Inhibition of G alpha i2 activation by G alpha i3 in CXCR3-mediated signaling

Abstract

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) 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 G alpha(i/o) protein family.

FIGURE 1

Chemotactic responses in the presence or absence of Gαi2 or Gαi3. A-E. Gαi2 and Gαi3 have opposing effects on CXCR3-stimulated chemotaxis. Activated T cells prepared from wild type (WT), Gαi2-/-, and Gαi3-/- mice were either left untreated (A-B, and E) or treated with 100 ng/ml PTX overnight (C and D), and then added to the upper chamber. CXCR3 agonists at indicated concentrations were added to the lower chamber in triplicate. After 4 hr incubation, the migrated cells in the lower chamber were collected and counted. Data are presented as mean percentages ± standard derivations (SD) of cell migration relative to cell input. One representative experiment in A-D and cumulative data from at least five independent experiments (n = 15) in E are shown. Chemokine concentrations used in E were the optimal concentrations for stimulating maximal migration toward CXCL9 (100ng/ml), CXCL10 (10ng/ml), CXCL11 (100ng/ml). Medium controls are designated as 100% and mean percentages ± SD of cell migration relative to the control are shown. * and **, statistical significance (p < 0.05) or (p < 0.01) respectively, in the presence vs. absence of a specific Gαi protein. F-H. Varying effects of Gαi2 and Gαi3 on T cell chemotaxis induced by different chemokines. The migration was assayed as above, except that non-stimulated T cells were used in place of activated T cells in chemotactic responses to CXCL12 and CCL19 in F and G. Data are presented as mean percentages ± SD of cell migration relative to cell input as A and B. One representative result of three independent experiments with each performed in triplicate.

FIGURE 1

Chemotactic responses in the presence or absence of Gαi2 or Gαi3. A-E. Gαi2 and Gαi3 have opposing effects on CXCR3-stimulated chemotaxis. Activated T cells prepared from wild type (WT), Gαi2-/-, and Gαi3-/- mice were either left untreated (A-B, and E) or treated with 100 ng/ml PTX overnight (C and D), and then added to the upper chamber. CXCR3 agonists at indicated concentrations were added to the lower chamber in triplicate. After 4 hr incubation, the migrated cells in the lower chamber were collected and counted. Data are presented as mean percentages ± standard derivations (SD) of cell migration relative to cell input. One representative experiment in A-D and cumulative data from at least five independent experiments (n = 15) in E are shown. Chemokine concentrations used in E were the optimal concentrations for stimulating maximal migration toward CXCL9 (100ng/ml), CXCL10 (10ng/ml), CXCL11 (100ng/ml). Medium controls are designated as 100% and mean percentages ± SD of cell migration relative to the control are shown. * and **, statistical significance (p < 0.05) or (p < 0.01) respectively, in the presence vs. absence of a specific Gαi protein. F-H. Varying effects of Gαi2 and Gαi3 on T cell chemotaxis induced by different chemokines. The migration was assayed as above, except that non-stimulated T cells were used in place of activated T cells in chemotactic responses to CXCL12 and CCL19 in F and G. Data are presented as mean percentages ± SD of cell migration relative to cell input as A and B. One representative result of three independent experiments with each performed in triplicate.

FIGURE 1

Chemotactic responses in the presence or absence of Gαi2 or Gαi3. A-E. Gαi2 and Gαi3 have opposing effects on CXCR3-stimulated chemotaxis. Activated T cells prepared from wild type (WT), Gαi2-/-, and Gαi3-/- mice were either left untreated (A-B, and E) or treated with 100 ng/ml PTX overnight (C and D), and then added to the upper chamber. CXCR3 agonists at indicated concentrations were added to the lower chamber in triplicate. After 4 hr incubation, the migrated cells in the lower chamber were collected and counted. Data are presented as mean percentages ± standard derivations (SD) of cell migration relative to cell input. One representative experiment in A-D and cumulative data from at least five independent experiments (n = 15) in E are shown. Chemokine concentrations used in E were the optimal concentrations for stimulating maximal migration toward CXCL9 (100ng/ml), CXCL10 (10ng/ml), CXCL11 (100ng/ml). Medium controls are designated as 100% and mean percentages ± SD of cell migration relative to the control are shown. * and **, statistical significance (p < 0.05) or (p < 0.01) respectively, in the presence vs. absence of a specific Gαi protein. F-H. Varying effects of Gαi2 and Gαi3 on T cell chemotaxis induced by different chemokines. The migration was assayed as above, except that non-stimulated T cells were used in place of activated T cells in chemotactic responses to CXCL12 and CCL19 in F and G. Data are presented as mean percentages ± SD of cell migration relative to cell input as A and B. One representative result of three independent experiments with each performed in triplicate.

FIGURE 2

Gαi and chemokine receptor expression A. Gαi expression in T cells by immunoblotting analysis. Cell membranes of unstimulated (upper panel) or stimulated (lower panel) T cells prepared from indicated mice were analyzed by immunoblotting using anti-Gαi2 specific monoclonal Ab (left panel). Cell membranes with Gαi2 depletion were analyzed by anti-Gαi3 Ab (right panel). The blots were stripped and re-probed with anti-G protein pan β subunit Ab for equal protein loading controls. One representative result of four (left) or two (right) experiments performed is shown. B. Analysis of chemokine receptor expression on T cells. T cells prepared from indicated mice were either left unstimulated for staining CCR7 and CXCR4 receptors or stimulated with Con A for 4 days for measuring CCR7 and CXCR3 expression on a flow cytometer. The numbers within the panels indicate the percentages of positive cells on gated CD3+ T cell population. Representative results of four (CXCR3) and three (CXCR4 and CCR7) experiments performed are shown.

FIGURE 2

Gαi and chemokine receptor expression A. Gαi expression in T cells by immunoblotting analysis. Cell membranes of unstimulated (upper panel) or stimulated (lower panel) T cells prepared from indicated mice were analyzed by immunoblotting using anti-Gαi2 specific monoclonal Ab (left panel). Cell membranes with Gαi2 depletion were analyzed by anti-Gαi3 Ab (right panel). The blots were stripped and re-probed with anti-G protein pan β subunit Ab for equal protein loading controls. One representative result of four (left) or two (right) experiments performed is shown. B. Analysis of chemokine receptor expression on T cells. T cells prepared from indicated mice were either left unstimulated for staining CCR7 and CXCR4 receptors or stimulated with Con A for 4 days for measuring CCR7 and CXCR3 expression on a flow cytometer. The numbers within the panels indicate the percentages of positive cells on gated CD3+ T cell population. Representative results of four (CXCR3) and three (CXCR4 and CCR7) experiments performed are shown.

FIGURE 3

CXCR3 agonist-stimulated G protein activation in the presence or absence of Gαi2 or Gαi3. A. CXCR3 agonist-stimulated GTPγS incorporation in activated T cells. Cell membranes equivalent to 20μg protein prepared from activated T cells with or without Gαi2 or Gαi3 were stimulated in triplicate with 100ng/ml CXCL9, 10ng/ml CXCL10, 100ng/ml CXCL11, or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS as described in materials and methods. Percentages ± SD of specific ³⁵S-GTPγS incorporation relative to background controls are shown with unstimulated cell membranes designed as 100%. The result represents four separate experiments. * and ** are statistical significance (p < 0.05) or high significance (p < 0.01), respectively, in the presence vs. absence of a specific Gαi protein. B. Inhibition of G protein activation by PTX treatment. Activated T cells prepared from indicated mice were treated with 10ng/ml PTX for 1 hr before the cell membranes were prepared and assayed for GTPγS incorporation induced by either 10ng/ml CXCL10 or 100ng/ml CXCL11 as Figure 3A. Data represent three separate experiments with each sample in triplicate as Figure 3A. C. A blockade of G protein activation in Gαi3-/- T cell membrane by anti-Gαi2 Ab. G protein activation in cell membrane prepared from Gαi3-/- T cells was assayed in the presence of either 3 μg anti-Gαi2 monoclonal Ab or isotype-matched control Ab (Control Ab) during chemokine stimulation as in A. The data represent three independent experiments with each sample in triplicate as A. ** statistical significance (p < 0.01) in the presence vs. absence of a specific anti-Gαi2 Ab. D. Inhibition of Gαi2 activation in Gαi3-/- T cell membranes by exogenous Gαi3 protein. Cell membranes prepared from Gαi3-/- T cells were pre-incubated with indicated concentrations of in vitro synthesized Gαi3 or Gαo protein before stimulation of the membrane by 100 ng/ml CXCL11 and assaying G protein activation as in A. The data represent three separate experiments with each sample in triplicate. * and **, statistical significance (p < 0.05) or (p < 0.01), respectively, in the presence of Gαi3 vs. Gαo protein.

FIGURE 3

CXCR3 agonist-stimulated G protein activation in the presence or absence of Gαi2 or Gαi3. A. CXCR3 agonist-stimulated GTPγS incorporation in activated T cells. Cell membranes equivalent to 20μg protein prepared from activated T cells with or without Gαi2 or Gαi3 were stimulated in triplicate with 100ng/ml CXCL9, 10ng/ml CXCL10, 100ng/ml CXCL11, or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS as described in materials and methods. Percentages ± SD of specific ³⁵S-GTPγS incorporation relative to background controls are shown with unstimulated cell membranes designed as 100%. The result represents four separate experiments. * and ** are statistical significance (p < 0.05) or high significance (p < 0.01), respectively, in the presence vs. absence of a specific Gαi protein. B. Inhibition of G protein activation by PTX treatment. Activated T cells prepared from indicated mice were treated with 10ng/ml PTX for 1 hr before the cell membranes were prepared and assayed for GTPγS incorporation induced by either 10ng/ml CXCL10 or 100ng/ml CXCL11 as Figure 3A. Data represent three separate experiments with each sample in triplicate as Figure 3A. C. A blockade of G protein activation in Gαi3-/- T cell membrane by anti-Gαi2 Ab. G protein activation in cell membrane prepared from Gαi3-/- T cells was assayed in the presence of either 3 μg anti-Gαi2 monoclonal Ab or isotype-matched control Ab (Control Ab) during chemokine stimulation as in A. The data represent three independent experiments with each sample in triplicate as A. ** statistical significance (p < 0.01) in the presence vs. absence of a specific anti-Gαi2 Ab. D. Inhibition of Gαi2 activation in Gαi3-/- T cell membranes by exogenous Gαi3 protein. Cell membranes prepared from Gαi3-/- T cells were pre-incubated with indicated concentrations of in vitro synthesized Gαi3 or Gαo protein before stimulation of the membrane by 100 ng/ml CXCL11 and assaying G protein activation as in A. The data represent three separate experiments with each sample in triplicate. * and **, statistical significance (p < 0.05) or (p < 0.01), respectively, in the presence of Gαi3 vs. Gαo protein.

FIGURE 3

CXCR3 agonist-stimulated G protein activation in the presence or absence of Gαi2 or Gαi3. A. CXCR3 agonist-stimulated GTPγS incorporation in activated T cells. Cell membranes equivalent to 20μg protein prepared from activated T cells with or without Gαi2 or Gαi3 were stimulated in triplicate with 100ng/ml CXCL9, 10ng/ml CXCL10, 100ng/ml CXCL11, or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS as described in materials and methods. Percentages ± SD of specific ³⁵S-GTPγS incorporation relative to background controls are shown with unstimulated cell membranes designed as 100%. The result represents four separate experiments. * and ** are statistical significance (p < 0.05) or high significance (p < 0.01), respectively, in the presence vs. absence of a specific Gαi protein. B. Inhibition of G protein activation by PTX treatment. Activated T cells prepared from indicated mice were treated with 10ng/ml PTX for 1 hr before the cell membranes were prepared and assayed for GTPγS incorporation induced by either 10ng/ml CXCL10 or 100ng/ml CXCL11 as Figure 3A. Data represent three separate experiments with each sample in triplicate as Figure 3A. C. A blockade of G protein activation in Gαi3-/- T cell membrane by anti-Gαi2 Ab. G protein activation in cell membrane prepared from Gαi3-/- T cells was assayed in the presence of either 3 μg anti-Gαi2 monoclonal Ab or isotype-matched control Ab (Control Ab) during chemokine stimulation as in A. The data represent three independent experiments with each sample in triplicate as A. ** statistical significance (p < 0.01) in the presence vs. absence of a specific anti-Gαi2 Ab. D. Inhibition of Gαi2 activation in Gαi3-/- T cell membranes by exogenous Gαi3 protein. Cell membranes prepared from Gαi3-/- T cells were pre-incubated with indicated concentrations of in vitro synthesized Gαi3 or Gαo protein before stimulation of the membrane by 100 ng/ml CXCL11 and assaying G protein activation as in A. The data represent three separate experiments with each sample in triplicate. * and **, statistical significance (p < 0.05) or (p < 0.01), respectively, in the presence of Gαi3 vs. Gαo protein.

FIGURE 3

CXCR3 agonist-stimulated G protein activation in the presence or absence of Gαi2 or Gαi3. A. CXCR3 agonist-stimulated GTPγS incorporation in activated T cells. Cell membranes equivalent to 20μg protein prepared from activated T cells with or without Gαi2 or Gαi3 were stimulated in triplicate with 100ng/ml CXCL9, 10ng/ml CXCL10, 100ng/ml CXCL11, or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS as described in materials and methods. Percentages ± SD of specific ³⁵S-GTPγS incorporation relative to background controls are shown with unstimulated cell membranes designed as 100%. The result represents four separate experiments. * and ** are statistical significance (p < 0.05) or high significance (p < 0.01), respectively, in the presence vs. absence of a specific Gαi protein. B. Inhibition of G protein activation by PTX treatment. Activated T cells prepared from indicated mice were treated with 10ng/ml PTX for 1 hr before the cell membranes were prepared and assayed for GTPγS incorporation induced by either 10ng/ml CXCL10 or 100ng/ml CXCL11 as Figure 3A. Data represent three separate experiments with each sample in triplicate as Figure 3A. C. A blockade of G protein activation in Gαi3-/- T cell membrane by anti-Gαi2 Ab. G protein activation in cell membrane prepared from Gαi3-/- T cells was assayed in the presence of either 3 μg anti-Gαi2 monoclonal Ab or isotype-matched control Ab (Control Ab) during chemokine stimulation as in A. The data represent three independent experiments with each sample in triplicate as A. ** statistical significance (p < 0.01) in the presence vs. absence of a specific anti-Gαi2 Ab. D. Inhibition of Gαi2 activation in Gαi3-/- T cell membranes by exogenous Gαi3 protein. Cell membranes prepared from Gαi3-/- T cells were pre-incubated with indicated concentrations of in vitro synthesized Gαi3 or Gαo protein before stimulation of the membrane by 100 ng/ml CXCL11 and assaying G protein activation as in A. The data represent three separate experiments with each sample in triplicate. * and **, statistical significance (p < 0.05) or (p < 0.01), respectively, in the presence of Gαi3 vs. Gαo protein.

FIGURE 4

Similar structural requirement for Gαi3 binding to the CXCR3 receptor and for Gαi2 activation. A. Analysis of CXCR3 expression in transfected Cos-7 cells. Cos-7 cells transfected with WT or mutated CXCR3 receptors were stained with anti-CXCR3 Ab conjugated with Alexa and analyzed by flow cytometry. Percentages of CXCR3⁺ cells on gated viable cell populations are indicated within the panels, where the filled profiles are negative controls obtained by isotype-matched control Ab conjugated with Alexa. One representative result of three experiments performed is shown. B. Interaction of Gαi3 with the CXCR receptor at the G protein-binding domain. Cell membranes prepared from Cos-7 cells transfected with constructs harboring either WT or CXCR3 receptor variants were incubated with ³⁵S-labeled Gαi2 or ³⁵S-labeled Gαi3 protein for 2 hrs and then stimulated with 100ng/ml CXCL11. The associated Gαi proteins were immunoprecipitated by anti-CXCR3 Ab and quantified by scintillation counting on a 1450 MicroBeta scintillation counter (left panel). Association of ³⁵S-Gαi2 with the S1P₁ receptor in unstimulated cell membrane was also shown as a ³⁵S-Gαi2 binding control (right panel). Data represent three separate experiments with two samples per assay (n = 6). **, statistical significance (p < 0.01) obtained by comparing mutated CXCR3 receptors with the WT one. C. Requirement of the G protein-binding motif for Gαi2 activation. Cell membranes prepared from Cos-7 cells transfected with indicated constructs as in A were stimulated with 100ng/ml CXCL11 or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS and the resultant ³⁵S-GTPγS incorporation was measured as Figure 3A. The data represent three separate experiments with each sample in triplicate (n = 9). **, statistical significance (p < 0.01) obtained by comparing mutated CXCR3 receptors with the WT receptor.

FIGURE 4

Similar structural requirement for Gαi3 binding to the CXCR3 receptor and for Gαi2 activation. A. Analysis of CXCR3 expression in transfected Cos-7 cells. Cos-7 cells transfected with WT or mutated CXCR3 receptors were stained with anti-CXCR3 Ab conjugated with Alexa and analyzed by flow cytometry. Percentages of CXCR3⁺ cells on gated viable cell populations are indicated within the panels, where the filled profiles are negative controls obtained by isotype-matched control Ab conjugated with Alexa. One representative result of three experiments performed is shown. B. Interaction of Gαi3 with the CXCR receptor at the G protein-binding domain. Cell membranes prepared from Cos-7 cells transfected with constructs harboring either WT or CXCR3 receptor variants were incubated with ³⁵S-labeled Gαi2 or ³⁵S-labeled Gαi3 protein for 2 hrs and then stimulated with 100ng/ml CXCL11. The associated Gαi proteins were immunoprecipitated by anti-CXCR3 Ab and quantified by scintillation counting on a 1450 MicroBeta scintillation counter (left panel). Association of ³⁵S-Gαi2 with the S1P₁ receptor in unstimulated cell membrane was also shown as a ³⁵S-Gαi2 binding control (right panel). Data represent three separate experiments with two samples per assay (n = 6). **, statistical significance (p < 0.01) obtained by comparing mutated CXCR3 receptors with the WT one. C. Requirement of the G protein-binding motif for Gαi2 activation. Cell membranes prepared from Cos-7 cells transfected with indicated constructs as in A were stimulated with 100ng/ml CXCL11 or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS and the resultant ³⁵S-GTPγS incorporation was measured as Figure 3A. The data represent three separate experiments with each sample in triplicate (n = 9). **, statistical significance (p < 0.01) obtained by comparing mutated CXCR3 receptors with the WT receptor.

FIGURE 4

Similar structural requirement for Gαi3 binding to the CXCR3 receptor and for Gαi2 activation. A. Analysis of CXCR3 expression in transfected Cos-7 cells. Cos-7 cells transfected with WT or mutated CXCR3 receptors were stained with anti-CXCR3 Ab conjugated with Alexa and analyzed by flow cytometry. Percentages of CXCR3⁺ cells on gated viable cell populations are indicated within the panels, where the filled profiles are negative controls obtained by isotype-matched control Ab conjugated with Alexa. One representative result of three experiments performed is shown. B. Interaction of Gαi3 with the CXCR receptor at the G protein-binding domain. Cell membranes prepared from Cos-7 cells transfected with constructs harboring either WT or CXCR3 receptor variants were incubated with ³⁵S-labeled Gαi2 or ³⁵S-labeled Gαi3 protein for 2 hrs and then stimulated with 100ng/ml CXCL11. The associated Gαi proteins were immunoprecipitated by anti-CXCR3 Ab and quantified by scintillation counting on a 1450 MicroBeta scintillation counter (left panel). Association of ³⁵S-Gαi2 with the S1P₁ receptor in unstimulated cell membrane was also shown as a ³⁵S-Gαi2 binding control (right panel). Data represent three separate experiments with two samples per assay (n = 6). **, statistical significance (p < 0.01) obtained by comparing mutated CXCR3 receptors with the WT one. C. Requirement of the G protein-binding motif for Gαi2 activation. Cell membranes prepared from Cos-7 cells transfected with indicated constructs as in A were stimulated with 100ng/ml CXCL11 or 10 μM adenosine in the presence of 0.3 nM ³⁵S-GTPγS and the resultant ³⁵S-GTPγS incorporation was measured as Figure 3A. The data represent three separate experiments with each sample in triplicate (n = 9). **, statistical significance (p < 0.01) obtained by comparing mutated CXCR3 receptors with the WT receptor.