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

CXC Chemokine Receptor 3 Alternative Splice VariantsSelectively Activate Different Signaling Pathways s

Yamina A. Berchiche1 and Thomas P. SakmarLaboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York (Y.A.B.; T.P.S.); andDepartment of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Center for Alzheimer Research, KarolinskaInstitutet, Huddinge, Sweden (T.P.S)

Received June 3, 2016; accepted August 9, 2016

ABSTRACTThe G protein-coupled receptor (GPCR) C-X-C chemokinereceptor 3 (CXCR3) is a potential drug target that mediatessignaling involved in cancer metastasis and inflammatory dis-eases. The CXCR3 primary transcript has three potential alter-native splice variants and cell-type specific expression resultsin receptor variants that are believed to have different func-tional characteristics. However, the molecular pharmacologyof ligand binding to CXCR3 alternative splice variants and theirdownstream signaling pathways remain poorly explored. Tobetter understand the role of the functional consequences ofalternative splicing of CXCR3, we measured signaling in re-sponse to four different chemokine ligands (CXCL4, CXCL9,CXCL10, and CXCL11) with agonist activity at CXCR3. BothCXCL10 andCXCL11 activated splice variant CXCR3A.Whereas

CXCL10 displayed full agonistic activity for Gai activation andextracellular signal regulated kinase (ERK) 1/2 phosphorylationand partial agonist activity for b-arrestin recruitment, CXCL9triggered only modest ERK1/2 phosphorylation. CXCL11 in-duced CXCR3B-mediated b-arrestin recruitment and little ERKphosphorylation. CXCR3Alt signaling was limited to modestligand-induced receptor internalization and ERK1/2 phosphory-lation in response to chemokines CXCL11, CXCL10, andCXCL9.These results show that CXCR3 splice variants activate differentsignaling pathways and that CXCR3 variant function is notredundant, suggesting a mechanism for tissue specific biasedagonism. Our data show an additional layer of complexity forchemokine receptor signaling that might be exploited to targetspecific CXCR3 splice variants.

IntroductionChemokine receptors are members of the G protein-coupled

receptor (GPCR) family that bind peptidic chemotactic cyto-kines also known as chemokines. One hallmark of chemo-kine receptors is that they tend to have multiple endogenousagonist ligands. Conversely, some chemokines can activatemultiple separate chemokine receptor subtypes. Accumulat-ing in vitro and in vivo evidence suggests that this promiscuityresults in a diversity of specific chemokine receptor signaling,rather than mere functional redundancy (Schall and Proud-foot, 2011). Furthermore, chemokine receptor signaling is alsocontrolled by chemokine binding to proteoglycans (Brady andLimbird, 2002; Proudfoot et al., 2003; Groom and Luster,2011b; Zweemer et al., 2014), posttranslational modifications,and chemokine dimerization (Ludeman and Stone, 2014).Another potential mechanism controlling receptor function

is the expression of alternative splice variants, which remainspoorly explored (Wise, 2012). Because many chemokine re-ceptors and chemokines are coexpressed by the same celltypes or are present in the same cellular environment duringinflammation, it is challenging to assess differences in thefunction of chemokine receptors and their alternative splicevariants in response to different ligands in in vivo settings.Inflammatory chemokine receptor CXCR3 is of increasing

clinical interest because it controls leukocyte chemotaxis and isinvolved in inflammatory disorders such as atherosclerosis aswell as in cancer metastasis (Hancock et al., 2000; Murphyet al., 2000; Groom and Luster, 2011a,b; Li et al., 2015; Lleoet al., 2015; Zhu et al., 2015). CXCR3 is expressed at the surfaceof a plethora of cells, includingmonocytes, lymphocytes, naturalkiller, and endothelial cells (García-López et al., 2001). CXCR3displays classic receptor-ligand promiscuity and binds fourchemokine ligands: CXCL4, CXCL9, CXCL10, and CXCL11(Loetscher et al., 1996; Cole et al., 1998; Mueller et al., 2008).CXCR3 couples to Gai/o heterotrimeric G proteins, which arepertussis toxin (PTX)-sensitive, and can also activate signalingpathways including MAPK, intracellular calcium flux, actinpolymerization, and chemotaxis in response to chemokines(Loetscher et al., 1996; Smit et al., 2003; Kouroumalis et al.,

This work was supported by a Fonds de la Recherche en Santé du Québecfellowship, Québec, Canada (Y.A.B.).

1Current affiliation: B cell Molecular Immunology Section, Laboratory ofImmunoregulation, National Institute of Allergy and Infectious Diseases, USNational Institutes of Health, Bethesda, MD.

dx.doi.org/10.1124/mol.116.105502.s This article has supplemental material available at molpharm.

aspetjournals.org.

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

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2005; Thompson et al., 2007). The CXCR3 receptor displaysthree alternative splice variants. After the identification of thecanonical receptor CXCR3A (Loetscher et al., 1996), twonaturally occurring alternative splice variants, CXCR3B andCXCR3Alt, were also identified (Lasagni et al., 2003; Ehlertet al., 2004). Compared with CXCR3A, CXCR3B has an addi-tional 51 amino acids at its N-terminal tail, whereas CXCR3Althas a major truncation of intracellular loop three and trans-membrane (TM) helices six and seven, which results in a ma-ture protein with possibly only five TM helices and a shortcytoplasmic C-terminal tail (Supplemental Figs. 1 and 2).The expression patterns and function of CXCR3 splice

variants in normal and disease states remain largely unex-plored. Tissue specific expression of CXCR3 alternative var-iants has been mainly quantified using reverse-transcriptionpolymerase chain reaction, and variant-specific antibodiesthat might allow measurement of expression levels remainunavailable. Nevertheless, in prostate cancer specimensCXCR3A mRNA levels are upregulated, whereas CXCR3BmRNA levels are downregulated (Wu et al., 2012). Thesedifferences receptor variant expression levels are reportedto alter the migration and invasion capabilities of prostatecancer cells (Wu et al., 2012). In addition, mRNA levels ofCXCR3A are decreased, whereas CXCR3Alt mRNA levelsare increased in CD31 peripheral blood lymphocytes inpatients suffering from Crohn’s disease, suggesting a variantspecific role of CXCR3Alt with Crohn’s disease (Manousouet al., 2008).We set out to determine whether CXCR3 alternative splice

variants activated different signaling pathways in response totheir chemokine ligands. We dissected signaling profilesacross multiple pharmacological assays when CXCR3variants were individually expressed in an human embry-onic kidney (HEK) 293T expression system. We carefullyquantitated receptor expression levels and measuredchemokine-induced Gai activity, b-arrestin recruitment,receptor internalization, and ERK1/2 phosphorylation todemonstrate that the nature of the activated signaling path-ways depends on both the alternative receptor variant and thechemokine tested. For example, nearly all ligands inducedCXCR3 internalization, whereas selected ligands inducedb-arrestin recruitment in a PTX-insensitive manner, support-ing a possible Gai-independent mechanism of b-arrestin re-cruitment to CXCR3A and CXCR3B. Moreover, our resultsstrongly suggest that b-arrestin2 is recruited to both CXCR3AandCXCR3B in a ligand-independentmanner. In summary,weshow that CXCR3 splice variants have different signalingprofiles in response to different ligands, which could lead todifferent pathophysiological roles.Our data support the conceptof biased agonism in CXCR3 variant-mediated signaling andsuggest that individual CXCR3 variants might prove to beviable drug targets.

Materials and MethodsMaterials. Recombinant chemokines were from PeproTech, Inc.

(Rocky Hill, NJ). Coelenterazine 400A for BRET2 experiments wasfrom Biotium (Hayward, CA). Forskolin, pertussis toxin (PTX), andpoly-D-lysine were from Sigma (St. Louis, MO), and the anti-CXCR3mAb (clone 1C6) directly coupled to phycoerythrin was from R&DSystems (Minneapolis, MN). Dulbecco’s modified Eagle’s mediumGlutamax, 1% penicillin-streptomycin, and Lipofectamine 2000 were

from Life Technologies (Carlsbad, CA). bovine serum albumin(BSA) fraction V, fatty acid free was from EMD Millipore (Bedford,MA), and 96-well white microplates with clear bottom and 384-wellblack microplates with clear bottom plates were from Corning(Woburn, MA).

Plasmids. The CXCR3A (Uniprot identifier P49682-1) plasmidwas purchased from the Missouri S&T cDNA Resource Center (Rolla,MO: www.cdna.org). The cDNA of splice variants CXCR3B (Uniprotidentifier P49682-2) and CXCR3Alt (Uniprot identifier P49682-3)were synthesized by Bio Basic (Markham, Canada) and subclonedinto pcDNA3.11 using with BstEII and XbaI restriction sites. ThecAMP EPA2 sensor was a gift from Michel Bouvier (Université deMontréal, Canada). CXCR3A-GFP10, CXCR3B-GFP10, and CXCR3Alt-GFP10 were constructed by ligating the coding sequence of CXCR3isoforms to GFP10, amplified by PCR from the cAMP EPAC reporterusing XhoI and XbaI, or NotI and XhoI, or NotI and XbaI, respec-tively. The amino acid sequence of the linker region between theterminal receptor residue and the fluorophore was described earlier(Percherancier et al., 2005; Berchiche et al., 2011). b-Arrestin1-Rluc3and b-arrestin2-Rluc3 sensors were constructed by C-terminallyfusing the coding sequence of b-arrestin to Rluc3, amplified from thecAMP EPAC reporter including the linker region, using HindIII andXbaI unique restriction sites (Leduc et al., 2009). All sequences wereverified by direct sequencing.

Cell Culture and Transfection. HEK293T cells (passage num-ber 5 to 15, ATCC, Manassas, VA) were maintained in Dulbecco’smodified Eagle’s medium Glutamax, 1% penicillin-streptomycin, and10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA).Transient transfection was performed in six-well plates using thepolyethylenimine method as described previously (Percherancier et al.,2005). Transient high-throughput in-plate transfections were performedin 384-well plates using Lipofectamine 2000 according tomanufacturer’sinstructions with some modifications. Briefly, cells were trypsinized,counted, and mixed with the DNA-Lipofectamine 2000 complex thendirectly plated in 0.01% poly-D-lysine coated 384-well plates at a densityof 20,000 cells/well. The total amount of transfected DNA was keptconstant at 2mg/well for six-well plates and 30 ng/well for 384-well platesby adding empty vector pcDNA3.11. Transfected DNA amounts wereadjusted to obtain comparable cell surface expression among theunfusedand GFP10- fused CXCR3 splice variants in all experiments.

Flow Cytometry. HEK293T cells transfected in six-well plateswithCXCR3alternative variantswere detached in ice-cold phosphate-buffered saline. Human peripheral blood mononuclear cells (PBMC)obtained by leukapheresis (Gift from Prof. Michel Nussenzweig, TheRockefeller University) and purified T cells (Gift from Dr. Helen Su,National Institute of Health/National Institute of Allergy and In-fectious Diseases, NIH/NIAID) obtained using a Pan T cell isolationkit (Miltenyi Biotec, San Diego, CA) were stimulated with 10 mg/mlphytohemagglutinin and cultured for 10 days in RPMI containing 10%fetal bovine serumand 50 IU/ml interleukin-2. Cells were labeledwithmonoclonal 1C6 phycoerythrin-conjugated antibody (BD Biosciences,San Jose, CA) that recognizes all three CXCR3 alternative variants for30 minutes at 4°C in BRET buffer [phosphate buffered saline (PBS)containing 0.5 mM MgCl2 and 0.1% BSA]. Cells were then washedthree times in ice-cold PBS. Cell surface expression was quantified byflow cytometry using the Accuri C6 flow cytometer (BD Biosciences).We also determined receptor surface expression of transfected CXCR3variants in HEK293T and the total CXCR3 quantities in bothactivated PBMC and T cells using the Quanti-BRITE standardizationbeads (BD Biosciences). Receptor quantities typically reached around1.3 to 2.5 � 104 antibody-binding sites per cell for both HEK293Ttransfected CXCR3 alternative variants and endogenous CXCR3expressed in activated PBMCs and purified T cells.

BRET Measurements. HEK293T cells transfected in six-wellplates were seeded in 0.01% poly-D-lysine coated 96-well, whitemicroplates with clear bottom 24 hours after transfection at a densityof 90,000 cells/well. Forty-eight hours posttransfection, media wasreplaced with BRET buffer. Coelenterazine 400A was added at a final

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concentration of 5 mM followed by a 5 minutes incubation at roomtemperature (RT). Luminescence and fluorescence readings werecollected using the Synergy NEO plate reader from Biotek (Winooski,VT) and Gen5 software. BRET2 readings between Rluc3 and GFP10were collected by sequential integration of the signals detected in the365 to 435 nm (Rluc3) and 505 to 525 nm (GFP10) windows. BRET2

ratios were calculated as described previously (Leduc et al., 2009;Berchiche et al., 2011). All BRET experiments were performed whilecells remained attached to the 96-well plates.

Adenylyl Cyclase Activity. cAMP was determined by using theRluc3-EPAC-GFP10, a BRET2 cAMP sensor, as described previously(Leduc et al., 2009). Briefly, cells cotransfected with 1.0 mg CXCR3Aor 1.5 mg CXCR3B or 1.0 mg CXCR3Alt and 0.03 mg Rluc3-EPAC-GFP10 reporter were seeded into poly-D-lysine coated 96-well plates24 hours after transfection. Coelenterazine 400Awas added to the cellsfollowed by a 5-minute incubation at RT. Cells were then stimulatedwith ligand in the presence of 5 mMof forskolin at RT for 3minutes. Forexperiments involving PTX, 100 ng/ml of PTX was added to the mediaand cells were treated with PTX for 16 hours before stimulation withforskolin and chemokines.

Arrestin Recruitment. b-Arrestin recruitment was measuredby BRET2 by cotransfection of 1.0 mg of CXCR3A-GFP10 or 1.5 mgof CXCR3B-GFP10 or 0.5 mg CXCR3Alt-GFP10 with 0.05 mg ofb-arrestin2-Rluc3 or b-arrestin1-Rluc3. Transfected cells were seededinto poly-D-lysine coated 96-well plates. We first determined the opti-mal donor (RLuc3) quantity and then performed acceptor (GFP10)titrations experiments with optimal donor quantity to determineBRETmax ratios of each -GFP10 and -Rluc3 fusion pair as describedpreviously (Berchiche et al., 2007; Kalatskaya et al., 2009; Bonneterreet al., 2016). For dose-response experiments, cells expressing the -GFP10and -Rluc3 fusion proteins at BRET2

max ratios were stimulated for5 minutes at 37°C with increasing concentrations of the indicatedligand before the addition of the substrate, unless specified other-wise. The values were corrected to net BRET by subtracting the back-ground BRET2 signal detected when the -Rluc3 construct wasexpressed alone.

Endocytosis. HEK293T transfected with 1.0 mg CXCR3A or1.5 mg CXCR3B or 1.0 mg CXCR3Alt were incubated with 100 nM ofchemokines at 37°C with gentle agitation, followed by immediateincubation on ice to stop the reaction. Surface bound chemokines wereremovedwith acidwashing (50mMglycine, pH 2.7, 150mMNaCl) andcells subsequently washed twice with ice-cold PBS. Cells were thenlabeled with anti-CXCR3 1C6 phycoerythrin-conjugated antibody for30 minutes on ice following manufacturer’s instructions. Antibody in-cubation was followed by two washes with ice-cold PBS. Cell surfacereceptor expression was quantified by flow cytometry using the AccuriC6 flow cytometer (BD Biosciences). Surface expression of CXCR3 afterligand incubation at 37°C was expressed as percentage of CXCR3expression comparedwith a sample drawnbefore addition of the ligandsand kept on ice before the acid wash and antibody staining.

Erk1/2 Phosphorylation. Chemokine-induced MAPK pathwayactivation was determined by InCell Western assay (Li-COR). Briefly,cells were transfected in a high-throughput in-plate transfectionmanner with 10 ng of CXCR3A, 15 ng of CXCR3B, or 10 ng ofCXCR3Alt. After 24 hours, the media was removed and replaced with1� Hank’s Balanced Salt Solution-20 mM HEPES pH 7.4 supple-mented with 0.2% BSA (HBSS-H 0.2% BSA). Transfected cells werethen stimulated with 100 nM of chemokines at 37°C for differentperiods of time (2, 5, 7, 10, and 15 minutes) or in the presence ofincreasing concentration of ligand. ERK activity was stopped by fixingcells with a solution of PBS containing 3.7% formaldehyde for20 minutes at RT. Detection of phosphor-ERK1/2 and total ERK2proteins were performed as described previously (Wong, 2004). Plateswere scanned using the Li-COR Odyssey infrared reader.

Data Analysis. Data were analyzed using Prism 6.0 software(GraphPad Software, San Diego, CA). Statistical significance of thedifferences between the various conditions was determined usingone-way analysis of variance with Tukey’s post t test or Bonferroni’s

post t test when appropriate. When indicated, differences of top orbottom values were also determined using sigmoidal dose responsesimultaneous curve fitting.

ResultsGai Activity of CXCR3 Is Limited to Specific Alter-

native Splice Variants. We evaluated the ability of CXCR3alternative splice variants to activate Gai signaling in re-sponse to chemokines (Fig. 1). Receptor cDNA quantities wereadjusted to obtain comparable cell surface expression for allCXCR3 variants, which were kept constant across all assays(Supplemental Fig. 3). HEK293T cells coexpressing the EPACcAMP biosensor previously described (Leduc et al., 2009) withCXCR3 alternative variants were stimulated with increasingconcentrations of chemokines in the presence of forskolin inlive cells and the inhibition of forskolin induced cAMP pro-duction as a consequence of Gai activation was measured.Chemokine receptor CXCR3A inhibited cAMP production

when stimulated with CXCL10 and CXCL11, and both chemo-kines had similar efficacies and potencies (Fig. 1A, Table 1).These results are in line with previous reports (Scholten et al.,2012). Yet, chemokines CXCL9 and CXCL4 failed to triggersignificant Gai activity even at chemokine concentrations ashigh as 100 nM. Furthermore, CXCR3B-mediated Gai activa-tion was only detected after stimulation with 100 nM ofCXCL11 but not at lower concentrations or in the presenceof the other chemokine ligands (Fig. 1B). In addition, weassessed the ability of chemokines to trigger Gas activationvia CXCR3B using the EPAC biosensor in the absence offorskolin (Leduc et al., 2009). As opposed to previously re-ported Gas activity in human microvascular endothelial cellline-1 (HMEC-1) stably expressing CXCR3B (Lasagni et al.,2003), we found that in our assays it failed to activate Gasupon treatment with its chemokine ligands (data not shown).Chemokine receptor variant CXCR3Alt failed to inducesignificant Gai activation in response to all chemokines tested(Fig. 1C), although the truncated receptor was expressedat the cell surface as measured by flow cytometry. Resultsobtained with CXCR3Alt were similar to our results obtainedwhen cells were cotransfected with the EPAC biosensor andempty vector pcDNA3.11 (Fig. 1D).CXCR3 Splice Variants Differentially Recruit

b-Arrestins. Wemeasured b-arrestin recruitment to CXCR3in live cells using a BRET proximity assay, which was exten-sively used to study receptor interaction with arrestins, ligand-induced receptor/arrestin conformational changes, as well asreceptor dimerization (Hamdan et al., 2005; Kalatskaya et al.,2009; Berchiche et al., 2011). We performed acceptor/donortitration experiments by coexpressing increasing quantities ofCXCR3-GFP10 acceptors with fixed quantities of b-arrestin1-Rluc3 or b-arrestin2-Rluc3 donors (Fig. 2). Surprisingly, wemeasured a significant and saturating basal BRET signal in theabsence of ligands for CXCR3A with both b-arrestins andCXCR3B with b -arrestin2, which further increased after stim-ulation with 100 nM CXCL11 (Fig. 2, A–C). As for CXCR3B/b-arrestin1, acceptor/donor titration in the absence of ligandresulted in a bystander curve,most likely due to randomcollisionof coexpressed BRET pairs (Bonneterre et al., 2016). However,stimulation with CXCL11 resulted in a saturating curve sup-porting ligand-induced b-arrestin1 recruitment to CXCR3B(Fig. 2D). This suggests a preference of CXCR3B forb-arrestin2

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in the absence of ligand. Curve fitting of the data obtainedfrom the titration experiments allowed us to determine bothBRETmax and BRET50 values in the absence and presence of100 nM CXCL11 (Table 2). BRETmax corresponds to the best

acceptor anddonor concentrations determinedat saturationwithoptimal sensitivity, which we used to assess ligand-inducedCXCR3/b-arrestin interaction. Furthermore, BRET50 corre-sponds to the acceptor/donor ratio giving 50% of the maximal

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 cAMPRluc3-EPAC-GFP10 sensor in HEK293T cells. Cells wereexposed to 5 mM forskolin with increasing concentrations of the indicated ligands at room temperature for 5 minutes before readings. Results areexpressed as the % of inhibition of forskolin-induced cAMP production. Data are reported as mean values of 4–6 independent experiments performed intriplicate 6 S.E.M.

TABLE 1Summary 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 (ERK1/2phosphorylation and Gai activity) units. Errors are presented as 6 S.E.M.

CXCR3A CXCR3B

CXCL11 CXCL10 CXCL11

cAMP inhibitionN 4 4 6IC50 (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-arrestin2N 3 3 3EC50 (nM) 8 71 61pEC50 6 S.E.M. 28.2 6 0.1 27.3 6 0.2 27.2 6 0.1BRET2

max 6 S.E.M. 0.11 6 0.01 0.07 6 0.01 0.026 6 0.001a

b-arrestin1N 3 3 3EC50 (nM) 16 32 33pEC50 6 S.E.M. 27.8 6 0.1 27.5 6 0.1 27.6 6 0.2BRET2

max 6 S.E.M. 0.06 6 0.01a 0.025 6 0.003a 0.01 6 0.001ERK1/2 phosphorylation

N 4 4 6EC50 (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.

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signal and reflects the propensity of BRET partners tointeract.Stimulation of CXCR3A/b-arrestin1, CXCR3A/b-arrestin2, as

well as CXCR3B/b-arrestin2 pairs with 100 nMCXCL11 resultedin an increased BRETmax (Fig. 2, A–C, and Table 2) comparedwith basal conditions. This increase is a direct consequence ofchanges in the distance and/or orientation between the -Rlucdonor and -GFP10 acceptor fusions. This strongly suggests thatCXCL11 induces conformational changes in these receptor/b-arrestinpreformedcomplexes. Inaddition,CXCL11stimulationalso decreasedBRET50 values, shifting the saturation curve to theleft for these receptor/arrestin pairs, which indicates a higherpropensity of CXCR3AandB variants to interactwithb-arrestinsin the presence than in the absence of CXCL11 (Table 2).In contrast, titration of the CXCR3Alt-GFP10 acceptor

with both b-arrestins donors showed no ligand-dependent or

-independent association, indicating that CXCR3Alt does notinteract with b-arrestins (Fig. 2, E and F).Moreover, we assessed the specificity of the interactions we

measured to ascertain whether select CXCR3 variants in-teract with b-arrestins in the absence of ligand. We cotrans-fected cells expressing the BRET2 b-arrestin donor andCXCR3 acceptor fusions with the corresponding FLAG-b-arrestin and measured its impact on the basal BRETsignal. We observed a transfected FLAG-b-arrestin quantity-dependent decrease of the basal BRET signal for CXCR3Aand both b-arrestins as well as CXCR3B and b-arrestin2,whereas CXCR3B and b-arrestin1 remained unaffected (Fig.3). A decrease in a dose-dependent manner of the basal BRETsignal indicates a competition between the donor fusedb-arrestin and the corresponding FLAG-tagged b-arrestin,which in turn supports a specific ligand-independent

Fig. 2. CXCR3 variants constitutively interact with select b-arrestins. Acceptor/donor titration curves of CXCR3 splice variants fused with GFP10 andb-arrestin-Rluc3 were obtained after transfection of HEK293T cells with increasing quantities of CXCR3-GFP10 fusion with 0.05 mg b-arrestin-Rluc3fusions 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 nMCXCL11. Data were plotted and fittedto a one-site binding hyperbola equation. BRET50 and BRETmax values were calculated for each BRET2 couple. Results obtained from at least threeindependent experiments, carried out in triplicate 6 S.E.M are shown.

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interaction between select CXCR3 alternative variants andb-arrestin.Next, we quantified ligand-mediated b-arrestin recruitment

to CXCR3 at BRETmax by performing kinetic experiments.These were carried out at RT to fully capture the details ofrecruitment kinetics after chemokine stimulation (100 nM).CXCR3A treatment with CXCL11 or CXCL10 resulted in arapid b-arrestin2 recruitment reaching a plateau in 5 to7 minutes (Fig. 4A). Similar results were obtained withb-arrestin1 (Supplemental Fig. 4). A small but statisticallysignificant elevation in b-arrestin2 recruitment to CXCR3Aafter stimulation with CXCL9 or CXCL4 was also observed(Fig. 4A, Supplemental Fig. 5). In contrast, CXCL9 or CXCL4failed to induce b-arrestin1 recruitment to CXCR3A (Supple-mental Fig. 4A). Our data show that CXCR3A and CXCR3Bacceptor fusions are functional and are able to recruit bothb-arrestins in response to ligands. In addition, these results

indicate a receptor preference for specific b-arrestins, which isselectively influenced by chemokines.Also, CXCL11 increased b-arrestin2 recruitment to

CXCR3B with the signal reaching a plateau within the first5 minutes of stimulation (Fig. 4B). CXCL10, CXCL9, andCXCL4, a chemokine suggested to be a selective high affinityligand of CXCR3B (Lasagni et al., 2003), failed to triggerb-arrestin2 recruitment to CXCR3B. Similar results wereobtained with b-arrestin1 (Supplemental Fig. 4B). As forCXCR3Alt, all chemokines failed to induce both b-arrestin1and b-arrestin2 recruitment to this C-terminally truncatedalternative variant (Fig. 4C, Supplemental Fig. 4C).To catalog the pharmacology of CXCR3 variants in response

to their chemokines, we measured the effect of increasingconcentrations of ligands on b-arrestin recruitment toCXCR3A and CXCR3B (Table 1). Our results obtained withCXCL11 and CXCL10 are consistent with previously reported

Fig. 3. Constitutive b-arrestin interaction is receptorvariant specific. HEK293T cells transfected with the in-dicated receptor variant/b-arrestin at BRETmax, in the ab-sence (control) or presence of increasing levels of C-terminallyFLAG-taggedb-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 ofmicrograms 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 increasinglevels of coexpressed C-terminally FLAG-tagged b-arrestin:*P, 0.05, **P, 0.01, ***P, 0.001, ****P, 0.0001 [one-wayanalysis of variance (ANOVA), Bonferroni’s multiple compar-ison test]. Results obtained for three independent experi-ments carried out in triplicate 6 S.E.M are shown.

TABLE 2Fitted curve parameters of CXCR3 (acceptor)/b-arrestin (donor) titration curves. BRET50 and BRETmaximal values are given in absolute unitsErrors are presented as 6 S.E.M.

Receptor Variant

CXCR3A-GFP10 CXCR3B-GFP10

Basal CXCL11 (100 nM) Basal CXCL11(100 nM)

b-Arrestin2-Rluc3N 4 4 3 3BRET50 6 S.E.M 24 6 5 5 6 1 9.5 6 0.4 7.9 6 0.3BRETmax 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-Rluc3N 4 4 3 3BRET50 6 S.E.M 47 6 12 2 6 1 ND 9 6 3BRETMax 6 S.E.M 0.034 6 0.002 0.07 6 0.01 ND 0.023 6 0.001

N.D., not detectable.

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observations obtained with a BRET1 arrestin proximity assay(Scholten et al., 2012). Indeed, CXCL11 acts as a full agonistwith a potency of 8 nM, whereas CXCL10 acts as a partialagonist with a potency of 70 nM (Fig. 5A) on the b-arrestin2recruitment to CXCR3A. A similar rank order of potencies wasobserved with b-arrestin1 (Table 1, Supplemental Fig. 6A). Asfor CXCR3B, CXCL11 caused arrestin recruitment withpotencies of 61 and 33 nM for b-arrestin2 and b-arrestin1,respectively (Fig. 5B, Table 1, Supplemental Fig. 6B). Theseresults suggest that the longer N terminus of CXCR3B issufficient to change the receptor’s ability to respond to chemo-kines and recruit b-arrestins. Moreover, these data suggestthat although the N-terminal tail of CXCR3Alt is identicalto that of CXCR3A, not surprisingly differences in the C

terminus of CXCR3Alt are sufficient to perturb its ability torecruit b-arrestins.Chemokine Triggered b-Arrestin Recruitment to

CXCR3 is PTX Insensitive. To determine whether ligand-induced b-arrestin recruitment to CXCR3A and CXCR3Bare linked to Gai signaling, we tested their sensitivity toGai/o-inactivating PTX treatment (Fig. 6). Ligand-mediatedb-arrestin2 (Fig. 6, A-C) and b-arrestin1 (Supplemental Fig. 7)recruitment to CXCR3A and CXCR3B were resistant to PTXtreatment. As expected, PTX treatment inhibited ligand-induced Gai activity mediated by CXCR3A (Fig. 6A). Thisresult suggests that b-arrestin recruitment to CXCR3A occursindependently from Gai signaling in response to CXCL11 andCXCL10. PTX treatment also impaired CXCL11 triggered

Fig. 4. Receptor variants selectively recruit b-arrestin2in response to 100 nM of chemokines. HEK293T cellstransiently coexpressing the indicated CXCR3 variantGFP10 fusion with b-arrestin2-Rluc3 at BRETmax wereincubated with 100 nM of the indicated ligands at roomtemperature. A, CXCR3A/b-arrestin2; B, CXCR3B/b-arrestin2; C, CXCR3Alt/b-arrestin2. BRET was mea-sured immediately after ligand addition. Data are re-ported asmean values of four independent experimentsperformed in duplicate 6 S.E.M.

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CXCR3BGai activity, even if it was only detected with 100 nMof chemokine (Fig. 6C) comparable to CXCR3A.Chemokines Induce Internalization of All CXCR3

Splice Variants. We stimulated HEK293T cells expressingCXCR3 variants with chemokine ligands (100 nM) for varioustime periods at 37°C to assess ligand-induced receptor in-ternalization. Excess chemokine was removed by acid washand remaining surface receptors were quantified by flowcytometry. Stimulation with chemokines induced internaliza-tion of all receptor variants (Fig. 7). Chemokine CXCL11induced CXCR3A internalization was modest in comparison

with the other chemokines tested for this receptor variant, yetour results with CXCL11 remain comparable to previouslyreported observations (Scholten et al., 2012). Moreover, nearly40% of CXCR3A receptors internalized within the first10 minutes of stimulation with CXCL10 and CXCL4, com-pared with CXCL9, which required 30 minutes of stimulationto reach 40% internalization. Similarly, nearly 50% of CXCR3Breceptors internalized during the first 10minutes after incubationwith CXCL10, CXCL9, and CXCL4.Yet CXCL11 only induced moderate receptor internaliza-

tion in the same fashion as CXCL11-induced CXCR3A

Fig. 5. CXCR3A and CXCR3B recruit b-arrestin2 in the presence of increasing concentrations of chemokine. HEK293T cells transiently coexpressingA, CXCR3A-GFP10 or B, CXCR3B-GFP10 with b-arrestin2-Rluc3 at BRETmax were incubated with increasing concentrations of the indicated ligands for5 minutes at 37°C. BRET was measured 5 minutes after ligand addition. Data are reported as the mean values of three independent experimentsperformed in triplicate 6 S.E.M (see Table 1 for curve fitting parameters).

Fig. 6. PTX treatment only affects chemokine induced Gai activity of CXCR3A and CXCR3B. HEK293T cells transfected with CXCR3 and the EPACcAMP 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 orpresence of PTX. The forskolin induced cAMP production measured in the absence of chemokine and PTX was set to 100% (white bars). b-Arrestin2recruitment to CXCR3A (B) and CXCR3B (D) induced with 100 nM of the indicated ligands in the absence or presence of PTX. Results represent dataobtained from three to five independent experiments performed in triplicate 6 S.E.M.

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internalization. Interestingly, CXCL11, CXCL9, and CXCL4induced a robust and rapid CXCR3Alt internalization,whereas CXCL10 had little to no effect. Surprisingly, in ourassay CXCL4 (100 nM), a CXCR3B ligand, induced robustreceptor internalization of all three CXCR3 variants. CXCL4chemokine is reported to bind to CXCR3A and triggerchemotaxis at high chemokine concentrations of 500 to 750 nM(Mueller et al., 2008; Korniejewska et al., 2011). Our resultsindicate that all ligands (100 nM) are able to bind CXCR3variants expressed at the cell surface and thus trigger theirfunctionCXCR3 Splice Variants Display Different ERK1/2

Phosphorylation Profiles. We quantitatively measuredligand-induced ERK1/2 phosphorylation in HEK293T cellsexpressing CXCR3 variants. Transfected cells were stimu-lated with chemokines (100 nM) at 37°C for various timeperiods (Fig. 8, A, C, and D). ERK1/2 phosphorylation wasquantified using an InCell Western approach. This methodwas previously used to measure ERK1/2 phosphorylation ofcannabinoid receptor CB1, a member of the GPCR familycoupled to Gai/o heterotrimeric G proteins (Daigle et al.,2008). Three out of the four chemokines tested inducedCXCR3A-mediated ERK1/2 phosphorylation (Fig. 8A). In cellsexpressing CXCR3A, CXCL11 induced the longest and most

significant response starting as early as 2 minutes afterligand addition (2946 37% of basal) and lasting 10 minutes(247 6 21% of basal), with a peak phosphorylation mea-sured at 5 minutes (4026 37% of basal). In comparison withCXCL11, CXCL10-mediated ERK1/2 phosphorylation throughCXCR3A was shorter and more modest. Its signal was de-tected between 2 (250 6 60% of basal) and 5 minutes (199 625% of basal) after stimulation. Chemokine CXCL9 alsotriggered a moderate elevation of ERK1/2 phosphorylationstarting at 5 minutes (225 6 24% of basal), which declinedafter 10 minutes (217 6 26% of basal). In contrast, CXCR3Bactivation by chemokines showed a different ERK1/2 phos-phorylation profile than for CXCR3A (Fig. 8B). Indeed,CXCL11 triggered a modest ERK1/2 phosphorylation startingat 5 minutes (203 6 18% of basal) and lasted 10 minutes(170 6 13% of basal). Moreover, CXCL9 also induced a shortERK1/2 phosphorylation, peaking at 10 minutes (192 6 22%of basal) and a short and weak, yet statistically significant,signal at 5 minutes (159 6 22% of basal) after stimulationwith CXCL4. Surprisingly, cells expressing CXCR3Alt variantalso responded to ligands and we measured ERK1/2 phosphor-ylation after stimulation with CXCL11, CXCL10, and CXCL9(Fig. 8D). Treatment with CXCL11 increased ERK1/2phosphorylation between 5 to 10 minutes, with a peak

Fig. 7. Chemokines induce CXCR3 splice variant’s internalization. HEK293T cells expressing CXCR3A; CXCRB, and CXCR3Alt were incubated with100 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 acidwashing, and remaining surface CXCR3 was measured by flow cytometry. Data are mean of three to four independent experiments 6 S.E.M.

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phosphorylation detected after 7 minutes (195 6 16% ofbasal). We also quantified modest ERK1/2 phosphorylationfrom 5 to 15 minutes after treatment with CXCL10. Incontrast, CXCL9 triggered ERK1/2 phosphorylation at asingle time point of 10 minutes (155 6 14% of basal).Compared with CXCR3A, chemokines induced weaker

ERK1/2 phosphorylation through CXCR3B and CXCR3Alt.However, these changes were statistically significant com-paredwith cells transfectedwith the receptor and treatedwithassay buffer only. Furthermore, to ensure that the signalswere indeed the result of CXCR3 alternative variants, allexperiments were performed in parallel with cells transfectedwith empty vector pcDNA3.11. These cells failed to respond tochemokines (data not shown), indicating that the responsesmeasured for CXCR3 were ligand induced and receptor-variant specific. We also collected pharmacological data forCXCR3A by taking advantage of favorable dynamic range andthe quantitative nature of the ERK1/2 phosphorylation InCellWestern assay.Wemeasured ERK1/2 phosphorylation in cellstransfected with CXCR3A in response to increasing concen-trations of CXCL11 and CXCL10 (Fig. 8B). Our resultsindicate that both CXCL11 and CXCL10 induce ERK1/2phosphorylation with similar efficacy and potency and arefull agonists for this pathway (Table 1).

DiscussionThe goal of this study was to explore the specific intracel-

lular signaling pathways activated by CXCR3 splice variantsin response to their chemokine ligands. We report that

different CXCR3 splice variants induce quantitatively distinc-tive signaling responses after ligand stimulation and thatreceptor signaling efficacy depends on the splice variant/chemokine pair assessed. Receptor variant/chemokine pairsand the corresponding pathways activated are summarized inTable 3. The ability of ligands to activate different intracellu-lar signaling pathways via the same receptor to differentextents is a widely observed phenomenon and has beendocumented for many GPCRs, including chemokine receptors.This characteristic has been given several names includingbiased agonism, stimulus trafficking, and functional selectiv-ity. Biased agonism of GPCRs supports the existence ofmultiple active receptor conformations that ligands maydifferentially stabilize, leading to the activation of specificsignaling pathways (Kenakin, 2007, 2009). The nonredundantsignaling responses we report for CXCR3 alternative splicevariants could be explained if chemokines stabilized distinctreceptor splice variant conformations leading to the activationof different intracellular signaling pathways.Indeed, CXCR3A-mediated Gai activity was only detectable

in response to CXCL11 and CXCL10, which are in line withthe Gai activity previously reported for this canonical receptor(Scholten et al., 2012). Interestingly, CXCL11 also inducedCXCR3B-mediated Gai activity, yet only at a saturatingconcentration of 100 nM. Sulfation of Tyr27 and Tyr29 inthe N-terminal region of CXCR3A is essential for chemokinebinding and thus receptor function (Colvin et al., 2006). Thelonger N terminus of CXCR3B contains two additionalpotential sulfation sites (Tyr6 and Tyr40). These Tyr residuescould also be sulfated or influence the sulfation of the Tyr

Fig. 8. CXCR3 variants induced ERK1/2 phosphorylation with different intensities after stimulation with chemokines. HEK293T cells transientlyexpressing CXCR3 variants were incubated at 37°C with the indicated ligands CXCR3A (A), CXCR3B (C), CXCR3Alt (D) with 100 nM of chemokines atthe 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 thedifferences between stimulated and control condition: *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 (one-way ANOVA, Bonferroni’s multiplecomparison test).

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residues common to both variants, and therefore cotransla-tional Tyr sulfation might explain the Gai activity measuredfor CXCR3B.As expected, Gai activation of CXCR3A was inhibited by

PTX treatment. Surprisingly, CXCR3B-induced Gai activa-tion was also inhibited by PTX treatment, suggesting that thisGai activity is specific. Relatedly, stimulation of CXCR3AwithCXCL11 or CXCR10 and of CXCR3B with CXCL11 furtherincreased basal b-arrestin recruitment to the receptor andwas PTX insensitive. Taken together, our results indicate thatligand-mediated Gai signaling and b-arrestin recruitmentoccur independently from each other. Our findings alsosuggest that chemokine receptor variant/ligand pairs selec-tively determine the receptor/b-arrestin conformation as wellas the efficacy of Gai activity. Similar to the ligand-initiatedb-arrestin recruitment and Gai signaling, we report that theCXCR3-mediated ERK1/2 phosphorylation intensity and sig-nal duration measured depend on the splice variant/chemo-kine pair assessed. For example, CXCL11-mediated ERK1/2phosphorylation through CXCR3A was stronger and lastedlonger than the one mediated through both CXCR3B andCXCR3Alt.Ligand-independent b-arrestin recruitment was previ-

ously reported for chemokine receptor CCR1 (Gilliland et al.,2013), atypical chemokine receptor ACKR2/D6 (McCullochet al., 2008), as well as a chimeric receptor composed ofACKR3/CXCR7 with its C-terminal tail swapped with CXCR4(Gravel et al., 2010). C-terminal tails of both D6 and CCR1show basal phosphorylation, which plays an important role intheir ligand independent activity. Our data are the first toindicate that ligand-independent b-arrestin recruitment canalso differentially occur for the different CXCR3 alternativesplice variants. Such constitutive b-arrestin recruitment toCXCR3A and CXCR3B might also arise from low levels ofbasal receptor phosphorylation. Indeed, the C-terminal tail ofCXCR3A andCXCR3B contains nine Ser residues and one Thrresidue that might allow for some basal level of Ser/Thrphosphorylation, favoring the interaction with b-arrestins.An earlier report of a low level of phosphorylation measuredfor transfected CXCR3 further supports this hypothesis(Colvin et al., 2004).Receptor internalization may occur in both b-arrestin-

dependent and -independent fashion. Here, we demonstratethat the internalization profiles of all three CXCR3 alterna-tive splice variants fail to correlate with ligand-inducedb-arrestin recruitment, supporting a b-arrestin-independentinternalization mechanism. Similarly, Rajagopal et al. (2013)recently showed that CXCL11-induced b-arrestin recruitmentto the canonical CXCR3 receptor does not correlate with

CXCL11-induced internalization. Likewise, CXCL11 andCXCL10 provoked receptor internalization in a b-arrestin-independent fashion when CXCR3A was expressed in L1.2murine cells (Meiser et al., 2008). Furthermore, b-arrestinsare well recognized for their role as scaffold proteins fordownstream signaling phosphorylation cascades (DeWireet al., 2007). ERK1/2 phosphorylation induced by CXCR3splice variants does not correlate either with ligand-inducedb-arrestin recruitment. This indicates that b-arrestin maynot be required for ERK1/2 phosphorylation provoked byspecific receptor variant/ligand pairs such as CXCR3B/CXCL9and CXCR3Alt/CXCL10. Nevertheless, b-arrestins mayplay a role in ERK1/2 phosphorylation provoked by theCXCR3A/CXCL11 pair. Similarly, only CXCR3A/CXCL11 andCXCR3A/CXCL10 pairs provoke Gai activity that correlateswith their ERK1/2 phosphorylation profile, supporting thepossibility that ERK1/2 phosphorylation may be a conse-quence of Gai activation.Many GPCRs with highly truncated alternative splice

variants lacking some TM helices are reported in the litera-ture (Wise, 2012). Alternative splice variant CXCR3Alt is apredicted five TM domain receptor with a short C-terminalend (Ehlert et al., 2004). CXCR3Alt lacks the third intracel-lular loop important for G-protein interaction and subse-quent activation of intracellular components (Thelen andThelen, 2008). Stimulation of this splice variant resulted inweak but statistically significant ERK1/2 phosphorylationand chemokine-induced receptor internalization. Neverthe-less, CXCR3Alt failed to induce Gai activation and b-arrestinrecruitment in the absence or presence of chemokines. Com-pared with CXCR3A and CXCR3B, the C terminus of CXCR3Altlacks the Ser and Thr phosphorylation sites typically phos-phorylated by GRKs and required for b-arrestin recruitment.This fundamental difference of CXCR3Alt and its limitedsignaling further supports the idea that CXCR3 splice vari-ants play different roles in fine tuning chemokine-inducedsignaling responses.It may be speculated that CXCR3Alt splice variant behaves

as an atypical chemokine receptor. Its role could be toscavenge chemokines and contribute to the establishment ofa chemokine gradient, similarly to chemokine receptorCXCR7, which does not activate Gai heterotrimeric G protein,yet still recruits b-arrestin and induces ERK1/2 phosphoryla-tion and internalization in response to CXCL12 (Boldajipouret al., 2008; Kalatskaya et al., 2009; Levoye et al., 2009).Another explanation for our results could be that the role ofCXCR3Alt is to modulate the function of a chemokine receptorsuch as CXCR3A, because these variants can be coexpressedin cells (Aksoy et al., 2006) and because truncated GPCRs

TABLE 3Summary 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, CXCL4ERK1/2 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.

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could act as dominant negative of CXCR3A (Wise, 2012). Thelater possibility is further supported by the atypical andpredicted five TM arrangement of CXCR3Alt. An additionalpossibility could be that activation of CXCR3Alt leads to otherfunctional consequences we did not assess. For example,coupling to Ga12/13 (Kouroumalis et al., 2005) or Gai proteinsisoforms i2 and i3 (Thompson et al., 2007) are also reported forCXCR3.We cannot rule out that our observations are cell-type

specific, yet CXCR3 natural ligands behave as perfect biasedligands (Kenakin and Christopoulos, 2013), which activateonly specific signaling pathways but not others. For example,CXCL4 induces internalization of all variants, modestlystimulates ERK1/2 phosphorylation via CXCR3B and yethas no other effects on any of the receptor variants in thepathways assessed. Lack of signaling in our assays of specificCXCR3 variant/chemokine pairs could also be explained bythe need of a GPCR modifying protein such as CXCR3A splicevariant to induce signaling, which in turn could contribute tofurther diversify CXCR3-mediated signaling. Another possi-bility is that specific CXCR3 alternative splice variants that donot signal in our assays play a role to internalize specificchemokines and help establish gradients in specific tissues.Nevertheless, the differences we report support that CXCR3

ligands stabilize different conformations of CXCR3 splicevariants. As a consequence, each CXCR3 variant/chemokinepair is likely to fulfill different functions in vivo. Althoughother chemokines receptors, such as CCR2 and (Charo et al.,1994) CCR9 (Yu et al., 2000), possess alternative splicevariants with some differences expression and signaling levels(Wong et al., 1997; Sanders et al., 2000), our work is the first toquantify the function of chemokine receptor CXCR3 alterna-tive splice variants in response to its four natural ligandsacross multiple four signaling pathways.In addition, our work provides insight into the differences in

the signaling abilities of CXCR3 alternative splice variantsthat are a direct consequence of their differences in the N- andC-terminal regions and intracellular loops. Our finding thatchemokine receptor CXCR3 splice variants are able to selec-tively stimulate specific signaling pathways in response todifferent chemokines supports the idea that the chemokinesystem is not functionally redundant. Instead, the differentactivation patterns of CXCR3 splice variants indicate that thechemokine system displays an additional layer of complexityfine-tuned by receptor splice variants. The differences inCXCR3 variant/chemokine pair signaling must be taken intoaccount in the context of targeting CXCR3 with smallmolecules. Furthermore, these differences could form a basisto design smallmolecules that selectivelymodulate in a biasedmanner specific receptor alternative splice variants in diseasesettings.

Acknowledgments

The authors thank: the Bouvier laboratory for the gift of the EPACcAMP reporter; Michel Nussenzweig for the gift of human PBMC aswell as Dr. Helen Su for the gift of purified human T cells; Dr.CassandraKoole andDr. Alexandre Fürstenberg for critical reading ofthemanuscript andManija Kazmi as well as the other members of theSakmar laboratory; and the High-Throughput and SpectroscopyResource Center (HTSRC) and the Flow Cytometry Resource Center(FCRC) at The Rockefeller University for advice and access toinstrumentation.

Authorship Contributions

Participated in research design: Berchiche and Sakmar.Conducted experiments: Berchiche.Performed data analysis: Berchiche.Wrote or contributed to the writing of the manuscript: Berchiche

and Sakmar.

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Address correspondence to: Thomas P. Sakmar, Laboratory of ChemicalBiology and Signal Transduction, Box 187, RRB 510, 1230 York Ave., NewYork, NY, 10065. E-mail: sakmar@rockefeller.edu

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