The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shi, G.-X.
Right arrow Articles by Kehrl, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shi, G.-X.
Right arrow Articles by Kehrl, J. H.
The Journal of Immunology, 2004, 172: 5175-5184.
Copyright © 2004 by The American Association of Immunologists

Toll-Like Receptor Signaling Alters the Expression of Regulator of G Protein Signaling Proteins in Dendritic Cells: Implications for G Protein-Coupled Receptor Signaling

Geng-Xian Shi1, Kathleen Harrison1, Sang-Bae Han1, Chantal Moratz and John H. Kehrl2

B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G{alpha}i- and G{alpha}q-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1–/– mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)3 function as the sentinels of the immune system (reviewed in Refs. 1 and 2). Immature DCs (iDC) traffic from the blood to inflamed tissues where they capture Ag, and differentiate into mature DC (mDC). Subsequently, they move to the draining lymphoid nodes to prime naive T cells. iDC are highly endocytic and well adapted for the capture of Ag, but they function poorly as APCs. In contrast, mDC are efficient APCs and important modulators of T cell function. Many pathogen-derived substances are efficient inducers of iDC maturation, and do so predominantly by the engagement of Toll-like receptors (TLRs) (reviewed in Refs. 3 and 4). In humans, 10 TLR homologs have been identified, the majority displayed by DCs. TLR contain two major domains, an extracellular domain characterized by leucine-rich repeats and an intracellular Toll-like domain. TLR signaling leads to NF-{kappa}B activation, a requirement for the differentiation of iDC to mDC (5).

iDCs express the chemokine receptors CCR1, CCR2, CCR5, and CXCR1, and respond to their respective ligands, chemokines often expressed in inflamed tissues (6, 7). In addition, iDCs migrate in response to other inflammatory mediators that couple to G protein-coupled receptors (GPCRs) including histamine (8), sphingosine-1-phosphate (S-1P) (9), lysophosphatidic acid (LPA) (10), and ATP (11). Maturing DCs lose their migratory response to many of these inflammatory chemoattractants by either receptor down-regulation or receptor desensitization, and acquire responsiveness to CC chemokine ligand (CCL)19 and CCL21 via the acquisition of high levels of CCR7 (6, 7). CCL19 and CCL21 have significant roles in the accumulation of Ag-loaded DCs in T cell-rich areas of draining lymph nodes. Exposure of maturing DCs to histamine, S-1P, LPA, or ATP no longer induces a chemotactic response, but rather down-regulates IL-12 and enhances IL-10 production (8, 9, 10, 11). A number of prior studies have demonstrated that signaling via chemokine and other GPCRs can modulate DC IL-12 production (reviewed in Ref. 12). For example, the production of IL-12 by CD8{alpha}+ murine DCs can be triggered by CCR5 signaling (13).

Ligand-activated GPCRs such as chemokine receptors act as a guanine nucleotide exchange factor for G{alpha} subunit of the heterotrimeric G protein (reviewed in Refs. 14 and 15). Once the G{alpha} subunit exchanges GDP for GTP, it dissociates from the G{beta}{gamma} heterodimer, thereby allowing both G{alpha} and G{beta}{gamma} to activate downstream effectors. However, G{alpha} subunits have an intrinsic GTPase activity that limit the duration that they remain GTP bound and thus able to signal. In addition, GTPase-activating proteins (GAPs) for G{alpha} subunits termed regulator of G protein signaling (RGS) proteins can further accelerate the intrinsic GTPase activity of G{alpha} subunits (reviewed in Ref. 16). Genetic studies in yeast, Aspergillus nidulans, and Caenorhabditis elegans initially identified such proteins (17, 18, 19). Providing evidence that they function by interacting with G{alpha} subunits, a yeast two-hybrid screen with G{alpha}i3 identified a mammalian RGS protein originally termed GAIP and now RGS19 (20). Cementing the functional relationship between the yeast and mammalian proteins, several human RGS proteins substituted for Sst2p, a protein involved in the desensitizing of pheromone signaling, a G protein-coupled signaling pathway in yeast (21). Rapidly thereafter, RGS proteins were shown to possess GAP activity for Gi and Gq subfamily members (22, 23, 24). Coding regions for ~25 human RGS proteins have now been identified. Two Rho guanine exchange factors, which possess divergent RGS domains, selectively act as GAPs for G{alpha}12 and G{alpha}13 (25, 26). Experimentally, the introduction of expression vectors for RGS1, RGS3, and RGS4 into B lymphocyte cell lines dramatically impairs chemokine-induced cell migration (27, 28, 29). Furthermore, the lack of Rgs1 results in aberrant responses to the chemokines CXCL12 and CXCL13 in murine B cells.4

Differential expression of RGS proteins in iDC and mDC may also contribute to the regulation of DC trafficking and regulate responses to other GPCR ligands. We have examined the expression of RGS proteins by analyzing mRNA expression in iDC and mDCs derived from human monocytes and mouse bone marrow (BM). Maturation is accompanied by a marked reduction in RGS14 (Rgs14) and RGS18 (Rgs18) levels and the induction of RGS1 (Rgs1) and RGS16 (Rgs16). Because the effects of RGS18 on cell migration had not been previously studied, we examined whether RGS18 modulates CXCL12-, CCL19-, and CCL21-induced cell migration. In addition, we provide evidence for the functional importance of Rgs1 by examining the chemoattractant responses of Rgs1-deficient mouse DCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and reagents

The coding regions of human RGS18 and mouse RGS18 were isolated by PCR from human BM and mouse spleen Marathon-ready cDNA library (BD Clontech, Palo Alto, CA) and then subcloned into the EcoRI/BamH1 sites of p3XFLAG-CMV-14 (Sigma-Aldrich, St. Louis, MO) or pEGFP-N1 (BD Clontech, Palo Alto, CA). The coding region of CXCR5 was isolated by RT-PCR using RNA prepared from HS-Sultan cells and subcloned into EcoRI site of pAAV-MCS. The coding region of human and mouse CCR7 were isolated by PCR from human and mouse spleen Marathon-ready cDNA libraries (BD Clontech) and then subcloned into TA-cloning vector pCR3.1 (Invitrogen, Carlsbad, CA). The Abs against the following were purchased: FLAG (Sigma-Aldrich), phospho-p42/44 extracellular signal-regulated kinase (ERK) (Cell Signaling, Beverly, MA), p42/44 ERK, anti-G{alpha}s (Santa Cruz Biotechnology, Santa Cruz, CA), CD14, CD11c, CD40, CD95 (BD PharMingen, San Diego, CA), and anti-RGS1 (Novus Biologicals, Littleton, CO). Rabbit anti-RGS14 was raised against recombinant mouse RGS14 and cross-reacts with human RGS14.

Human GM-CSF, IL-4, IL-15, CXC chemokine ligand (CXCL)12, CXCL13, CCL19, and CCL21 were purchased from R&D Systems (Minneapolis, MN), and LPS, poly(I:C), and L-{alpha}-LPA were from Sigma-Aldrich.

Cell lines and cell cultures

293T, CHO-K1, COS, and HeLa were obtained from the American Type Culture Collection (Manassas, VA). All of the cell lines were maintained in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% FCS (HyClone, Logan, UT) except CHO-K1 cells, which were maintained in RPMI 1640 (Life Technologies) supplemented with 10% FCS.

Generation of human monocyte-derived DCs

PBMC were obtained from heparinized blood of healthy donors by Ficoll density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden). The isolated PBMC were cultured in RPMI 1640 at 37°C in 100-mm plate (Falcon, Franklin Lakes, NJ) for 3 h, and the nonadherent cells were discarded, and the adherent cells were washed with PBS for three times. After this procedure, the resulting cell population was represented by >98% CD14+ monocytes, as assessed by flow cytometry using FITC-CD14 Ab. Alternatively, elutriated monocytes prepared from leucopaks were used as the starting population. The monocytes were maintained in RPMI 1640 medium supplemented with 10% FCS in the presence of GM-CSF (100 ng/ml) and IL-4 (50 ng/ml). After 4–6 days of culture, nonadherent and loosely adherent cells were collected and used for subsequent experiments. The purity of the recovered DCs exceeded 95% as assessed by flow cytometry using PE-CD11c Ab.

Isolation of mouse BM-derived DCs

DCs were generated from BM cells from 8- to 10-wk-old C57BL/6 female mice (30). Briefly, BM cells were flushed out from the femurs and tibias. After lysis of RBC, whole BM cells (2 x 105 cells/ml) were cultured in 100-mm2 culture dishes in 10 ml/dish complete medium containing 2 ng/ml GM-CSF. At day 3, another 10 ml of fresh complete medium containing 2 ng/ml GM-CSF was added. On day 6 of the culture, half of the medium was changed. On day 8 of the culture, nonadherent DCs and loosely adherent DCs were harvested by gently pipetting and used as iDC. iDCs recovered from these cultures were generally ~85–90% CD11c+ and MHC class IImed-high, CD80med, and CD86low-med. Maturation of iDC was accomplished by treating with LPS at 1 µg/ml for the last 24 h of culture. mDCs were MHC class IIhigh, CD80high, and CD86high.

Luciferase reporter gene assay

For the muscarinic type 1 (M1) receptor-mediated serum response element (SRE) and NF-{kappa}B activation, HeLa cells were cotransfected with M1 receptor gene constructs (0.25 µg), SRE (50 ng), or NF-{kappa}B (50 ng) luciferase reporter gene, and {beta}-galactosidase gene (100 ng) in the absence or presence of RGS18-3XFlag (0.5, 1.0, or 2.0 µg), RGS3-Flag (2.0 µg), or C3 (0.5 µg). After 24 h, the cells were stimulated with 100 µmol/L carbachol (Sigma-Aldrich) for 5–6 h while starving cells with fresh DMEM without FCS and then were lysed in reporter lysis buffer (Promega, Ann Arbor, MI). After removing the cell debris, the luciferase and {beta}-galactosidase activity were measured using a luminometer (Analytical Luminescence Laboratory, San Diego, CA).

Measurement of inositol phosphates

The COS cells maintained in the inositol-free DMEM in 12-well plate were transfected with 0.1 µg of M1 receptor gene construct in the absence or presence of RGS18-3XFlag (0.25, 0.5, or 1.0 µg) or RGS3-Flag (1.0 µg). Twenty-four hours after transfection, the culture medium was replaced with inositol-free DMEM containing 5% FCS and 1 mM sodium pyruvate for 2 h, after which 2 µCi/ml myo-[2-3H]inositol (Amersham Pharmacia Biotech) was added and, 15 min later, 10 mM LiCl. The cells were incubated for an additional 14 h and then stimulated with 100 µmol/L carbachol for 15 min before washing with PBS, followed by the addition of 0.5 ml of 20 mM formic acid. Thirty minutes later, the supernatant was collected, and a second extraction was performed. Each 1-ml extract was neutralized to pH 7.5 with 7.5 mM HEPES and 150 mM KOH. The supernatants were centrifuged for 2 min at 15,000 x g and collected, and each was loaded onto a 0.5-ml Dowex AG-X8 column (Bio-Rad, Richmond, CA) that had been previously washed with 2 ml of 1 M NaOH, 2 ml of 1 M formic acid, and then five washes of 5 ml of water. After loading the sample, the column was washed with 5 ml of water and 5 ml of 5 mM borax and 60 mM sodium formate. The columns were eluted with 3 ml of 0.9 M ammonium formate and 0.1 M formic acid. A volume of 0.2 ml of each elution was added to 10 ml of CytoScint and analyzed via scintillation counting.

Immunoblotting and immunoprecipitations

Cell lysates were prepared using an appropriate lysis buffer plus protease inhibitors for 30 min on ice. The detergent-insoluble materials were removed by microcentrifugation for 10 min at 4°C. Equal amounts of proteins from each sample were fractionated by 10% SDS-PAGE and transferred to pure nitrocellulose. Membranes were blocked with 5% BSA in Tween 20 plus TBS (TTBS) for 1 h and then incubated with an appropriate dilution of the primary Ab in 5% BSA in TTBS for 2 h or overnight. The blots were washed three times with TTBS before the addition of a biotinylated Ab (DAKO, Carpinteria, CA) diluted 1/5,000 in TTBS containing 5% BSA for 1 h and then incubated with streptavidin conjugated to HRP (DAKO) diluted 1/10,000 in TTBS containing 5% BSA for 1 h. The signal was detected by ECL according to the manufacturer’s instruction (Amersham Pharmacia Biotech).

Mitogen-activated protein kinase (MAPK) assay or ERK activation

COS cells were transfected with appropriate receptor expression construct (0.5 µg, respectively) in presence or absence of RGS18-3XFlag, RGS3-Flag, or RGS1-Flag (2.0 µg, respectively) using Superfect (Qiagen, Valencia, CA). Pertussis toxin (PTX; Calbiochem, Darmstadt, Germany) treatment for 6 h at concentration of 100 ng/ml was used as positive control. Twenty-four hours after transfection, the cells were starved with fresh DMEM without FCS for 6 h and then stimulated with LPA (30 µmol/L), CXCL12 (100 ng/ml), CXCL13 (250 ng/ml), or CCL19 (250 ng/ml) for varying durations, and then lysed with 300 µl of kinase lysis buffer. MAPK (ERK) activation was detected by immunoblotting with anti-phospho-p42/44 ERK mAb using detergent-soluble fraction of lysates after fractionation by SDS-PAGE.

RT-PCR and Northern blot analysis

Total RNA was isolated using TRIzol reagents. For RT-PCR, 500 ng of total RNA was used for reverse transcription (Qiagen). The PCR primers used for the PCR are listed in Table I.


View this table:
[in this window]
[in a new window]
  		Table I. Primers used for the PCR

 
For the quantitative RT-PCR, a Roche LightCycler was used with a LightCycler Fast Start-DNA Master Syber Green 1 kit (no. 2239264; Roche, Indianapolis, IN). Melting curve analysis was performed to control the specificity of PCR product fluorescence. Value of the crossing point was determined for each gene and sample during real-time PCR. The value of crossing point represents the number of cycles where fluorescence levels of each sample are the same. Plasmids with the appropriate PCR insert subcloned served as the control templates. For the Northern blot analysis, RNA was size fractionated and transferred to nitrocellulose. The membranes were hybridized with a 708-bp RGS18 cDNA fragment or a 1137-bp CCR7 cDNA fragment labeled with [{alpha}-32P]dCTP using Prime-It RmT Random Primer Labeling kit (Stratagene, La Jolla, CA) as a probe. {beta}-Actin expression was used as a control. Hybridization was performed at 68°C for 2 h using QuickHyb (Stratagene), washed three times in 2x SSC/0.1% SDS for 15 min each at room temperature, and then in 0.1x SSC/0.1% SDS at 60°C for 30 min.

Migration assay

For CHO cell migration, CHO cells were transfected with or without 0.5 µg of expression vector for CCR7 in the presence of 2 µg of RGS18-GFP, RGS1-GFP, or pEGFP-N1 as control. After 36 h, the transfected cells were harvested and loaded into upper 8-µm-pore polycarbonate six-well chamber (Corning, Cambridge, MA). CXCL12 (100 ng/ml), CCL19 (250 ng/ml), and CCL21 (250 ng/ml) were diluted in serum-free medium and added to the lower compartment. After 6-h incubation at 37°C, the migrated cells were collected and counted with FACS at high speed for 1 min. The 100 ng/ml PTX-treated pEGFP-N1-transfected cells were used as positive control. For monocyte-derived DC migration, the recovered DCs were transfected with empty pEGFP-N1, hRGS-18-GFP, hRGS13-GFP, and hRGS1-CT-GFP using Human Dendritic Cell Nucleofector kit I (Amaxa Biosystems, Gaithersburg, MD). The cells were incubated in presence of 100 ng/ml GM-CSF and 50 ng/ml IL4 at 37°C for 48–60 h and then harvested for migration assay using 5-µm pore polycarbonate filter in 24-well Transwell chambers (Corning) with or without chemokines in lower well at concentration of SDF1{alpha}/CXCL12 (100 ng/ml), MIP-3{beta}/CCL19 (250 ng/ml), or 6Ckine/CCL21 (250 ng/ml) for 3 h. The PTX-treated (100 ng/ml) pEGFP-N1-transfected cells (3 h) was used as positive control. The migrated cells were harvested, and the green fluorescent protein (GFP)-positive cells were counted with FACS for 1 min at high flow speed. The initial cell pools were counted as control of loaded cell number. The percentage of migration is calculated by dividing migrated GFP-positive cell number with loaded GFP-positive cell number. The migration assays with the murine BM-derived DCs were performed similar to those performed with the human DCs.

Generation of Rgs1–/– mice

The targeting construct was designed by replacing the small Xbal fragment located at the end of exon 1 with the neomycin gene. For negative selection of nonhomologous recombination, the thymidine kinase gene was placed in opposite transcriptional orientation upstream of exon 1. Following electroporation with the linearized targeting, ES cells were selected with G418 and resistant clones screened for homologous recombination. Resultant ES clones were injected into C57BL/6J blastocysts. Chimeric mice were bred, and germline transmission was documented by Southern blotting. Screening for homozygous Rgs1–/– mice was performed by PCR analysis of genomic DNA using Rgs1-specific primers. The Rgs1 mutation was backcrossed onto a C57BL/6 background six times. Mice were housed in specific pathogen-free conditions and used in accordance to the guidelines of the Institutional Animal Care Committee at the National Institutes of Health.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RGS protein expression in human monocytes and monocytes-derived DCs

We reverse-transcribed RNAs extracted from purified human monocytes, iDC (monocytes cultured with GM-CSF and IL-4 for 4–6 days), and iDC stimulated with LPS, poly(I:C), or CD95 for either 4 or 24 h, and amplified the resulting DNA with specific primers for RGS1–14, RGS16–20, or {beta}-actin (Fig. 1A). We detected very low levels or no mRNA expression of RGS3–9, RGS11, RGS13, or RGS17 in monocytes, iDC, or stimulated iDC (data not shown). The purified monocytes contained modest amounts of RGS2, RGS10, RGS14, and RGS19, and lower amounts of RGS1, RGS16, and RGS18. In comparison, the iDCs had much less RGS1 and RGS16 expression and a higher level of RGS18. Signaling through the TLRs, TLR3 and TLR4, markedly enhanced RGS1, RGS16, and RGS20 expression, but down-regulated that of RGS14 and RGS18. The analysis of the same RNAs by Northern blotting revealed the same pattern of RGS18 expression as detected by RT-PCR as well as verified the efficacy of the LPS and poly(I:C) signaling, because both stimuli rapidly induced mRNA expression for the chemokine receptor CCR7 (Fig. 1A, and data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 1. Expression of RGS proteins in DCs. A, Monocyte-derived DCs were cultured in medium for 4 or 24 h or stimulated with LPS, CD95, or poly(I:C) for similar durations (lanes 1–8). RNA was extracted and subjected to RT-PCR with primers specific for various RGS proteins or {beta}-actin. In addition, RNAs from monocytes (lane 9) were similarly analyzed. Lane 10 is from RNA not subjected to reverse transcription before amplification (no RT control). PCR products were fractionated on an agarose gel followed by ethidium bromide staining. At the bottom of the figure, a Northern blot is shown, which documents RGS18, CCR7, and {beta}-actin expression in similar RNAs. B, Quantitative RT-PCR analysis was performed with the same RNAs to determine changes in RGS18, RGS16, or RGS1 relative to {beta}-actin levels. The amounts are expressed as mRNA expression relative to {beta}-actin. The RGS18 results (x10) are shown. Because the RGS16 peak expression level did not exceed 10–4, the RGS16 results are not shown on the graph. C, Western blot analysis of RGS1, RGS14, and G{alpha}s expression. Human iDCs were cultured with medium for 24 h or stimulated with LPS for 24 or 48 h. Cell lysates were immunoblotted using a 1/300 dilution of anti-RGS1, 1/1000 dilution of anti-RGS14, or 1/500 dilution of anti-G1{alpha}s. The approximate molecular masses of the identified bands are indicated.

 
Next, we established quantitative PCR assays for the analysis of RGS1, RGS16, RGS18, and {beta}-actin expression. We normalized the result from the RGS proteins to that of {beta}-actin and expressed the data as nanograms per microliter based on standard curves generated from plasmid DNA containing the appropriate inserts. This approach allowed for a quantitative comparison between the different samples (Fig. 1B). We found that the iDC expressed modest levels of RGS18 (~50% higher than monocytes) and low levels of RGS1 (5-fold less than monocytes) and RGS16 (7-fold less than monocytes). Stimulation of iDC with LPS led to a ~24-fold increase in RGS1 levels, a 6-fold increase in RGS16, and a 100-fold decrease in RGS18 expression, whereas poly(I:C) stimulation caused a 32-fold increase in RGS1, 3-fold increase in RGS16, and a similar drop in RGS18, as did LPS.

Although high-quality Abs for many of the RGS proteins are lacking, an RGS1 Ab raised against a peptide from the C terminus of RGS1 readily identified an LPS-inducible band at the appropriate molecular mass in human monocyte-derived DCs (Fig. 1C). Similar lysates immunoblotted with affinity-purified Abs raised against the C terminus of RGS18 failed to identify a band at the appropriate molecular mass, which decreased with stimulation (data not shown). Although this antiserum recognized overexpressed RGS18, it did so poorly, suggesting that the failure to detect endogenous RGS18 may be secondary to a relatively low affinity. An RGS14-specific Ab (31) documented the fall in RGS14 expression following LPS signaling, whereas a G{alpha}s-specific Ab demonstrated no significant change in G{alpha}s levels, and an actin-specific Ab revealed similar actin levels (Fig. 1C, and data not shown).

G{alpha} expression in human BM-derived DCs

Signaling through the yeast pheromone receptor causes a significant increase in the expression of the yeast RGS homolog SST2 as well as the yeast G{alpha} homolog GPA1 (32, 33). To determine whether the changes in RGS protein expression in DCs stimulated with TLRs was accompanied by the altered expression of G{alpha} subunits, we examined RNAs prepared from iDCs and iDCs stimulated with LPS or poly(I:C) (Fig. 2) We found that monocytes and iDC expressed significant amounts of G{alpha}s, G{alpha}i2, and G{alpha}16, which did not change significantly following TLR3 or TLR4 stimulation. iDC expressed low levels of G{alpha}i3, which were reduced by TLR3 and TLR4 signaling. iDC also expressed low levels of G{alpha}q and G{alpha}13; however, in contrast to G{alpha}i3, TLR4 and, even more so, TLR3 signaling caused a significant increase in their expression levels. We did not detect PCR products arising from either G{alpha}i1 or G{alpha}o. Despite the pronounced induction of RGS20, we detected only a very low level of G{alpha}z, which did not change following TLR signaling (data not shown). Immunoblotting with G{alpha}-specific Abs revealed no changes in G{alpha}s (above), G{alpha}16, G{alpha}12, or G{alpha}i2 following LPS stimulation of iDCs (data not shown).



View larger version (89K):
[in this window]
[in a new window]
  		FIGURE 2. Levels of G{alpha} subunits in monocytes, monocyte-derived DCs, and following TLR stimulation. RNAs extracted from monocytes (lane 9) or monocyte-derived DCs cultured in medium for 4 or 24 h or stimulated with LPS, poly(I:C), or CD95 for similar durations were subjected to RT-PCR (33 cycles) to analyze the expression of G{alpha} subunits. The RT-PCR products were size fractionated on agarose gels and visualized by ethidium bromide staining. The G{alpha} subunits are referred to by their GenBank names. There was no detectable expression of GNAI1, GNAO, or GNA14, and low levels of GNAZ relative to the other G{alpha} subunits.

 
Comparison of RGS18 with RGS3 on M1 receptor and chemokine receptor signaling

The pronounced alteration in RGS18 during DC maturation prompted a comparison between RGS18 and several other RGS proteins on known GPCR signaling pathways (Figs. 3 and 4). The activities of numerous RGS proteins have been assessed using the M1 receptor, which signals through Gq and G12/13. To monitor Gq signaling, we measured the production of inositol 1,4,5-trisphosphates (IP3), and used the activation of a SRE reporter gene to assess both Gq and G12/13 signaling (34, 35). We first compared RGS18 to RGS3, because RGS3 is among the most potent of the RGS proteins in inhibiting GPCR signaling (34). We transfected 293 cells with the M1 receptor and the following day stimulated the cells with M1 receptor ligand carbachol and measured the generation of IP3 and the activation of the SRE reporter (Fig. 5). Both RGS3 and RGS18 blunted to a similar extent the induction of IP3 by M1 receptor signaling. In contrast, RGS3 much more significantly interfered with the M1 receptor-mediated activation of the SRE reporter than did RGS18. M1 receptor signaling also activates an NF-{kappa}B-dependent reporter gene, probably also via Gq and G12/13 signaling (36). When we compared the effects of RGS3 and RGS18 on M1 receptor, both inhibited, although the effect of RGS3 again exceeded that of RGS18.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3. RGS18 inhibits M1 receptor signaling. A, IP3 production. COS cells were transfected with 0.2 µg of M1 receptor gene constructs in the presence or absence of constructs that express RGS18 or RGS3. IP3 production was measured as described in Materials and Methods. The cells were stimulated with 100 µmol/L carbachol for 6 h after serum starving the cells. B, SRE activation. HeLa cells were cotransfected with 50 ng of a SRE reporter gene construct and 0.25 µg of M1 receptor gene construct in the presence or absence of RGS18 or RGS3. A construct that expresses the Clostridium botulinum C3 exozyme served as a control. The cells were stimulated as above. Luciferase activity was measured and normalized to a control plasmid that expressed {beta}-galactosidase. C, NF-{kappa}B activation. HeLa cells were cotransfected with 0.2 µg of a M1 receptor construct and 100 ng of a NF-{kappa}B reporter gene construct in the presence or absence of constructs that express RGS18 or RGS3. Luciferase activity normalized to {beta}-galactosidase activity is shown.

 


View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4. Comparision of RGS18, RGS1, and RGS3 on signaling through the LPA receptor, CXCL12, CXCL13, and CCR7. A, Inhibition of LPA induced ERK activation. COS cells were transfected with or without 2 µg of vectors that express RGS18 (lanes 2, 5, 8, and 11) or RGS3 (lane 3, 6, 9, and 12). The cells (lanes 4–12) were stimulated with LPA (30 µmol/L) for 2, 5, or 10 min. The amount of phosphorylated ERK1 (pERK1) induced was detected with a specific Ab by immunoblotting. The levels of RGS3 and RGS18, and of ERK1 and ERK2 in the cell lysates are shown. B, Inhibition of CXCL12 induced ERK activation. COS cells were transfected with 0.5 µg of expression vector for CXCR4 (lanes 1–12) in the presence or absence of 2 µg of expression vectors for RGS3 or RGS18. The cells (lanes 4–12) were stimulated with CXCL12 for 2, 5, or 10 min. Similar immunoblotting was performed as in the first panel. C, Inhibition of CXCL13 induced ERK activation. Similar experiment as shown in second panel except the cells were transfected with CXCR5 rather than CXCR4. The cells were stimulated with CXCL13 at final concentration of 250 ng/ml for 5, 10, or 15 min. D, Inhibition of CCL19 induced ERK1 activation. COS cells were transfected with 0.5 µg of expression vector of CCR7 (lanes 1–15) in the presence or absence of 2 µg of expression vectors for RGS18 (lanes 2, 7, and 12), RGS1 (lanes 3, 8, and 13) or RGS3 (lanes 4, 9, and 14). The cells treated with PTX (lanes 5, 10, and 15; 100 ng/ml) for 6 h was used as control. The cells were stimulated with CCL19 (lanes 6-15) at final concentration of 250 ng/ml for 2 or 5 min. Similar immunoblotting was performed as in the above panels.

 


View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of CHO cell and monocyte-derived DC migration in response to CXCL12, CCL19, and CCL21. A, RGS18 inhibits CHO cell migration to CCL19 and CCL21. CHO cells were transfected with 0.5 µg of expression construct for CCR7 in the presence or absence of RGS18-GFP, RGS1-GFP, or GFP for 36 h, and then collected for migration assay as described in Materials and Methods. CCL19 and CCL21 were used at 250 ng/ml in the lower chamber. PTX treatment (100 ng/ml) for 6 h was used as control. B, RGS18 inhibits human monocyte-derived DC cell migration. Recovered DCs were transfected with RGS18-GFP, RGS13-GFP, RGS1-GFP, or GFP vector for 48 h, and then collected for migration assay as described in Materials and Methods. The data are represented as X ± SD from one experiment performed in triplicate. Medium or medium plus either CXCL12 (100 ng/ml) or CCL21 (250 ng/ml) were placed in the bottom chamber. Where indicated, the cells were treated with PTX (100 ng/ml) for 6 h before the assay. The experiments were performed three times with similar results.

 
LPA is a bioactive lipid mediator, which signals through the LPA1, LPA2, and LPA3 receptors, all of which are expressed by iDC and mDC (10). LPA stimulates iDC actin polymerization and chemotaxis, through a PTX-sensitive pathway. To assess the effect of RGS18 on LPA signaling, we transfected COS-7 cells with RGS18 or RGS3 and measured MAPK/ERK activation using phospho-specific Abs following exposure of COS-7 cells to LPA (Fig. 6). In this experiment, LPA signaled through the endogenous LPA receptors on COS-7 cells. We found that both RGS18 and RGS3 inhibited LPA-mediated ERK activation, although RGS3 reduced ERK activation slightly more than did RGS18. Next, we compared RGS18 and RGS3 on signaling through two chemokine receptors, CXCR4 and CXCR5 (Fig. 6). Both iDC and mDC express CXCR4, whereas a subset of DC that home to primary lymphoid follicles express CXCR5. Using a similar approach as with the other receptors, we transfected 293 cells with either CXCR4 or CXCR5 in the presence or absence of expression vectors for RGS18 or RGS3. We again monitored ERK activation using phospho-specific Abs at various time points following exposure to the appropriate chemokine. Both RGS3 and RGS18 significantly inhibited CXCL12- and CXCL13-induced ERK activation. Finally, we examined signaling through the CCR7, a chemokine receptor induced on mDC (Fig. 6). We compared RGS1, RGS3, and RGS18. We did not detect a significant difference among the three RGS proteins. PTX blocked the CCR7-induced ERK activation, a result consistent with the known role of Gi in chemokine signaling. Overall, the signaling data did not reveal any significant difference between RGS18 and either RGS1 or RGS3 in modulating Gi or Gq signaling.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 6. Rgs expression in murine BM-derived DCs. A, Rgs expression following LPS stimulation. BM-derived DCs were stimulated or not for 2 or 48 h; RNA was extracted and subjected to RT-PCR with Rgs-specific primers listed in Table I (30–33 cycles). PCR products were fractionated on an agarose gel followed by ethidium bromide staining. B, Effect of CXCL12 on Rgs1 and Rgs18 expression. BM-derived DCs were stimulated with LPS or CXCL12 for 2, 4, or 24 h. The levels of Rgs1, Rgs18, and actin expression at the various time points are shown.

 
Comparison of RGS18 and RGS on chemokine-induced cell migration

We also tested whether RGS18 like RGS1 and RGS3 can inhibit chemokine-induced cell migration. First, we transfected CHO cells with CXCR4 or CCR7 in the presence of expression vectors for GFP, RGS18-GFP, or RGS1-GFP, and measured the ability of the cells to respond to either CXCL12 (data not shown), CCL19, or CCL21 (Fig. 7) using a standard filter-based assay. We found that stimulation of the cells with the appropriate chemokine led to an increase in CHO cell migration, and that both the RGS proteins significantly inhibited the chemokine-induced enhancement. Finally, we performed a similar experiment using DCs transfected with RGS18-GFP, RGS1-GFP, or RGS13-GFP, inducing cell migration by stimulation through endogenous chemokine receptors. Although the transfection procedure had a deleterious effect upon the migratory capacity of the DCs, each of the RGS-GFP fusion proteins significantly reduced DC migration in response to CXCL12, CCL19 (not shown), and CCL21 when compared with GFP alone. The decreased migratory capacity following transfection was not due to expression of GFP, but rather secondary to the transfection procedure itself (K. Harrison, unpublished observation).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 7. Migratory response of BM-derived DCs from wild-type and Rgs1–/– mice. A, Rgs1 and Rgs18 expression in wild-type and Rgs1–/– mice. RNA extracted from BM-derived DCs stimulated with LPS for 24 h (mDC) or not (iDC) from wild-type and Rgs1–/– was subjected to RT-PCR to detect Rgs1, Rgs18, and actin expression levels. B, Migration of iDCs to CXCL12 or CCL19. Wild-type and Rgs1–/– BM-derived iDCs were subjected to a standard chamber chemotaxis assay using increasing concentrations of either CXCL12 or CCL19. Data are shown as percentage of migration and are representative of one of three experiments performed. C, Migration of mDCs to CXCL12. Wild-type and Rgs1–/– BM-derived DCs were stimulated to mature by treating with LPS for 24 h. Percentage of migration of wild-type and Rgs1–/–-deficient mDCs in response to increasing concentrations of CXCL12 is shown. Representative of one of three experiments performed.

 
Rgs expression in mouse BM-derived DCs

Next, we examined the Rgs protein expression in mouse BM-derived DCs induced to mature with LPS for 2 or 48 h or not (Fig. 2A). A similar pattern of Rgs protein expression occurred, although we noted some differences. Like the human DCs, the levels of Rgs2, Rgs10, and Rgs19 remained unchanged following stimulation, Rgs1 and Rgs16 levels rose, and Rgs18 and Rgs14 levels declined, although by 48 h the levels of Rgs14 had begun to return toward the level observed in the immature cells. In contrast to the human cells, the mouse DCs expressed Rgs11, Rgs12, and Rgs17, but not Rgs20.

Because CXCL12 signaling enhanced RGS1 expression in human monocyte-derived DCs (K. Harrison, unpublished observation), we also examined the expression of Rgs1 and Rgs18 following stimulation of mouse BM-derived DCs with CXCL12 and compared it to LPS. Again, the stimulation of BM-derived DCs resulted in an up-regulation of Rgs1 and down-regulation of Rgs18; however, in contrast to the human DCs, signaling through CXCR4 had no effect on Rgs1 and Rgs18 expression (Fig. 2B). Also, several other Rgs proteins including Rgs11, Rgs14, and Rgs16 did not change following exposure to CXCL12 (data not shown).

Migration of Rgs1-deficient mouse BM-derived DCs

The availability of mice in which Rgs1 has been disrupted allowed us to directly test the effect of Rgs1 on the migratory response of DCs to chemokines. Mice homozygous for the Rgs1 mutation have no readily apparent abnormalities, although many of the B cell follicles in their spleens have germinal centers even in the absence of immune stimulation. Furthermore, antigenic stimulation of a Rgs1–/– mouse leads to an exaggerated splenic germinal center reaction, partial disruption of the normal architecture of the spleen and Peyer’s patches, and abnormal trafficking of Ab-secreting cells.4 Although many of these abnormalities likely result from improper trafficking of Rgs1–/– B cells, DC defects may also contribute. We first verified that the Rgs1–/– iDCs and mDCs lacked Rgs1 mRNA. We prepared BM-derived DCs from wild-type and Rgs1–/– mice, stimulated them with LPS or not, and checked Rgs1 expression. As expected, Rgs1–/– DCs lacked Rgs1 expression, but possessed levels of Rgs18 similar to that of wild-type mice, indicating that the disruption of Rgs1 did not effect Rgs18 expression (Fig. 7A). Rgs18 resides near to Rgs1 on chromosome 1. Next, we analyzed the response of wild-type and Rgs1–/– BM-derived iDCs in a migration assay, using varying concentrations of either CXC12 or CC19 in the bottom well of the chemotaxis chamber. At every concentration that we tested, nearly twice as many Rgs1–/– iDCs migrated in response to CXCL12 and to CCL19 as compared with the wild-type iDCs (Fig. 7B). Somewhat surprisingly, the absence of Rgs1 had less effect on the ability of the mDCs to migrate in response to CXCL12 in the chemotaxis assay than it did in the iDCs. Nevertheless, the chemotactic response of the Rgs1–/– mDCs exceeded that of wild-type mice at every concentration tested.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both TLR and GPCR receptor signaling have substantive roles in the regulation of DC function. Signaling through either TLR3 or TLR4 induces the maturation of iDC and alters the expression of chemokine receptors, i.e., induces CCR7 and CXCR4, and diminishes CCR5 and CCR6. TLR signaling significantly alters the expression of RGS proteins in human monocyte-derived DCs, decreasing RGS18 and RGS14, but augmenting RGS1, RGS16, and RGS20. In addition, TLR signaling induces changes in the expression of several G{alpha} subunits including increasing the expression of Gq{alpha} and G{alpha}13 in these cells. This provides a mechanism whereby TLR signaling can regulate signaling through chemokine receptors and other GPCRs. Consistent with human monocyte-derived DC data, mouse BM-derived iDCs expressed a similar pattern of Rgs proteins, whose expression levels responded similarly to LPS signaling. Of the RGS proteins modulated by TLR signaling, we focused on RGS1 and RGS18 because of their substantial regulation and the availability of Rgs1–/– mice.

We showed that iDC cells express RGS18 and that TLR receptor signaling potently down-regulates it. Three previous reports documented strong RGS18 expression in megakaryocytes (37, 38, 39), and another report found that hemopoietic stem cells express high amounts of Rgs18 (40). These reports also demonstrated that Jurkat, K562, platelets, and CD14+ peripheral blood cells expressed RGS18. RGS18 acted as a GAP for Gi{alpha} and Gq{alpha} and localized in the cytosol of megakaryocytes. It also inhibited angiotensin-induced IP3 production in 293 cells and CCR2 signaling (37, 38, 40). We performed a wider range of functional studies of RGS18 than previously reported, revealing that it inhibits Gi and/or Gq signaling through the CXCR4, CXCR5, CCR7, LPA receptors, and the M1 receptor. In most instances, RGS18 behaved similar to RGS3 in its effect on GPCR signaling, and our data suggests that RGS18 is a particularly potent inhibitor of Gq signaling. In addition, we demonstrated that RGS18 inhibited chemokine-induced DC migration; however, its role in DC function will require further study.

Besides decreasing RGS18, TLR signaling also decreased RGS14 levels in human monocyte-derived iDC and Rgs14 and Rgs11 in mouse BM-derived DCs. RGS14 possesses G{alpha}i and G{alpha}o GAP activity, G{alpha}i guanine nucleotide dissociation inhibitor activity, and a small GTPase binding domain (31, 41, 42). A recent microarray study of DCs exposed to various pathogens (43) also indicates that RGS14 levels decreases in DCs following exposure to Leishmania major or Toxoplasma gondii. Rgs11, which contains a conserved DEP (Dishevelled/EGL-10/Pleckstrin) and a GGL (G protein {gamma}-like) domain, is prominently expressed in the brain and not previously reported to be expressed in any BM-derived cell type.

Although TLR signaling reduced RGS18 and RGS14 expression, RGS1 and RGS20 and to a lesser extent RGS16 were induced. The two original reports of RGS20 documented a largely brain-specific expression pattern (44, 45). However, RGS20 expressed sequence tags suggest a broader range of tissue expression, because expressed sequence tags have been found in cDNA libraries from placenta, liver, melanocytes, and several tumors. The level of RGS20 expression we detected in poly(I:C)-treated DCs was similar to those observed with RNA from brain (K. Harrison, unpublished observation). Because RGS20 has potent Gz GAP activity, the question arose whether TLR signaling altered DC G{alpha}z expression. However, we detected only low levels of G{alpha}z, which did not change with TLR3 or TLR4 signaling. A recent study documented a role for G{alpha}z in maintenance of the Golgi apparatus. The overexpression of RGS20 caused the dissolution of the Golgi complex in HeLa cells (46). Because DCs are known to tightly control the compartmentalization and transport of MHC class I and class II molecules (47), perhaps Gz and RGS20 have some role in the regulation of MHC transport through the Golgi complex. RGS16 expression was also up-regulated in response to TLR signaling, although much more in human than in mouse cells. RGS16 reportedly regulates signaling through CXCR4, CCR3, and CCR5 in T cells, while having little effect on CCR2 and CCR7 signaling (48).

Human monocyte-derived iDCs express low levels of RGS1, and TLR signaling markedly increases RGS1 expression and RGS1 protein levels. Similarly, mouse BM-derived iDCs express lower amounts of Rgs1 than do mDCs stimulated with LPS to mature. Analysis of the chemotactic response of iDCs from Rgs1–/– mice revealed a heightened sensitivity to both CXCL12 and CCL19, arguing that Rgs1 functions in iDCs to set a threshold for chemokine-triggered cell migration. Those cells with a lower level respond while those cells with a higher level do not.

A higher percentage of BM-derived mouse mDCs migrated to CXCL12 than did iDCs at each concentration tested, despite the normal up-regulation of Rgs1 expression that occurs in wild-type mDCs. Although the lack of Rgs1 further enhanced the chemotactic response of mDCs to CXCL12, the difference was not as striking as between wild-type and Rgs1–/– iDCs. Because G{alpha}i expression did not significantly change during DC maturation, an increased availability of G{alpha}i is an unlikely explanation for their robust CXCL12-triggered migratory response. What else might explain the enhanced migratory response of wild-type mDCs? One possibility is that some of the intracellular pool of RGS1 protein is unavailable to interfere with chemokine signaling. For example, a posttranslational modification triggered by TLR signaling might interfere with the intracellular localization or function of RGS1. Another possibility is that the overall balance of RGS proteins in mDCs favors enhanced signaling: although Rgs1 expression increases, Rgs18 and Rgs14 expression falls. Finally, the enhanced chemotaxis of mDCs may be secondary to alterations in the levels of CXCR4 or other components in the signaling apparatus. Future studies should be able to delineate among these possibilities.

Several studies have observed differences in the responsiveness of human monocyte-derived iDC and mDC to GPCR signaling. Although both iDC and mDC have a similar array of S-1P receptors, S-1P stimulated a PTX-sensitive (Gi-sensitive) increase in actin polymerization and chemotaxis of iDC, but those responses were lost by DCs matured with LPS. In mDCs, S-1P inhibited the secretion of TNF-{alpha} and IL-12, and it enhanced secretion of IL-10 (9). The differential effect of S-1P on iDCs and mDCs suggests a prominent Gi response or Gs response, respectively. Although none of the five S1P receptors, S1P1–5, functionally couple to Gs, recently S-1P has been shown to be a ligand for GPR3, GPR6, and GPR12, receptors that do couple to Gs (49). Studies of LPA, ATP, and histamine signaling suggest a similar pattern, Gi-coupled responses in iDC and Gs-coupled responses in mDCs (10, 11, 12). Although a change in receptor expression did not account for the changing pattern of responses (9, 10, 11, 12), an altered RGS protein expression could explain the apparent switch from a prominent Gi response to a Gs response. Two important factors distinguish the chemokine receptors from the GPCR receptors for the above ligands. First, the LPA, ATP, histamine, and likely the S-1P receptors have subtypes that couple to Gs, whereas chemokine receptors do not. Therefore, a modest reduction in Gi signaling mediated by an RGS protein may facilitate a Gs-mediated response in those ligands that have Gs- and Gi-coupled receptors. In contrast, no Gs-coupled response is unmasked with chemokine receptors. Second, mDCs markedly increase their CXCR4 and CCR7 expression levels (6, 7), whereas the expression of the nonchemokine receptors remain stable. The high receptor levels and large amounts of chemokines may overcome some of the inhibitory effects of the RGS proteins, whereas those GPCRs expressed at lower levels remain sensitive.

In conclusion, TLR signaling dramatically altered RGS expression in human and murine iDCs, increasing RGS1 (Rgs1) and RGS16 (Rgs16), and decreasing RGS14 (Rgs14) and RGS18 (Rgs18). One consequence of the enhanced RGS1, RGS16, and RGS20 expression may be to shunt a prominent Gi response to a Gs response to those ligands that have both Gi- and Gs-coupled receptors. Consistent with that hypothesis is the known shift in GPCR signaling that occurs during DC maturation. Studies of the Rgs1–/– iDCs indicate that RGS1 sets a threshold for chemoattractant responses in these cells. The ability of mDCs to respond to chemoattractants despite their significant up-regulation of RGS1 argues for another level regulation, which likely has an important physiological role in DC function.


   Acknowledgments
 
We thank Mary Rust for her editorial assistance and Dr. Anthony Fauci for his continued support.


   Footnotes
 
1 G.-X.S., K.H., and S.-B.H. contributed equally to the completion of this study. Back

2 Address correspondence and reprint requests to Dr. John H. Kehrl, B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. E-mail address: jkehrl{at}niaid.nih.gov Back

3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, mature DC; TLR, Toll-like receptor; GPCR, G protein-coupled receptor; S-1P, sphingosine-1-phosphate; LPA, lysophosphatidic acid; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; GAP, GTPase-activating protein; RGS, regulator of G protein signaling; BM, bone marrow; ERK, extracellular signal-regulated kinase; med, medium; M1, muscarinic type 1; SRE, serum response element; TTBS, Tween 20 plus TBS; MAPK, mitogen-activated protein kinase; PTX, pertussis toxin; IP3, inositol 1,4,5-trisphosphate; GFP, green fluorescent protein. Back

4 C. Moratz, J. R. Hayman, H. Gu, and J. H. Kehrl. Abnormal B cell responses to chemokines, disturbed plasma cell localization and distorted tissue architecture in Rgs1–/– mice. Submitted for publication. Back

Received for publication September 29, 2003. Accepted for publication February 13, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. T. Liu, B. Pulendran, K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767.[Medline]
  2. Sallusto, F., A. Lanzavecchia. 2000. Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression. Immunol. Rev. 177:134.[Medline]
  3. Janeway, C. A., R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20:197.[Medline]
  4. Kaisho, T., S. Akira. 2003. Regulation of dendritic cell function through Toll-like receptors. Curr. Mol. Med. 3:373.[Medline]
  5. Rescigno, M., M. Martino, C. L. Sutherland, M. R. Gold, P. Ricciardi-Castagnoli. 1998. Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188:2175.[Abstract/Free Full Text]
  6. Sozzani, S., W. Luini, A. Borsatti, N. Polentarutti, D. Zhou, L. Piemonti, G. D’Amico, C. A. Power, T. N. Wells, M. Gobbi, et al 1997. Receptor expression and responsiveness of human dendritic cell to a defined set of CC and CXC chemokines. J. Immunol. 159:1993.[Abstract]
  7. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Quin, A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28:2760.[Medline]
  8. Idzko, M., A. la Sala, D. Ferrari, E. Panther, Y. Herouy, S. Dichmann, M. Mockenhaupt, F. Di Virgilio, G. Girolomoni, J. Norgauer. 2002. Expression and function of histamine receptors in human monocyte-derived dendritic cells. J. Allergy Clin. Immunol. 109:839.[Medline]
  9. Idzko, M., E. Panther, S. Corinti, A. Morelli, D. Ferrari, Y. Herouy, S. Dichmann, M. Mockenhaupt, P. Gebicke-Haerter, F. Di Virgilio, et al 2002. Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses. FASEB J. 16:625.[Free Full Text]
  10. Panther, E., M. Idzko, S. Corinti, D. Ferrari, Y. Herouy, M. Mockenhaupt, S. Dichmann, P. Gebicke-Haerter, F. Di Virgilio, G. Girolomoni, J. Norgauer. 2002. The influence of lysophosphatidic acid on the functions of human dendritic cells. J. Immunol. 169:4129.[Abstract/Free Full Text]
  11. Idzko, M., S. Dichmann, D. Ferrari, F. Di Virgilio, A. la Sala, G. Girolomoni, E. Panther, J. Norgauer. 2002. Nucleotides induce chemotaxis and actin polymerizaton in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors. Blood 100:925.[Abstract/Free Full Text]
  12. Braun, M. C., B. L. Kelsall. 2001. Regulation of interleukin-12 production by G-protein-coupled receptors. Microbes Infect. 3:99.[Medline]
  13. Aliberti, J., C. ReiseSousa, M. Schito, S. Hieny, T. Wells, G. B. Huffnagle, A. Sher. 2000. CCR5 provides a signal for microbial induced production of IL-12 by CD8{alpha}+ dendritic cells. Nat. Immunol. 1:83.[Medline]
  14. Hepler, J. R., A. G. Gilman. 1992. G proteins. Trends Biochem. Sci. 17:383.[Medline]
  15. Neer, E. J.. 1995. Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80:249.[Medline]
  16. Kehrl, J. H.. 1998. Heterotrimeric G protein signaling: roles in immune function and fine-tuning by RGS proteins. Immunity 8:1.[Medline]
  17. Dietzel, C., J. Kurjan. 1987. Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. Mol. Cell. Biol. 7:4169.[Abstract/Free Full Text]
  18. Koelle, M. R., H. R. Horvitz. 1996. EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 84:115.[Medline]
  19. Lee, B. N., T. H. Adams. 1994. Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development. Mol. Microbiol. 14:323.[Medline]
  20. De Vries, L., M. Mousli, A. Wurmser, M. G. Farquhar. 1995. GAIP, a protein that specifically interacts with the trimeric G protein G{alpha}i3, is a member of a protein family with a highly conserved core domain. Proc. Natl. Acad. Sci. USA 92:11916.[Abstract/Free Full Text]
  21. Druey, K. M., K. J. Blumer, V. H. Kang, J. H. Kehrl. 1996. Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family. Nature 379:742.[Medline]
  22. Berman, D. M., T. M. Wilkie, A. G. Gilman. 1996. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G proteins {alpha} subunits. Cell 86:445.[Medline]
  23. Watson, N., M. E. Linder, K. M. Druey, J. H. Kehrl, K. J. Blumer. 1996. RGS family members: GTPase-activating proteins for heterotrimeric G-protein {alpha}-subunits. Nature 383:172.[Medline]
  24. Hunt, T. W., T. A. Fields, P. J. Casey, E. G. Peralta. 1996. RGS10 is a selective activator of G{alpha}i GTPase activity. Nature 383:175.[Medline]
  25. Mao, J., H. Yuan, W. Xie, D. Wu. 1998. Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein {alpha} subunit G{alpha}13. Proc. Natl. Acad. Sci. USA 27:12973.
  26. Kozasa, T., X. Jiang, M. J. Hart, P. M. Sternweis, W. D. Singer, A. G. Gilman, G. Bollag, P. C. Sternweis. 1998. p115 RhoGEF, a GTPase activating protein for G{alpha}12 and G{alpha}13. Science 280:2109.[Abstract/Free Full Text]
  27. Bowman, E. P., J. J. Campbell, K. M. Druey, A. Scheschonka, J. H. Kehrl, E. C. Butcher. 1998. Regulation of chemotactic and proadhesive responses to chemoattractant receptors by RGS (regulator of G-protein signaling) family members. J. Biol. Chem. 273:28040.[Abstract/Free Full Text]
  28. Moratz, C., V. H. Kang, K. M. Druey, C. S. Shi, A. Scheschonka, P. M. Murphy, T. Kozasa, J. H. Kehrl. 2000. Regulator of G protein signaling 1 (RGS1) markedly impairs Gi{alpha} signaling responses of B lymphocytes. J. Immunol. 164:1829.[Abstract/Free Full Text]
  29. Reif, K., J. G. Cyster. 2000. RGS molecule expression in murine B lymphocytes and ability to down-regulate chemotaxis to lymphoid chemokines. J. Immunol. 164:4720.[Abstract/Free Full Text]
  30. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of DC from mouse bone marrow cultures supplemented with GM-CSF. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
  31. Cho, H., T. Kozasa, K. Takekoshi, J. De Gunzburg, J. H. Kehrl. 2000. RGS14, a GTPase-activating protein for Gi{alpha}, attenuates Gi{alpha}- and G13{alpha}-mediated signaling pathways. Mol. Pharmacol. 58:569.[Abstract/Free Full Text]
  32. Jahng, K. Y., J. Ferguson, S. I. Reed. 1988. Mutations in a gene encoding the {alpha} subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling. Mol. Cell. Biol. 8:2484.[Abstract/Free Full Text]
  33. Dohlman, H. G., J. Song, D. Ma, W. E. Courchesne, J. Thorner. 1996. Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein {alpha} subunit). Mol. Cell. Biol. 16:5194.[Abstract]
  34. Scheschonka, A., C. W. Dessauer, S. Sinnarajah, P. Chidiac, C. S. Shi, J. H. Kehrl. 2000. RGS3 is a GTPase-activating protein for Gi{alpha} and Gq{alpha} and a potent inhibitor of signaling by GTPase-deficient forms of Gq{alpha} and G11{alpha}. Mol. Pharmacol. 58:719.[Abstract/Free Full Text]
  35. Shi, C. S., S. Sinnarajah, H. Cho, T. Kozasa, J. H. Kehrl. 2000. G13{alpha}-mediated PYK2 activation: PYK2 is a mediator of G13{alpha}-induced serum response element-dependent transcription. J. Biol. Chem. 275:24470.[Abstract/Free Full Text]
  36. Shi, C. S., J. H. Kehrl. 2001. PYK2 links Gq{alpha} and G13{alpha} signaling to NF-{kappa}B activation. J. Biol. Chem. 276:31845.[Abstract/Free Full Text]
  37. Nagata, Y., M. Oda, H. Nakata, Y. Shozaki, T. Kozasa, K. Todokoro. 2001. A novel regulator of G-protein signaling bearing GAP activity for G{alpha}i and G{alpha}q in megakaryocytes. Blood 97:3051.[Abstract/Free Full Text]
  38. Yowe, D., N. Weich, M. Prabhudas, L. Poisson, P. Errada, R. Kapeller, K. Yu, L. Faron, M. Shen, J. Cleary, et al 2001. RGS18 is a myeloerythroid lineage-specific regulator of G-protein-signalling molecule highly expressed in megakaryocytes. Biochem. J. 359:109.[Medline]
  39. Gagnon, A. W., D. L. Murray, R. J. Leadley. 2002. Cloning and characterization of a novel regulator of G protein signalling in human platelets. Cell. Signal. 14:595.[Medline]
  40. Park, I. K., C. A. Klug, K. Li, L. Jerabek, L. Li, M. Nanamori, R. R. Neubig, L. Hood, I. L. Weissman, M. F. Clarke. 2001. Molecular cloning and characterization of a novel regulator of G-protein signaling from mouse hematopoietic stem cells. J. Biol. Chem. 276:915.[Abstract/Free Full Text]
  41. Kimple, R. J., L. De Vries, H. Tronchere, C. I. Behe, R. A. Morris, M. Faquhar, D. P. Siderovski. 2001. RGS12 and RGS14 GoLoco motifs are G{alpha}i interaction sites with guanine nucleotide dissociation inhibitor activity. J. Biol. Chem. 276:29275.[Abstract/Free Full Text]
  42. Siderovski, D. P., M. Diverse-Pierluissi, L. De Vries. 1999. The GoLoco motif: a G{alpha}i/o binding motif and potential guanine-nucleotide exchange factor. Trends Biochem. Sci. 24:340.[Medline]
  43. Chaussabel, D., R. T. Semnani, M. A. McDowell, D. Sacks, A. Sher, T. B. Nutman. 2003. Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites. Blood 102:672.[Abstract/Free Full Text]
  44. Glick, J. L., T. E. Meigs, A. Miron, P. J. Casey. 1998. RGSZ1, a Gz-selective regulator of G protein signaling whose action is sensitive to the phosphorylation state of Gz{alpha}. J. Biol. Chem. 273:26008.[Abstract/Free Full Text]
  45. Wang, J., A. Ducret, Y. Tu, T. Kozasa, R. Aebersold, E. M. Ross. 1998. RGSZ1, a Gz-selective RGS protein in brain: structure, membrane association, regulation by G{alpha}z phosphorylation, and relationship to a Gz GTPase-activating protein subfamily. J. Biol. Chem. 273:26014.[Abstract/Free Full Text]
  46. Nagahama, M., S. Usui, T. Shinohara, T. Yamaguchi, K. Tani, M. Tagaya. 2002. Inactivation of G{alpha}z causes disassembly of the Golgi apparatus. J. Cell. Sci. 115:4483.[Abstract/Free Full Text]
  47. Ackerman, A. L., P. Cresswell. 2003. Regulation of MHC class I transport in human dendritic cells and the dendritic-like cell line KG-1. J. Immunol. 170:4178.[Abstract/Free Full Text]
  48. Lippert, E., D. L. Yowe, J. A. Gonzalo, J. P. Justice, J. M. Webster, E. R. Fedyk, M. R. Hodge, C. Miller, J. C. Gutierrez-Ramos, F. Borrego, et al 2003. Role of regulator of G protein signaling 16 in inflammation-induced T lymphocyte migration and activation. J. Immunol. 171:1542.[Abstract/Free Full Text]
  49. Uhlembrock, K., H. Gassenhuber, E. Kostenis. 2002. Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors. Cell. Signal. 14:941.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
X. Li, T. Syrovets, S. Paskas, Y. Laumonnier, and T. Simmet
Mature Dendritic Cells Express Functional Thrombin Receptors Triggering Chemotaxis and CCL18/Pulmonary and Activation-Regulated Chemokine Induction
J. Immunol., July 15, 2008; 181(2): 1215 - 1223.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
G. Shi, S. Partida-Sanchez, R. S. Misra, M. Tighe, M. T. Borchers, J. J. Lee, M. I. Simon, and F. E. Lund
Identification of an alternative G{alpha}q-dependent chemokine receptor signal transduction pathway in dendritic cells and granulocytes
J. Exp. Med., October 29, 2007; 204(11): 2705 - 2718.
[Abstract] [Full Text] [PDF]

Home page
 J. Gen. Virol.Home page
R. T.-Y. Wong, C.-C. Hon, F. Zeng, and F. C.-C. Leung
Screening of differentially expressed transcripts in infectious bursal disease virus-induced apoptotic chicken embryonic fibroblasts by using cDNA microarrays
J. Gen. Virol., June 1, 2007; 88(6): 1785 - 1796.
[Abstract]