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J. Biol. Chem., Vol. 282, Issue 13, 9547-9555, March 30, 2007
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i2 Activation by Gi3 in CXCR3-mediated Signaling*
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From the
Wellman Center for Photomedicine, Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114, ¶NIEHS, Transmembrane Signaling Group, Laboratory of Signal Transduction, National Institutes of Health, Research Triangle Park, North Carolina 27709, and Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Received for publication, November 27, 2006 , and in revised form, January 16, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i2 and Gi3, two members of the Gi/o protein family, but whether these two Gi proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct Gi2 and Gi3 effects on chemokine receptor CXCR3-mediated signaling. Our data showed that Gi2 was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of Gi2 abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTP
S) incorporation. In sharp contrast, T cells isolated from Gi3 knock-out mice displayed a significant increase in both GTPS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPS incorporation was blocked by Gi3 protein in a dose-dependent manner. Gi3-mediated blockade of Gi2 activation did not result from Gi3 activation, but instead resulted from competition or steric hindrance of Gi2 interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only Gi2 activation induced by a CXCR3 agonist but also the interaction of Gi3 to the CXCR3 receptor. These findings reveal for the first time an interplay of Gi proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the Gi/o protein family. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 
, and subunits that couple to seven transmembrane receptors called G protein-coupled receptors (GPCRs).3 The G protein resides attached to the intracellular face of the plasma membrane in an inactive form consisting of the G subunit bound to GDP, a structure that is stabilized by interaction with the dimer. Agonist binding to the receptor provokes a conformation change that facilitates the interaction of the G subunit to the receptor. Upon interaction with the receptor, G protein becomes activated, causing GDP exchange for GTP. This activation to the high energy G-GTP results in dissociation of the and subunits and propagation of downstream signaling by both the G-GTP and the subunits. The GTP-binding proteins are classified by the signaling events they instigate, of which there are four major families as follows: Gi/o, Gq/11, G12/13, and Gs (1, 2). Members of the Gi/o family, including Gi1, Gi2, Gi3, Go, Ggust, and Gt, can all be irreversibly uncoupled from receptors by pertussis toxin (PTX) (3, 4). PTX catalyzes ADP-ribosylation of a specific cysteine residue at position –4 from the C terminus of the subunits, and thus blocks downstream pathways by preventing receptor interaction (5). The bacterial toxin has proven to be an excellent tool in dissecting the essential role of Gi proteins in chemotaxis, proliferation, and differentiation of various cells (3, 4, 6, 7). However, it cannot distinguish unique properties of individual Gi/o protein family members. Investigation of the details of GPCR and G protein interactions has drawn great attention in the past decade, because the interactions dictate the specificity and amplitude of a GPCR-stimulated cellular response, and GPCRs are targets for more than 30% of the current marketed drugs (8).
The primary PTX substrates evident in T lymphocytes are the 40–41-kDa subunits of the Gi2 and Gi3 proteins (9, 10). The importance of PTX-sensitive proteins in lymphocyte trafficking came initially from the unique phenotype of lck-PTX-transgenic mice that expressed the PTX catalytic S1 subunit in thymocytes and peripheral T cells under the control of an lck promoter (11). The transgenic mice had 2–4-fold more mature CD4+ and CD8+ thymocytes in the thymus than did control mice and few or no T cells in the peripheral lymphoid tissues because of a defect in thymic emigration (11). Thymocyte emigration appears to be controlled by either Gi3 or Gi2, because null mutation of either of them does not affect this process significantly (12, 13). In the periphery, however, their function differs considerably. Gi2-deficient mice display impairment in lymph node development in several anatomic locations and in Peyer's patch formation (12, 14). The mice develop inflammatory bowel disease, at least in part, due to a lack of transforming growth factor- responses in T cells that causes Th1-skewed hyperimmune responses in the colon (12, 15). In contrast, Gi3-deficient mice develop no overt phenotype (16).
In this study, we investigate unique properties of Gi2 and Gi3 in chemotaxis of activated T cells induced by CXCR3 ligands, because expression of the CXCR3 receptor has been associated with pathophysiology of Th1-type autoimmune diseases in both humans and experimental models (17–19). The receptor has the following three known ligands: monokine-induced IFN- (MIG)/CXCL9, IFN--inducible 10-kDa protein (IP-10)/CXCL10, and IFN--inducible T cell -chemoattractant/CXCL11. These three chemokines exhibited discernible biological effects in vivo and in vitro (20–24), but they all activated exclusively Gi2 through the CXCR3 receptor, as indicated by a lack of CXCR3-mediated responses in Gi2–/– T cells, regardless of the agonist employed. Interestingly, we found that Gi3 was negatively involved in the CXCR3-mediated signaling. This translated to cellular responses, where a lack of Gi3 significantly enhanced T cell migration toward CXCR3 agonists. Gi3 appears to hinder Gi2 activation via the N terminus of the second intracellular loop. A mutation in this region abolished both activation of Gi2 and the binding of Gi3 to the receptor. This work begins to unravel an interplay between individual Gi proteins that has previously been masked by the use of PTX.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i2-knock-out (KO), Gi3-KO, and wild type (WT) control mice on the mixed 129Sv/C57BL/6 background were generated by gene targeting and backcrossed with C57BL/6 (B6) mice for six times as described (12, 25). Both female and male mice were used at 4–6 weeks of age unless otherwise indicated. The mice were housed in conventional cages at the animal facilities of the Massachusetts General Hospital in accordance with institutional guidelines.
T Cell Preparation—Single-cell suspensions prepared from lymph nodes and spleens were treated with a mixture of rat anti-mouse monoclonal antibodies (Abs) against CD19, CD32, and CD16 followed by depletion of Ab-bound cells with BioMag goat anti-rat IgG (Polysciences Inc., Warrington, PA) per the manufacturer's instructions. This preparation isolated an 
90% pure population of T cells. CXCR3 expression on these T cells was induced by stimulation for 2 days at 37 °C and 5% CO2 with 2.0 µg/ml concanavalin A (ConA) in a complete RPMI 1640 medium (10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM -mercaptoethanol). The cells were continuously cultured for 2 more days in the medium with replenishment of 15% ConA supernatant, which gave rise to a maximal level of CXCR3 expression.
Cell Membrane Preparation—Cell membrane was prepared as described with some modifications (26). Briefly, T cells stimulated with or without ConA as above were collected, suspended in ice-cold hypotonic buffer (10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 3 mM EGTA, 1x protease inhibitor mixture, and 1 mM PMSF), and Dounce-homogenized. In some cases, the cells were treated with 10 ng/ml PTX for 1 h at 37°C before suspension in hypotonic buffer and homogenization. Disrupted cells were centrifuged at 500 x g at 4 °C for 10 min to remove nuclei and unbroken cells. The remaining supernatant was spun at 32,000 x g for 25 min at 4 °C to pellet the cell membrane that was then suspended in membrane solubilization buffer (20 mM glycine, pH 7.2, 1 mM MgCl2, 250 mM sucrose, 1x protease inhibitor mixture, and 1 mM PMSF). The suspended cell membrane was left on ice for at least 2 h with periodic vortexing and then frozen until use.
GTPS Assay—Cell membrane equivalents of 20 µg of protein were first incubated with the indicated concentrations of chemokines on ice for 1 h to prime Gi activation by the receptor (27). G protein activation was initiated by placing samples at room temperature for 2 h in the presence of 0.3 nM [35S]GTPS (1250 Ci/mmol; PerkinElmer Life Sciences) in a GTP-binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EGTA, and 5 mM MgCl2) supplemented with fresh 0.1% bovine serum albumin, 1 mM dithiothreitol, and 2 mM GDP (28). Activation of Gi proteins at room temperature instead of 37 °C minimizes background radioactive incorporation (28). The concentrations used for each CXCR3 ligand were empirically determined based on the most robust migratory response and our preliminary tests. All chemokines were obtained from PeproTech Inc. (Rocky Hill, NJ). Where indicated, a myristoylated Gi3 protein or control Go protein (Calbiochem) at varying concentrations was preincubated with the cell membrane for 1 h on ice to allow the added G protein to interact with the subunit in the membrane prior to chemokine stimulation (27). To determine the Gi protein in association with the receptor, 20 µg/ml monoclonal Ab specific for Gi2 (IgG2b, Chemicon, Temecula, CA) or an isotype-matched control Ab was added to the samples, along with the chemokine to block Gi2 protein activation (29). The samples were then transferred to a 96-well plate and suctioned through a glass fiber filter by a TomTec cell harvester (model MACH 3 M, Hamden, CT). The filters were washed five times with a cold GTP washing buffer (20 mM Tris, pH 8.0, 100 mM NaCl, and 25 mM MgCl2) and dried, and radioactive count was determined on a 1450 MicroBeta scintillation counter (Wallac, Turku, Finland). Percentages of specific GTPS incorporation in activated T cell membranes were calculated as ((counts/min of sample) – (counts/min of background control))/((counts/min control without a stimulus) – (counts/min of background control)) x 100, where background controls contained 1,000 times excess cold GTPS relative to radioactive [35S]GTPS.
Migration Assays—The Transwell system with 3-µm pore size polycarbonate membrane (Corning Glass, Corning, NY) was used for all transmigration assays, because T cells are relatively small in size. This pore size significantly reduced the basal levels of T cell migration in our study. T cells at 3 x 106 cells/ml in Aim-V medium (Invitrogen) were added to the upper chamber at 0.3 ml per well, with the indicated concentrations of chemokines in 0.6 ml of Aim-V medium in the lower chamber. Freshly isolated T cells or T cells activated by ConA for 4 days were allowed to migrate for 4–6 h at 37 °C and 5% CO2.Atthe end of the migration, the cells in the lower chamber were collected and counted on a hemocytometer.
Western Blot Analysis—To examine Gi2 expression in T cell membranes, T cells with or without stimulation were collected and lysed in cold nondenaturing lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, and 1x protease inhibitor mixture), followed by centrifugation at 500 x g for 10 min to remove nuclear fragments and unbroken cells. Crude cell membrane was pelleted from the supernatant by centrifugation at 32,000 x g for 25 min. Protein samples of 100 µg were separated on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with anti-Gi2 monoclonal Ab (Chemicon, Temecula, CA). Gi2 protein was visualized by incubation of the membrane with sheep anti-mouse Ab conjugated with horseradish peroxidase (Amersham Biosciences) followed by reaction with SuperSignal West Pico ECL reagents (Pierce) and exposure to Kodak film (Eastman Kodak Co.). For assessment of Gi3 protein expression, Gi2 protein was first depleted by anti-Gi2-specific Ab because of cross-reaction of anti-Gi3 Ab with Gi2 protein and similar molecular mass of these two G proteins. Briefly, T cell membranes prepared above were incubated for 1 h on ice with anti-Gi2 Ab-conjugated protein G-agarose beads that had been treated previously with 1 mg/ml bovine serum albumin to block nonspecific binding. The beads were removed by centrifugation, and the depletion was repeated three times. The resultant Gi2-depleted T cell membranes were subjected to Western blotting analysis using anti-Gi3 Ab (BIOMOL International LP) as detailed above. The same membranes were re-probed, after stripping, with anti-G protein pan- subunit Ab (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY) for equal protein loading controls.
Flow Cytometry—To determine chemokine receptor expression, T cells with or without stimulation with ConA or transfected COS-7 cells were incubated with specific Abs followed by analysis on a FACSCalibur cytometer equipped with a CellQuest software (BD Biosciences). A murine CXCR3-specific Ab (clone 4c4) was a generous gift from Dr. Dominic Picarella (Millenium Pharmaceuticals) (30). Fluorescence-conjugated rat anti-mouse Abs specific for CCR7 or CXCR4 and mouse anti-human CXCR3 Ab were purchased from Biolegend (San Diego, CA), R&D Systems (Minneapolis, MN), or Pharmingen, respectively.
In Vitro Transcription and Translation—Gi2 and Gi3 proteins were synthesized and 35S-labeled by the TNT® Quick-Coupled in Vitro Transcription/Translation Systems per the manufacturer's instructions (Promega, Madison, WI). In brief, 1 µg of a Gi2 or Gi3 plasmid was mixed with the rabbit reticulocyte lysate provided, 100 µM myristic acid, and [35S]methionine (1000 Ci/mmol) for 75 min at 30 °C. The resulting product was verified by SDS-PAGE on the basis of the size and compared in the presence or absence of the specific plasmid.
Immunoprecipitation—To study CXCR3-associated Gi proteins, COS-7 cells were transfected with pcDNA plasmids containing either WT or mutated CXCR3 receptors or a control HA-tag·S1P1 (sphingosine 1-phosphate 1) receptor using Lipofectamine according to the manufacturer's instructions (Invitrogen). Three mutants were as follows: 1) an R149N-CXCR3 that had asparagine (Asn) substitution for arginine (Arg) at position 149; 2) LLL-CXCR3 generated by replacement of alanines for leucines (Leu) at positions 332–334 in the C terminus; and ST(–)-CXCR3 constructed by substitution of all serines (Ser) and threonines (Thr) in the C terminus with alanines (24, 31, 32). The transfected cells were lysed, and the cell membrane was prepared and suspended in a GPCR solubilization buffer (1 mM MgCl2, 250 mM sucrose, 1x protease inhibitor mixture, and 1 mM PMSF in phosphate-buffered saline) as described (24, 31, 32). Cell membranes with equivalent of 60 µg of protein were incubated with 3 µl of in vitro synthesized 35S-labeled Gi2 or Gi3 for 1 h on ice to allow the added Gi protein to interact with the subunit in the membrane, after which the cell membrane was either left unstimulated or stimulated for 2 h on ice by indicated chemokines and activated at room temperature as described (27). The stimulated membranes were then cross-linked for 2 h on ice with 20 µM dithiobis-sulfosuccinimidyl propionate (Pierce). The cross-linker was quenched by the addition of 50 µM Tris, pH 7.5, for 15 min on ice. To the chemokine-stimulated membranes, anti-CXCR3 Ab or control Ab was added, and the Ab-bound receptor was pulled down by pre-cleaned protein G-agarose beads (Invitrogen). For HA-S1P1-transfected cells, immunoprecipitation using anti-HA Ab was carried out in place of anti-CXCR3 Ab. The resultant beads were washed extensively with a cold GPCR washing buffer (1 mM EGTA, 5 mM MgCl2, and 0.1% bovine serum albumin in phosphate-buffered saline). Radioactivity of the resultant beads was quantified by scintillation counting on the 1450 MicroBeta scintillation counter to determine the amount of 35S-Gi2 or 35S-Gi3 in association with the CXCR3 receptor.
Statistical Analysis—The Student's two-tailed t test was used to analyze the significance of experimental groups compared with relevant controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i2-orGi3-deficient T Cells—CXCR3 agonists induce chemotactic responses of activated T cells in vitro with a potency of CXCL11 > CXCL10 > CXCL9 in a PTX-sensitive manner (24, 34). These three chemokines also mediate distinct biological effects in vivo (20, 21, 23, 35). In an attempt to understand whether the varying responses evoked by these agonists resulted from activation of different Gi proteins, we tested the role of Gi2 and Gi3 in the migration of activated T cells toward CXCL9, CXCL10, or CXCL11 (36). Stimulation of WT T cells with increasing concentration gradients of CXCR3 ligands elicited a weak chemotactic response to CXCL9 (data not shown) but vigorous responses to CXCL10 or CXCL11 in a dose-dependent manner (Fig. 1, A, B, and E), mirroring previous investigations (37). However, migration toward these three individual chemokines was all reduced to control levels in cells lacking Gi2 over a wide range of agonist concentrations tested (Fig. 1, A, B, and E). The lack of chemotactic responses toward CXCR3 agonists was not due to aberrant cell viability or migration of these cells, because they migrated similarly as WT counterparts in response to CCL19 or CCL21 (data not shown), two ligands for the CCR7 receptor (Fig. 1H). These results indicate that CXCR3-mediated migration depends predominantly on Gi2, irrespective of the ligands.
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i3 (Fig. 1, A and E). The migration toward CXCL10 was also increased, albeit to a lesser extent (Fig. 1, B and E). However, such increased chemotaxis could not be induced by CCL21 (data not shown) or CCL19 in the cells, regardless of whether or not the cells were activated (Fig. 1, G and H). We also observed no augmented chemotactic response in G –/–i3 cells stimulated with CXCL12, a ligand for another Gi-dependent receptor CXCR4. In fact, CXCL12-provoked chemotaxis was diminished in a lack of either Gi2 or Gi3 (Fig. 1F). The inflammatory chemokines MCP-1 and MIP-1 were unable to elicit a significant migratory response given our T cell activation conditions (data not shown). Together, these data stress that the increased migration toward CXCR3 agonists in the absence of Gi3 is receptor-specific.
Gi2 and CXCR3 Expression in T Cells—The opposing responses of Gi2- and Gi3-deficient T cells to CXCR3 ligands argue for Gi3 antagonizing the function of Gi2. To conclude this, we must rule out that the increased migration toward CXCR3 agonists in G –/–i3 cells resulted from increased expression of Gi2 that could potentially provide a compensatory mechanism to offset the lack of Gi3 (38). Fig. 2A clearly showed that Gi2 expression was not increased in activated G –/–i3 T cells compared with WT T cells (lanes 3 versus 1, lower panel), as was the case for Gi3 expression in activated G –/–i2 T cells (lanes 5 versus 1, lower panel). Neither Gi2 nor Gi3 expression was increased in freshly isolated KO T cells compared with WT T cells (Fig. 2A, top panel). We also detected comparable levels of the CXCR3 receptor on these three groups of T cells (Fig. 2B). The expression of other receptors, including CCR7 on stimulated and unstimulated T cells and CXCR4 on resting T cells, was also comparable in these cells, despite some variations (Fig. 2B). To exclude another possibility that the increased chemotactic response was ascribed to coupling of the CXCR3 receptor to other G protein family members in the absence of Gi3, the cells were treated with 100 ng/ml PTX overnight before they were assayed for chemotaxis. The pretreatment ablated CXCR3-mediated chemotactic responses in both WT and Gi3-deficient T cells (Fig. 1, C and D), confirming involvement of PTX-sensitive Gi2 protein only in the increased migratory response in the absence of Gi3. Our investigation thus concludes that Gi3 participates as an inhibitor in CXCR3-stimulated signaling, and null mutation of Gi3 directly accounts for the increased migration of G –/–i3 T cells toward CXCR3 ligands.
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S Incorporation in the Absence of Gi2 or Gi3—Our migration data show involvement of both Gi2 and Gi3 in CXCR3-mediated signaling, presumably with Gi2 activating downstream effectors to drive T cell migration. Yet Gi3-mediated inhibition of Gi2 activity in a receptor-specific fashion has never been explored. So in the subsequent studies, we set out to determine whether Gi3 directly blocked Gi2 activation or diverted signals downstream of Gi2. For downstream signaling disruption, we would expect Gi3 activation to be an indispensable receptor-stimulated event in the G protein-signaling cascade, which can be readily measured by a GTPS incorporation assay. All three CXCR3 chemokines at the concentrations optimal for cell migration were able to stimulate significant GTPS incorporation in WT T cell membranes as reported previously (Fig. 3A) (39). In contrast, T cells from G –/–i2 mice failed to incorporate GTPS significantly above controls following stimulation with CXCL9 or CXCL11 under similar conditions (Fig. 3A). A small increase in GTPS incorporation was seen in the cells stimulated with CXCL10, consistent with lesser Gi3 inhibition in response to this chemokine (Fig. 3A and Fig. 1B). The lack of GTPS incorporation in G –/–i2 T cell membranes was not a failure of these cells to incorporate the GTP analog, because adenosine elicited comparable GTPS incorporation in T cells isolated from all these mice (Fig. 3A) (40). Instead, the result argues strongly that Gi3 cannot be activated by CXCR3 agonists, despite its ability to inhibit Gi2 function. Moreover, GTPS incorporation in these T cells proportionally correlated with their migratory responses stimulated by CXCR3 agonists, increasing G protein activation in G –/–i3 T cells and lacking such activation in G –/–i2 T cells as compared with WT T cells. The increased GTPS incorporation in G –/–i3 cells was solely contributed by Gi2 activation, as demonstrated by complete abrogation of the response following PTX treatment (Fig. 3B). The findings not only indicate that Gi2 is the predominant G protein activated by the CXCR3 receptor, but also suggest that CXCR3 receptor activation cannot elicit exchange of GDP for GTP in Gi3 protein, arguing that Gi3 interferes with Gi2 signaling without being activated.
Inhibition of CXCR3-mediated Gi2 Activation by Gi3—The consistent and significant increases in GTPS incorporation in G –/–i3 T cell membranes and in the migration of these T cells argue strongly for inhibition of Gi2 activation by Gi3. Moreover, inhibition of Gi2 activation without a concomitant rise in Gi3 activation raises an intriguing possibility that Gi3 may interact with the receptor after ligand stimulation but remain inactive and continue its association with the receptor, by which it blocks Gi2 interaction with the receptor. To address this, we first used an Ab directed against the C terminus of the Gi2 protein to block the receptor-G protein interaction in the GTPS incorporation assay. As shown in Fig. 3C, pretreatment with Gi2-specific Ab but not control Ab completely abolished CXCL10- and CXCL11-stimulated GTPS incorporation in G –/–i3 T cell membranes. A specific and complete blockade of G protein activation by anti-Gi2 Ab further rules out activation of other G proteins by the CXCR3 receptor. Second, in vitro synthesized Gi3 protein was added to the GTPS incorporation assay prior stimulation with CXCL11 to block Gi2 activation. Cell membranes prepared from activated G –/–i3 T cells were used in the assay to ensure no competition from endogenous Gi3. As shown in Fig. 3D, the addition of 50 nM Gi3 resulted in a significant decrease in GTPS incorporation of CXCL11-stimulated membranes, with 100 nM exogenous Gi3 protein leading to complete inhibition of Gi2 activation. Under similar conditions, however, addition of Go, another member of the Gi/o family, did not affect Gi2 activation, indicating a specific blockage of CXCR3 activation by Gi3 protein.
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i3 with the CXCR3 Receptor—We next determined whether Gi3 could physically interact with the CXCR3 receptor following stimulation with CXCR3 ligands. Co-immunoprecipitation of an activated G protein with its receptor has rarely been documented due to transient interactions between the activated receptor and a G protein. Because Gi3 is not activated by the CXCR3 receptor, Gi3 would be retained by the receptor after stimulation. To test this possibility, COS-7 cells were transiently transfected with constructs encoding the CXCR3 receptor or its variants that have been shown to alter migration and GTPS incorporation (24). Cell surface expression of these receptors was compared by flow cytometry (Fig. 4A). The results showed comparable levels of the ST(–)-CXCR3 variant and WT CXCR3 and slightly but similarly decreased levels of the DNY-CXCR3 and LLL-CXCR3 variants on the cells. To evaluate the effects of these mutations on Gi3 binding, cell membrane fractions prepared from these transfected cells were incubated with in vitro synthesized 35S-labeled Gi2 or Gi3 and stimulated with or without CXCL11, as the chemokine induced the most robust response among the three CXCR3 agonists. Immunoprecipitation of the receptor using anti-CXCR3 Ab revealed Gi3 interacting with WT CXCR3 receptor after stimulation with CXCL11, with the presence of 35S-labeled Gi3 only in CXCL11-stimulated but not in nonstimulated immunoprecipitates (Fig. 4B). Co-immunoprecipitation of Gi3 with the C-terminal mutants LLL-CXCR3 and ST(–)-CXCR3 was also evident in CXCL11-stimulated cell membranes. However, the interaction between Gi3 and the receptor apparently did not occur when the G protein-binding motif DRY was mutated to DNY (R149N mutant) in the N-terminal portion of the second intracellular loop (Fig. 4B). The finding that this motif is required for interaction between Gi3 and the CXCR3 receptor recapitulates its critical role in proper migratory responses of the CXCR3 receptor and other chemokine receptors (24, 41). Therefore, "incomplete activated" Gi3 may prevent Gi2-mediated signaling via the DRY site.
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i2 as expected, presumably because of its rapid dissociation from the CXCR3 receptor upon activation. The 35S-Gi2 could interact well with the S1P1 receptor, another PTX-sensitive GPCR (Fig. 4B, right panel), ruling out that undetectable 35S-Gi2 in CXCR3 immunoprecipitates resulted from a lack of binding function of the protein. Furthermore, significant GTPS incorporation induced by CXCL11 was seen in the cell membranes prepared from COS-7 cells transfected with WT CXCR3 receptor and all variants, except for the R149N variant that was totally ineffective (Fig. 4C). The inability of the R149N mutant to activate Gi2 and to interact with Gi3 suggests that Gi3 may compete for or sterically interfere with Gi2 binding to the CXCR3 receptor directly or indirectly via the DRY domain, by which it controls the amplitude of a migratory response elicited by the receptor. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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i3 augments Gi2-mediated migratory responses toward CXCR3 ligands, whereas deletion of Gi2 ablates the response. The Gi3-mediated inhibition of CXCR3 signaling is not reliant on the exchange of GDP for GTP by Gi3, but it does require ligand-stimulated interaction between Gi3 and the CXCR3 receptor. Presumably, a conformational change of the receptor caused by ligand binding provides conditions that are optimal for Gi3 binding to the receptor. However, the receptor-bound Gi3 appears unable to undergo a conformational change required for dissociation of GDP and/or binding to GTP, thus preventing it from full activation and leaving the receptor. By occupancy of the receptor, Gi3 blocks Gi2 activation. It has been shown that the DRY motif at the N terminus of the second intracellular loop is key for proper G protein recognition and interaction (31). Our data suggest that this motif may be also essential for the blockade induced by Gi3, which requires further investigation. Substitution of Arg149 amino residue to Asn ablates not only activation of Gi2 but also binding of Gi3 to the receptor. Under similar conditions, receptors carrying mutations at the C terminus can activate Gi2 and preserve Gi3 interaction similarly to the WT CXCR3 receptor (Fig. 4B). Lack of Gi3 binding to and Gi2 activation by the R149N mutant cannot be ascribed to the levels of receptor expression on the cells. Although the R149N-CXCR3 mutant was expressed on the cells at levels slightly lower than the WT CXCR3 receptor, its expression was comparable with the LLL-CXCR3 mutant (Fig. 4A) (24). The latter could activate Gi2 and bind with Gi3 as the WT CXCR3 receptor (Fig. 4, B and C). Murine Gi2 and Gi3 are 83% identical in amino residue sequence. This structural similarity may be the basis for their competition for the same binding domain of the CXCR3 receptor or for Gi3 interference with Gi2 interaction of the receptor. A subtle difference in the receptor conformation induced by a specific agonist is likely to determine whether the activated receptor can interact and activate a given Gi protein or interact with a Gi protein without inducing its full activation. Therefore, function of a given Gi protein as an activator or inhibitor is receptor-specific, and the interplay between these two G proteins may be essential in the control of the amplitude of a GPCR-induced signal. To the best of our knowledge, this is the first description of a G protein interacting with a receptor after ligand binding for the sole purpose of diminishing the other G protein interaction with the receptor. This finding gives novel insight into regulation of GPCR signaling by heterotrimeric G proteins.
GPCRs initiate a signaling cascade through activation of heterotrimeric G proteins following stimulation by an extracellular agonist (43). To meet the complexity and versatility of cell migration in the body, many chemokine receptors have evolved to recognize more than one chemokine, such as the CXCR3 and CCR7 receptors, or one chemokine receptor can be activated by multiple chemokines like CCL5 (regulated on activation normal T cell expressed and secreted), CCL3 (MIP-1), MCP-2, and MCP-3 (44). Our finding that a migratory response is tied proportionally to the amplitude of G protein activation indicates that stoichiometry of G protein activation is directly linked to the propensity of a cell to migrate. The precise activation level of the Gi2 protein stimulated by ligation of the CXCR3 receptor is determined by the affinity of the ligand as well as the interplay of Gi2 and Gi3 with the receptor. In accordance with CXCR3 signaling dependent on Gi2 and inhibition of the signaling by Gi3, our in vivo study showed that similar to T cells from mice lacking the CXCR3 receptor, Gi2-deficient T cells failed to elicit an acute graft-versus-host defense reaction after being transfused into full major histocompatibility complex-mismatched Balb/c SB-17 severe combined immunodeficiency mice (30, 45).4 In contrast, transfer of Gi3-deficient T cells into the severe combined immunodeficiency mice stimulated an aggravating graft-versus-host defense response compared with WT T cells. Thus, an interplay among different heterotrimeric G proteins is likely to have a role to play in vivo, introducing another level of complexity in the control of cell migration systemically. The complex regulation of GPCR signaling at multiple levels is crucial in temporal and spatial regulation of cell migration in the body.
Determination of receptors linked to Gi proteins has been greatly aided by the use of PTX. PTX has also allowed insight into downstream effectors that are either partially or fully involved in propagating a signal by Gi proteins. Although PTX has provided extensive and important details about the agonist and signal transduction mechanisms, the specifics about individual Gi/o family members has gone largely unresolved because PTX blocks receptor interaction by several Gi/o family members. With the advent of the genetic manipulation of mice, we can tease out the specifics of Gi1, Gi2, and Gi3 in a migratory response stimulated by a specific receptor. Studies of B cell migration in G –/–i2 mice suggest an irreplaceable role of Gi2 in chemotactic responses to chemokines CXCL12 and CXCL13 (14). On the other hand, Gi2 and Gi3 are redundant in thymic emigration as indicated by no significant defects in thymic exportation in Gi2- and Gi3-deficient mice (12, 13). Although the proportions and the numbers of CD4 and CD8 single positive thymocytes were increased in G –/–i2 mice as lck-PTX-transgenic mice, the increases were not caused by a defect in thymic egress, rather by an accelerated transition from the double positive to single positive thymocytes as shown by our previous investigations (12, 46). In support, nearly normal numbers and percentages of CD4+ and CD8+ T cells were seen in the spleen of G –/–i2 mice (11–13). Likewise, mature thymocytes in G –/–i3 mice populated lymphoid tissues normally in the periphery, and there was no aberrant accumulation of single positive thymocytes in the mice (13). The redundancy of Gi2 and Gi3 in thymic egress was also consistent with our recent study showing that Gi2-KO and Gi3-KO T cells migrated indistinguishably from WT T cells in response to an increasing concentration of sphingosine 1-phosphate (S1P), a lipid mediator controlling T cell egress (33).5
How the redundancy works mechanistically is not fully understood at present. Association with the same subunit of G may be one of the common mechanisms whereby Gi2 and Gi3 can compensate for the absence of one to another. Hwang et al. (42) have shown that functional silence of either Gi2 or Gi3 by specific short interfering RNA has little effect on the migration of macrophages toward an increasing concentration gradient of C5a or C3a. Deletion of G2, however, ablated C5a- or C3a-provoked migration, suggesting that both Gi2 and Gi3 can transduce the migration signal as long as the G2 subunit is presented (42). On the contrary, the CXCR4 receptor requires both Gi2 and Gi3 for a full response. Absence of either gene impaired T cell migration induced by CXCL12 (Fig. 1F), although decreased expression of the CXCR4 receptor on these cells may also be partially involved. We should point out that a slight increase or decrease (10–15%) in the Gi2 expression levels in G –/–i3 T cells or Gi3 in G –/–i2 T cells was observed in some mice (Fig. 2A). However, the levels of Gi protein expression were not correlated with migratory changes for any of the studied chemokines. In particular, the slight decrease in Gi2 expression in Gi3-KO T cells was discordant with the increased activity for the CXCR3 receptor. Similarly, although the levels of Gi2 and Gi3 expression were slightly decreased in activated as compared with non-activated T cells, the decrease affects little their chemotactic response to CCL19 stimulation (Fig. 1, G and H). This is probably attributed to the following two reasons. First, Gi2 or Gi3 is a lot more abundant than any individual chemokine receptor in a cell. Second, constant recycling of Gi proteins from an active to inactive status further increases the size of a Gi protein pool that is already far in excess of any individual chemokine receptor. Therefore, distinct function of Gi2 and Gi3 as described in this study is unlikely ascribed to a slightly altered level of Gi2 or Gi3 protein. Nevertheless, this does not exclude that the absolute levels of Gi2 and Gi3 can affect activation of a given GPCR, when the relative levels of Gi2 and Gi3 differ drastically. Our data thus argue that Gi2 and Gi3 proteins may play overlapping, distinct, antagonizing, and additive roles depending on a specific receptor. The Gi2- and Gi3-null mutation mice provide a unique system to explore fundamental signaling differences of a specific GPCR coupling to Gi2, Gi3, or both and potential interplay between these Gi proteins.
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1 Supported in part by National Institutes of Health Training Grant T32 AR07098-31 from the Department of Dermatology, Harvard Medical School.
2 To whom correspondence should be addressed: Wellman Center of Photomedicine, Massachusetts General Hospital, 55 Fruit St., Edwards 222, Boston, MA 02114. Tel.: 617-726-1298; Fax: 617-726-1208; E-mail: mwu2{at}partners.org.
3 The abbreviations used are: GPCR, G protein-coupled receptor; Ab, antibody; GTPS, guanosine 5'-3-O-(thio)triphosphate; PTX, pertussis toxin; S1P1, sphingosine 1-phosphate 1; KO, knock-out; WT, wild type; IFN, interferon; PMSF, phenylmethylsulfonyl fluoride; HA, hemagglutinin; ConA, concanavalin A.
4 Y. Jin, K. H. Wu, Z. Zhou, and M. X. Wu, manuscript in preparation.
5 B. D. Thompson, Y. Jin, W. Hu, L. Charles, L. Birnbaumer, and M. X. Wu, unpublished data.
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