc3 at BRETmax were incubated with the indicated ligands at room temperature. BRET was measured immediately following ligand addition. Data are reported as mean values of three to four independent experiments performed in duplicate ± S.E.M. Authors: Yamina A. Berchiche & Thomas P. Sakmar Title: CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways Journal title: Molecular Pharmacology

CXCR3A/β-arrestin2 0.070

* *** *** * ** * ** ** ** * * * * * ** * * 0.065 * * * * *** * * * * * ** 100nM CXCL9 T2 * 100nM CXCL4 E 0.060 Control Ne t B R 0.055

0.050 0 5 10 15 20 25 Time (minutes)

Supplemental Figure 5. β-arrestin2 recruitement to CXCR3A in response to 100nM CXCL9 and CXCL4. These results are also shown in Fig4. of the manuscript on a different scale. HEK293T cells transiently co-expressing the indicated CXCR3 variant GFP10 fusion with β-arrestin2-Rluc3 at BRETmax were incubated with 100nM of the indicated ligands at room temperature. BRET was measured immediately following ligand addition in one minute intervals. Statistical significance of the differences between stimulated and control condition at a specific time: *, p< 0.05, **, p< 0.01, ***, p< 0.001, (Two-way ANOVA, Bonferroni’s multiple comparison test). Data are reported as mean values of four independent experiments performed in duplicate ± S.E.M. Authors: Yamina A. Berchiche & Thomas P. Sakmar Title: CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways Journal title: Molecular Pharmacology

A CXCR3A/β-arrestin1 CXCL10 0.08 0.025 CXCL11 CXCL10 0.020

0.015 0.06 0.010 T2 -12-11-10 -9 -8 -7 -6 E

0.04 Ne t B R

0.02

-12 -11 -10 -9 -8 -7 -6 -5 Log Chemokine [M]

B CXCR3B/β-arrestin1 0.012

0.010 T2 E

0.008 Ne t B R

0.006

0.004 -12 -11 -10 -9 -8 -7 -6 -5 Log Chemokine [M]

Supplemental Figure 6. CXCR3A and CXCR3B recruit β-arrestin1 in the presence of increasing concentrations of chemokine. HEK293T cells transiently co-expressing CXCR3A-GFP10 or CXCR3B-GFP10 with β-arrestin2-Rluc3 at BRETmax were incubated with increasing concentrations of the indicated ligands for 5 minutes at 37°C. BRET was measured five minutes following ligand addition. Data are reported as the mean values of three independent experiments performed in triplicate ± S.E.M (see Table 1 for curve fitting parameters). Authors: Yamina A. Berchiche & Thomas P. Sakmar Title: CXC Chemokine Receptor 3 Alternative Splice Variants Selectively Activate Different Signaling Pathways Journal title: Molecular Pharmacology

A B CXCR3A/β-arrestin1 CXCR3B/β-arrestin1 0.08 0.015 100nM Chemokine 100nM Chemokine + PTX 0.06 0.010 T2 T2

0.04 BR E BR E

t t e e N N 0.005 0.02

0.00 0.000 CXCL11 CXCL10 CXCL11

Supplemental Figure 7. PTX treatment does not inhibit chemokine induced β-arrestin1 recruitement to CXCR3A and CXCR3B.HEK293T cells transfected with CXCR3-GFP10 fusion with β-arrestin1-Rluc3 were incubated with 100ng/ml PTX for 16 hours at 37°C. β-arrestin1 recruitment to A, CXCR3A and B, CXCR3B induced with 100nM of the indicated ligands in the absence or presence of PTX. Results represent data obtained from three to four independent experiments performed in triplicate ± S.E.M.