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Background: Inhibition of G-protein βγ (Gβγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4 + T helper cells, suggesting that Gβγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4 + T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gβγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4 + T helper cells. Methods: Gallein, a small molecule inhibitor of Gβγ, and siRNA-mediated silencing of the G-protein β 1 subunit (Gβ 1 ) were used to test the effect of blocking Gβγ on mRNA levels of cytokines in primary human TCR-stimulated CD4 + T helper cells. Results: Gallein and Gβ 1 siRNA decreased interferon-γ (IFN-γ) and IL-17A mRNA levels in TCR-stimulated CD4 + T cells grown under TH1-promoting conditions. Inhibiting Gβγ also decreased mRNA levels of STAT4, which plays a positive role in TH1 differentiation and IL-17A production. Moreover, mRNA levels of the STAT4-regulated TH1-associated proteins, IL-18 receptor β chain (IL-18Rβ), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), lymphocyte activation gene 3 (LAG-3), natural killer cell group 7 sequence (NKG7), and oncostatin M (OSM) were also decreased upon Gβγ inhibition. Gallein also increased IL-4, IL-5, IL-9, and IL-13 mRNA levels in TCR-stimulated memory CD4 + T cells grown in TH2-promoting conditions. Conclusions: Inhibiting Gβγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn’s disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto’s thyroiditis (HT), in which both IFN-γ and IL-17A are elevated.

Background: The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gα s and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. Methods: The effect of blocking G s -coupled receptor (G s PCR)-mediated G s activation on TCR-stimulated IL-2 mRNA levels in CD4 + T cells was compared with that of knocking down Gα s expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gα s expression on TCR-stimulated cAMP accumulation was compared with that of blocking G s PCR signaling. Results: ZM-241385, an antagonist to the G s -coupled A 2A adenosine receptor (A 2A R), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4 + T helper cells and in Jurkat T cells. A dominant negative Gα s construct, Gα s DN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to G s PCR antagonists, Gα s DN3 blocked G s PCR-dependent activation of both Gα s and Gβγ. In contrast, Gα s siRNA and 2’,5’-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gα s siRNA, but not Gα s DN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. Conclusions: G s PCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gα s and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gα s does not appear to involve G s PCRs. These results suggest that the context of Gα s /cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

The specificity of G protein betagamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of betagamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta1gamma complexes containing gamma1, gamma2, gamma5, gamma7, gamma10, gamma11, or gamma12 to interact with alphas and of these gamma subunits to compete for interaction with beta1 in live human embryonic kidney (HEK) 293 cells. betagamma complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of alphas-CFP varied inversely with its expression level, and the abilities of YFP-N-beta1YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta1. When the intensities of coexpressed CFP-C-beta1CFP-N-gamma (cyan) and CFP-C-beta1YFP-N-gamma2 (yellow) complexes were compared under conditions in which CFP-C-beta1 was limiting, the CFP-N-gamma subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma2 for association with CFP-C-beta1. CFP-N-gamma12 and CFP-N-gamma1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for betagamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases.

Cells co-express multiple G protein β and γ subunit isoforms, but the extent to which individual subunits associate to form particular βγ complexes is not known. This issue is important because in vivo knockout experiments suggest that specific βγ complexes may have unique functions despite the fact that most complexes exhibit similar properties when assayed in reconstituted systems. This chapter describes how multicolor bimolecular fluorescence complementation (BiFC) can be used in living cells to study the association preferences of β and γ subunits. Multicolor BiFC determines the association preferences of these subunits by quantifying the two fluorescent complexes formed when β or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete for interaction with limiting amounts of a common γ or β subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). One means by which βγ complexes may differ from each other and thereby mediate unique functions in vivo is in the kinetics and patterns of their internalization responses to stimulation of G protein-coupled receptors (GPCRs). Methods are described for imaging and quantifying the internalization of pairs of βγ complexes in response to GPCR stimulation in living cells.

We have applied multicolor BiFC to study the association preferences of G protein beta and gamma subunits in living cells. Cells co-express multiple isoforms of beta and gamma subunits, most of which can form complexes. Although many betagamma complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate betagamma complex formation with functionality in intact cells by comparing the amounts of fluorescent betagamma complexes with their abilities to modulate effector proteins. The relative predominance of specific betagamma complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of beta and gamma subunits by simultaneously visualizing the two fluorescent complexes formed when beta or gamma subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common gamma or beta subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.

The G protein beta(5) subunit differs from other beta subunits in having divergent sequence and subcellular localization patterns. Although beta(5)gamma(2) modulates effectors, beta(5) associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate beta(5) complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 gamma subunits and RGS7 to compete for interaction with beta(5). Among the gamma subunits, beta(5) interacted preferentially with gamma(2), followed by gamma(7), and efficacy of phospholipase C-beta2 activation correlated with amount of beta(5)gamma complex formation. beta(5) also slightly preferred gamma(2) over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), beta(5) interacted similarly with gamma(2) and RGS7. Moreover, gamma(2) interacted preferentially with beta(1) rather than beta(5). These results suggest that multiple coexpressed proteins influence beta(5) complex formation. Fluorescent beta(5)gamma(2) labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas beta(5)RGS7 stained the cytoplasm diffusely. Coexpression of alpha(o) targeted both beta(5) complexes to the plasma membrane, and alpha(q) also targeted beta(5)gamma(2) to the plasma membrane. The constitutively activated alpha(o) mutant, alpha(o)R179C, produced greater targeting of beta(5)RGS7 and less of beta(5)gamma(2) than did alpha(o). These results suggest that alpha(o) may cycle between interactions with beta(5)gamma(2) or other betagamma complexes when inactive, and beta(5)RGS7 when active. Moreover, the ability of beta(5)gamma(2) to be targeted to the plasma membrane by alpha subunits suggests that functional beta(5)gamma(2) complexes can form in intact cells and mediate signaling by G protein-coupled receptors.

Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic microorganisms in the gastrointestinal (GI) tract, but the influence of IECs on the development and regulation of immunity to infection is unknown. Here we show that IEC-intrinsic IkappaB kinase (IKK)-beta-dependent gene expression is a critical regulator of responses of dendritic cells and CD4+ T cells in the GI tract. Mice with an IEC-specific deletion of IKK-beta show a reduced expression of the epithelial-cell-restricted cytokine thymic stromal lymphopoietin in the intestine and, after infection with the gut-dwelling parasite Trichuris, fail to develop a pathogen-specific CD4+ T helper type 2 (T(H)2) response and are unable to eradicate infection. Further, these animals show exacerbated production of dendritic-cell-derived interleukin-12/23p40 and tumour necrosis factor-alpha, increased levels of CD4+ T-cell-derived interferon-gamma and interleukin-17, and develop severe intestinal inflammation. Blockade of proinflammatory cytokines during Trichuris infection ablates the requirement for IKK-beta in IECs to promote CD4+ T(H)2 cell-dependent immunity, identifying an essential function for IECs in tissue-specific conditioning of dendritic cells and limiting type 1 cytokine production in the GI tract. These results indicate that the balance of IKK-beta-dependent gene expression in the intestinal epithelium is crucial in intestinal immune homeostasis by promoting mucosal immunity and limiting chronic inflammation.

Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic microorganisms in the gastrointestinal (GI) tract, but the influence of IECs on the regulation of immunity is unknown. Here we show that IEC-intrinsic IKKβ-dependent gene expression is a critical regulator of dendritic cell (DC) and CD4+ T cell responses in the gut. Following infection with the parasite Trichuris, littermate control mice develop pathogen-specific TH2 cytokine responses and eradicate infection while mice with an IEC-specific deletion of IKKβ (IkkbΔIEC mice) fail to do so. Further, IkkbΔIEC mice exhibit exacerbated production of DC-derived IL-12/23p40 and TNF-α, heightened levels of CD4+ T cell-derived IFN-γ and IL-17, and develop severe intestinal inflammation. Blockade of proinflammatory cytokines during Trichuris infection ablates the requirement for IKKβ in IECs to promote CD4+ TH2 cell-dependent immunity, identifying a critical role for IECs in limiting type 1 cytokine production in the GI tract. These results suggest that the balance of IKKβ-dependent gene expression is critical for intestinal immune homeostasis by promoting mucosal immunity and limiting chronic inflammation. This work is funded by the NIH, CCFA and the Irvington Institute.