RGS1) Markedly Impairs G Iα Signaling Responses of B Lymphocytes

Regulator of G Protein Signaling 1 (RGS1) Markedly Impairs G iα Signaling Responses of B Lymphocytes

This information is current as Chantal Moratz, Veronica H. Kang, Kirk M. Druey, of September 26, 2021. Chong-Shan Shi, Astrid Scheschonka, Philip M. Murphy, Tohru Kozasa and John H. Kehrl J Immunol 2000; 164:1829-1838; ; doi: 10.4049/jimmunol.164.4.1829

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Regulator of G Protein Signaling 1 (RGS1) Markedly Impairs

Gi␣ Signaling Responses of B Lymphocytes

Chantal Moratz,* Veronica H. Kang,* Kirk M. Druey,1* Chong-Shan Shi,* Astrid Scheschonka,2* Philip M. Murphy,† Tohru Kozasa,‡ and John H. Kehrl3*

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-␣. To determine the signaling pathways that RGS1 may regulate, we examined the speciﬁcity of RGS1 for various G␣ subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi␣ and

Gq␣ GTPase-activating protein and a potential G12␣ effector antagonist. Functional studies demonstrated that RGS1 impairs -platelet activating factor-mediated increases in intracellular Ca؉2, stromal-derived factor-1-induced cell migration, and the in Downloaded from duction of downstream signaling by a constitutively active form of G12␣. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells. The Journal of Immunology, 2000, 164: 1829–1838.

erminal centers within lymphoid tissue form specialized tors (GPCR)4, allowing members of the arrestin family to bind the microenvironments essential for the induction of thy- phosphorylated receptor, precluding subsequent G protein activa- G mus-dependent B cell Ab production, the affinity matu- tion. The levels of GPCR expression often decline following li- http://www.jimmunol.org/ ration of the humoral immune response, and the induction of B cell gand simulation, thereby reducing the number of available recep- memory. Although many of the genetic and biochemical mecha- tors for subsequent restimulation. G protein signaling can also be nisms that underlie these processes remain poorly understood, inhibited by proteins that accelerate the intrinsic GTPase activity some of the receptor-ligand interactions controlling these events of G␣ subunits. GTPase activators (GAPs), were first discovered activate heterotrimeric G proteins. For example, chemokine-che- for the small GTPase proteins Ras and EF-tu (reviewed in Refs. 11 mokine receptor interactions regulate lymphocyte migration pat- and 12). Recently, G␣ subunit GAPs have also been identified and terns as well as the establishment of germinal centers (1–4). Fur- termed regulator of G protein signaling proteins (RGS; reviewed in thermore, pretreatment of B lymphocytes with pertussis toxin, Refs. 13 and 14). by guest on September 26, 2021 which blocks Gi␣-mediated signaling, inhibits chemoattractant-in- Insights into the function of the RGS family members arose duced B cell migration (5–8). from the identification of evolutionarily conserved homologues in Heterotrimeric G proteins, which couple heptahelical receptors Saccharomyces cerevisiae (Sst2) and Caenorhabditis elegans to effectors in signal transduction pathways, consist of three sub- (Egl-10 and C05B5.7) (15, 16). Sst2p contains a split RGS domain units: ␣, ␤ and ␥. Each subunit has multiple isoforms; the ␣ sub- and in yeast is a key negative regulator of the mating response to unit isoforms are grouped into four families, Gi␣,Gs␣,Gq␣, and pheromone. In C. elegans, the Egl-10 protein contains an RGS ␣ G12/13␣ (reviewed in Ref. 9). Upon receptor activation, the sub- domain and functions in signal transduction pathways that regulate unit complex exchanges GTP for GDP and dissociates from the ␤␥ egg laying and movement (15). Mammalian RGS proteins also subunit. Both GTP-bound G␣ and the released ␤␥ subunit can regulate G protein-linked signal transduction pathways. Introduc- activate downstream effectors. Several mechanisms regulate the tion of RGS family members into yeast blunted the responses to duration and magnitude of G protein signaling (reviewed in Ref. pheromone and partially complemented an sst2 mutation while the 10). Protein kinases can phosphorylate G protein-coupled recep- same RGS proteins expressed in HEK 293 cells stably transfected with CXCR1 blunted the activation of mitogen activated protein kinase (MAPK) following IL-8 stimulation (16). RGS proteins inhibit signaling pathways that utilize either G or G as signal trans- *B Cell Molecular Immunology Section, Laboratory of Immunoregulation, and †Lab- i q oratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Na- ducers (reviewed in Refs. 13 and 14). Recently, p115 RhoGEF has tional Institutes of Health, Bethesda, MD 20892; and ‡Department of Pharmacology, been shown to have an RGS-like domain that has GAP activity for University of Texas Southwestern Medical Center, Dallas, TX 75235 G12/13␣ (17). Received for publication August 31, 1999. Accepted for publication December RGS proteins bind G␣ subunits and do so most efficiently in a 9, 1999. form that mimics a transition state in GTP hydrolysis (G␣ treated The costs of publication of this article were defrayed in part by the payment of page Ϫ charges. This article must therefore be hereby marked advertisement in accordance with GDP and AlF4 ) and possess GAP activity for Gi␣ and Gq␣ with 18 U.S.C. Section 1734 solely to indicate this fact. subfamily members (18–21). In the crystal structure of RGS4 1 Current address: Klinik und Poliklinik fuer Neurologie, Universitat Goettingen, 37075 Gottingen, Germany. 2 Current address: Molecular Signal Transduction Section, Laboratory of Allergic 4 Abbreviations used in this paper: GPCR, G protein-coupled receptor; RGS, regu- Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of lator of G protein signaling; PAF, platelet-activating factor; BCA-1, B cell-attracting Health, Bethesda, MD 20892. chemokine-1; MAPK, mitogen-activated protein kinase; SAPK, stress-activated pro- 3 Address correspondence and reprint requests to Dr. John H. Kehrl, National Institute tein kinase; SDF-1, stromal-derived factor-1; MFI, mean fluorescence intensity; GAP, of Allergy and Infectious Diseases, Building 10, Room 11B-13, 10 Center Drive, GTPase-activating protein; SRE, serum response element; KLH, keyhole limpet he- MSC 1876, Bethesda, MD 20892-1876. E-mail address: [email protected] mocyanin; ECL, enhanced chemiluminescence; LPA, lysophosphatidic acid.

Ϫ ␮ 32 complexed with Gi␣1-GDP-AlF4 , the RGS domain forms a four- min, and 400 l of supernatant containing Pi was counted by liquid scintillation spectrometry. Direct measurement of the k for GTPase ac- helix bundle that directly contacts Gi␣ at the three so-called cat tivity of G was assayed with the use of mutant G R183C (28). An “switch regions” (22), which undergo the greatest conformational q␣ q␣ analogous mutant of Gi␣,Gi␣R178C, has markedly reduced GTPase activ- change during the GTPase cycle. Mutagenesis studies of RGS4 ity but still responds to RGS proteins. The slow GTPase activity of ␥ 32 revealed that altering the contact residues identified in the crystal Gq␣R183C made it possible to load [ - P]GTP on Gq␣ without acceler- structure resulted in a loss of G␣ binding and an inability to inhibit ating GDP-GTP exchange by agonist bound receptor. Gq␣R183C was loaded with 10 ␮M[␥-32P]GTP in the presence of 50 mM HEPES (pH G␣ signaling (23, 24). Overall these studies indicate that RGS pro- 7.4), 0.1 mg/ml BSA, 1 mM DTT, 1 mM EDTA, 0.9 mM MgSO4,30mM teins stabilize Gi␣ in its transition state for GTP hydrolysis. Be- (NH4)2SO4, 4% glycerol, and 5.5 mM CHAPS at 20°C for 2 h. The reac- sides acting as GAPs for Gi␣ and Gq␣, certain RGS proteins also tion mixture was gel filtered through a Sephadex G50 spin column equil- act as effector antagonists, i.e., compete with effectors for binding ibrated with 50 mM HEPES (pH 7.4), 1 mM DTT, 1 mM EDTA, 0.9 mM MgSO , 0.1 mg/ml BSA, and 1 mM CHAPS. GTPase assays were initiated to Gq␣ (25). Despite these advances in our understanding of their 4 mechanisms of actions, the physiologic roles of most RGS proteins by addition of 1 mM GTP and the indicated amount of RGS proteins and incubation at 20°C. Aliquots (50 ␮l) were removed and processed as de- remain poorly defined. scribed above. Chemokine receptor signaling is a prime arena for regulation by RGS proteins. This type of regulation may help target cells to Cell lines particular sites and keep them localized there despite the continued The HS-Sultan (a B lineage human plasmacytoma), Molt-4 (a human T exposure to chemokines and chemoattractants. The initial studies lineage leukemia), Jurkat (a human acute T cell leukemia line), COS-7 (an of RGS1 indicated high levels of expression in tonsil germinal SV40-transformed fibroblast like kidney line), PC12 (a rat adrenal pheo- chromocytoma), Ramos (a human Burkitt’s B cell chronic lymphoma), and centers. Here we show the specificity of RGS1 for various G␣ K562 (a human chronic myelogenous leukemia) cell lines were obtained Downloaded from subtypes, discern some of the signals that induce RGS1 expression from the American Type Culture Collection (ATCC, Manassas, VA). The in B lymphocytes, and provide evidence that RGS1 down-regu- Burkitt lymphoma cell lines MC116 and CA46 were kind gifts of Dr. Ian lates signaling initiated by the platelet-activating factor (PAF) re- Magrath (National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD). The SuDHL5 and SuDHL6 B lymphoma cell lines ceptor and CXCR4 in stable RGS1-transfected lines. We also con- were kind gifts of Dr. Lazlo Krenacs (NCI, NIH, Bethesda, MD). The firm that RGS1 protein is specifically expressed in germinal center human pre-B cell line, NALM-6, was a kind gift of Dr. Thomas Tedder

B cells that, although they express the CXCR4 receptor, are re- (Duke University, Durham, NC). All the lymphoid cell lines were main- http://www.jimmunol.org/ fractive to stromal-derived factor (SDF)-1-induced migration. tained in RPMI 1640 supplemented with 5% to 10% FCS whereas the nonlymphoid cells were maintained in DMEM plus 10% FCS. HS-Sultan cells overexpressing RGS1 were generated using the retroviral vector Materials and Methods LXSN-RGS1 as previously described (16). Twelve separate pools of neo- Production of recombinant RGS1 mycin-resistant cells were isolated and tested for RGS1 expression; three pools that expressed RGS1 at high levels, termed TF7, TF8, and TF12, A bacterial expression vector for RGS1 and RGS4 were constructed using were used in these studies. A pool of LXSN vector-transfected and neo- Vent polymerase and the coding region of RGS1 or RGS4 to generate a mycin-resistant cells served as a control. PCR fragment ﬂanked by XhoI and BamHI restriction sites, which were subcloned into the corresponding sites of the His-tag fusion vector pET14b Isolation of tonsil B cells and tonsil B cell subsets

(Novagen, Madison, WI). The resulting constructs pET14b-RGS1 and by guest on September 26, 2021 pET14b-RGS4 were transformed into the bacterial strain BL21 (DE3) Tonsillar B cells were isolated as described in Current Protocols in Im- pLysS and induced with 1 mM isopropyl ␤-D-thiogalactoside (IPTG) for munology. First, lymphocytes were isolated by Ficoll-Hypaque (Amersham 3 h at 37°C. The His-tagged RGS proteins were purified from a 100-ml Pharmacia Biotech, Uppsala Sweden) density gradient centrifugation (29). culture by metal chelation chromatography as outlined by the manufacturer T cells were then depleted by rosetting with neuraminidase (Life Technol- (Novagen). The purified proteins were dialyzed against HED buffer (50 ogies, Gaithersburg, MD)-treated sheep RBC (NIH Media Unit, Frederick, mM Na-HEPES (pH 8.0), 1 mM EDTA, 1 mM DTT, and 10% glycerol). MD) and subsequent Ficoll-Hypaque density gradient centrifugation. The purity of tonsillar B cells was routinely greater than 95% using this method G protein-binding assays as defined by post separation immunofluorescense staining with anti-CD4, anti-CD8, and anti-CD19 (PharMingen, San Diego, CA). Subsequently, the 6 HS-Sultan cells (5 ϫ 10 ) were lysed in a 0.5 ml of buffer that consisted cells were washed and analysis was performed on a FACScalibur flow of 20 mM HEPES (pH 8.0), 1 mM EDTA, 6 mM MgCl2, 3 mM DTT, 380 cytometer with CELLQuest software (Becton Dickinson, Mountain View, mM NaCl, and 1% Triton X-100 for 20 min at 4°C. The lysates were CA). The purified B cells were stimulated with various reagents for 12 h in centrifuged at 14,000 ϫ g for 20 min to remove particulates and transferred RPMI 1640 and 10% FCS. IL-8, IL-10, and TNF-␣ were purchased from to fresh tubes before activation with 20 ␮M GDP, 20 ␮M GDP plus 30 ␮M R&D Systems (Minneapolis, MN), anti-CD40 from PharMingen, and PAF, Ϫ ␮ ␥ ϩ AlF4 ,or30 M GTP- S for 30 min at 30°C. RGS1 attached to Ni PMA, and sphingosine-1-phosphate were purchased from Sigma (St. 2NTA beads was added, and the mixture was incubated for another 90 min Louis, MO). B cell subsets were obtained from the B cell-enriched fraction at 4°C. The beads were washed four times with lysis buffer that included by cell sorting. In brief, separation of the naive, memory, and germinal the appropriate nucleotide, the bound G proteins were eluted with SDS- center B cells were isolated according to a schematic developed by Yong- sample buffer, and the samples were fractionated on SDS-PAGE before Jun Liu (30, 31). The cells were incubated with mAbs to CD19, IgD, and transfer and analysis by immunoblotting for various G␣ subunits (see CD38 (PharMingen) for 15 min and washed in FACS staining buffer (PBS below). without Ca2ϩ/Mg2ϩ plus 1% BSA). Using a FACStarPlus (Becton Dickin- son), the cells in the CD19-positive gate were sorted, based on their dif- G proteins and GAP assays ferential expression of CD38 and IgD, into naive, memory, and germinal center B cells. Reanalysis of each sorted population by flow cytometry G ␣,G␣ , and G ␣ were expressed in and purified from Escherichia coli s i 1 o indicated a population purity of greater than 97% for each subset sorted. (26). G ␣,G ␣, and G ␣R183C were expressed in Sf9 cells and purified as z 12 q Lysates of the sorted fractions were analyzed by immunoblotting (see described (27). Gi␣1,Go␣,Gs␣,Gz␣, and G12␣ (50 pM) were loaded with ␮ ␥ 32 ϳ below). 5–10 M[ - P]GTP ( 5000 Ci/mmol) at 20°C (for Gs␣) or 30°C (for Gi␣1,Go␣,Gz␣, and G12␣) for 20 to 30 min in the presence of 5 mM EDTA. Flow cytometry and migration assays Samples were then gel-filtered at 4°C through a Sephadex G-50 spin column equilibrated with buffer A (50 mM HEPES (pH 8.0), 1 mM DTT, 5 HS-Sultan-transfected cell lines were harvested and counted, and 1 ϫ 106 ␥ 32 mM EDTA,and 0.05% of the detergent C12E10) to remove free [ - P]GTP cells per well (96-well round-bottom plate, Costar, Cambridge, MA) were 32 and Pi. Hydrolysis of GTP was initiated by adding G␣ loaded with used for staining. The cells were washed in staining buffer (1% BSA frac- ␥ 32 [ - P]GTP in buffer A containing 8 mM MgSO4 and 1 mM GTP with the tion V (ICN Biomedicals, Aurora, OH) in PBS, blocked with 10% normal indicated amount of RGS proteins. The reaction mixture was incubated at rat serum (Cedarlane Laboratories, Acccurate Chemical & Scientific, ␮ 4°C (for Gi␣1,Go␣, and Gs␣) or 15°C (for Gz␣ and G12␣). Aliquots (50 l) Westbury, NY) in staining buffer for 10 min. After washing, the cells were were removed at the indicated times and mixed with 750 ␮l of 5% (w/v) stained with mAb, anti-CD4, anti-CXCR4, and anti-CD19 (PharMingen).

Norit in 50 mM NaH2PO4. The mixture was centrifuged at 2000 rpm for 5 Subsequently, the cells were washed, and analysis was performed on a The Journal of Immunology 1831

FACScalibur ﬂow cytometer with CellQuest software (Becton Dickinson). RGS domain, does not cross-react with other RGS proteins. For immuno- Cell migration was assessed in triplicate in 24-well 5-␮m pore size poly- blotting, the samples (25–100 ␮g protein, Bio-Rad Protein Assay, Bio-Rad, carbonate membrane ﬁlter transwell insert culture plates (Costar). Trans- Richmond, CA) were fractionated by SDS-PAGE and transferred to pure fected cell lines were harvested and resuspended at 1 ϫ 107 cells per ml in nitrocellulose. The membranes were blocked with 10% milk in TTBS for culture medium (RPMI 1640, penicillin (100 U/ml), and streptomycin (100 1 h, and then incubated with the appropriate dilution of Ab in 5% milk and ␮g/ml); all from Life Technologies), 10% FBS (Tissue Culture Biologi- 0.05% sodium azide in TTBS overnight (0.1% Tween 20, 100 mM Tris Cl cals, Tulare, CA). Transwell culture plates were set up. Six hundred mi- (pH 75), 0.9% NaCl). The blots were washed twice with TTBS before the croliters of medium or medium plus chemoattractant was placed in the addition of a biotinylated goat anti-rabbit Ig (DAKO, Carpinteria, CA) bottom of the well; then 100 ␮l of cells (1 ϫ 10Ϫ6 cells) were placed in the diluted 1:10,000 in TTBS containing 10% FCS. Following a 1-h incuba- upper chamber of the well. The chemokine SDF-1␣ (R&D Systems) was tion, the blot was washed twice with TTBS and then incubated with strepta- used at 10 ng/ml, 100 ng/ml, or 1000 ng/ml concentrations. The cultures vidin conjugated to HRP (DAKO). The signal was detected by enhanced

were incubated at 37°C, 5% CO2 for 4 h. Migrated cells were analyzed on chemiluminescence (ECL) following the recommendations of the manu- a FACScalibur. Results are shown as percentage of total input. Chemotaxis facturer (Amersham). The anti-RGS1 antiserum (no. 2247) was used at

studies with tonsil B cells were performed as described above except that 1:200 dilution; the rabbit Abs reactive with Gi␣1,Gi␣2,G12␣,orGi␣3 were migrated cells were harvested and stained with mAbs anti-CD19, anti-IgD, used as recommended (Santa Cruz Biotechnology, Santa Cruz, CA); and

and anti-CD38 for 15 min at 4°C, washed, and analyzed on a FACScalibur. the afﬁnity puriﬁed anti-peptide Ab reactive with Gi␣1 and Gi␣2 (AS/7) was Additionally, the studies used a dose of 50 ng/ml of SDF-1, which was used at a 1:1000 dilution (a kind gift of Dr. P. Goldsmith, NIDDK, NIH). determined to be optimal to induce maximal migration in a series of titra- tion experiments. Using Cellquest software, the percentage migration of each subpopulation of B cells was determined. Results RGS1 is expressed in B cell lines and normal B cells Determination of intracellular calcium levels

To examine RGS1 protein expression in B lymphocytes, we gen- Downloaded from Cultured cells were harvested, washed in HBSS buffer (HBSS (Biofluids, Rockville, MD), 10 mM HEPES, and 1% FBS), and resuspended at 1 ϫ erated RGS1-specific antisera for immunoblotting. Recombinant 107 cells/ml in HBSS buffer plus the fluorescent calcium probe Indo-1 RGS1 was produced in bacteria with a histidine tag to facilitate (indo-1/acetocxymethylester) (Sigma or Molecular Probes, Eugene OR) purification via metal chelation chromatography. The antisera were was added at a final concentration of 2 ␮g/ml plus Pleuronic detergent screened by immunoblotting recombinant RGS1 and by the detec- ␮ (Molecular Probes) at a final concentration of 300 g/ml (32). The cells tion of RGS1 in cell lysates from HS-Sultan cells stimulated with were incubated 30 min at 30°C while protected from light. The cells were

washed with HBSS buffer and resuspended to 1 ϫ 106 in HBSS buffer. phorbol esters. These cells are representative of mature B cells http://www.jimmunol.org/ Cells were warmed at 37°C for 3 min before stimulation. To stimulate, partially differentiated toward plasma cells. Phorbol esters had cells were loaded into the Time Zero module (Cyteck, Fremont, CA) and been previously shown to markedly increase RGS1 (BL34) mRNA run at 1000 cells/s. A baseline was collected for 30 s, and then a sham of expression in HS-Sultan and tonsillar B cells (33). The RGS1 an- 50 ␮l of HBSS buffer was injected. Finally at 60 s the stimulant was injected. The measurement for calcium flux was performed on a FACS- tisera recognized recombinant RGS1 via immunoblotting, and both Vantage flow cytometer (Becton Dickinson Immunocytometry Systems, the anti-recombinant RGS1 and the antiserum no. 2247 immuno- San Jose, Ca) equipped with an argon laser tuned to 488 nm and a krypton precipitated a 26-kDa protein in lysates prepared from HS-Sultan laser tuned to 360 nm. Indo-1 fluorescence was analyzed at 390/20 and cells stimulated with PMA (data not shown). This molecular mass 530/20 for bound and free probe, respectively. The data were analyzed agrees well with the predicted molecular mass of RGS1, and the using the FlowJo software (Tree Star, San Carlos, CA). Results are shown by guest on September 26, 2021 as ratio fluorescence (violet/blue). endogenous RGS1 protein comigrated with recombinant RGS1. Next, we analyzed RGS1 expression in human B cell lines rep- Stress-activated protein kinase (SAPK) and serum response resentative of various stages of B cell development as well as other element (SRE) reporter assays hemopoietic cell lines. Most of the cell lines had low or undetect- For the SAPK assays, COS cells were transfected via a DEAE-dextran able levels of RGS1, with the exception of a follicular B cell lym- method with the following plasmids: MT3-HA-SAPK-p46 (1 ␮g, provided phoma cell line SuDHL5, which expressed high levels (Fig. 1). ␮ by Dr. J. Kyriakis, Boston, MA), pcDNAG12␣-Q229L (2 g, provided by Two pre-B cell lines, NALM-6 and PB-697, failed to express Dr. S. Gutkind, NIH), or pcDNAG -Q226L (provided by Dr. S. Gut- 13␣ RGS1 (Fig. 1 and data not shown). One T cell line, MOLT-4, kind), and the presence or absence of varying concentrations of FLAG- pCMV2-RGS1 or pCR3-FLAG-RGS4. Transfected DNA levels were nor- contained low levels of RGS1 whereas the other T cell line, Jurkat, malized with control plasmids. Seventy-two hours following the was negative. Previously described HS-Sultan cell lines stably transfection, HA-immunoprecipitates were subjected to in vitro kinase as- transfected with RGS1 (TF8 andTF12) and recombinant RGS1 says using c-Jun (1-79) as a substrate, and the samples were size fraction- served as positive control (16). Further immunoblotting analysis of ated by SDS-PAGE. Following autoradiography, the c-Jun (1-79) bands were quantitated using NIH Image. For the SRE reporter assays, COS cells a panel of Burkitt lymphoma cell lines revealed that several of were transfected with pSRE-LUC (0.35 ␮g, Stratagene, San Diego, CA), them also constitutively expressed high levels of RGS1 (data not ␤ ␮ ␮ pCMV- gal (0.35 g), and pcDNAG12␣-Q229L (0.5 g) or pcDNAG13␣- shown). We also examined the kinetics of RGS1 induction in HS- Q226L in the presence or absence of FLAGpCMV2RGS1 using SuperFect Sultan cells following exposure to phorbol esters. PMA induced (Qiagen, Valencia, CA). Twenty-four hours later, the DMEM plus 5% FCS RGS1 expression within4hofstimulating the cells (Fig. 1). These was removed and replaced with DMEM plus 0.5% serum. The following day, the cells were harvested, and lysates were prepared in 100 ␮l reporter analyses of protein expression confirm that RGS1 expression is lysis buffer (Promega, Madison, WI). Using a luminometer (Analytical restricted and that it is not expressed in T cell, fibroblast cell, or Luminescence Laboratory, San Diego, CA), 10 ␮l of the supernatant was most B and pre-B cell lines. Additionally, they confirm that HS- tested for luciferase activity with a luciferase substrate (Promega), and 10 Sultan cells lack RGS1 unless stimulated by PMA, corroborating ␮l was tested for ␤-galactosidase activity with a galactan chemilumines- cent substrate (Tropix, Bedford, MA). The amount of luciferase activity data suggesting that B cells must be activated to express RGS1. was normalized to amount of ␤-galactosidase activity in each sample. Each In addition, we examined the effects of several other stimuli on transfection was done in either duplicate or triplicate. RGS1 expression. Rather than use B cell lines, we purified B cells from human tonsils and exposed them to media alone, anti-CD40 Immunoprecipitations and immunoblots mAb, anti-CD40 plus IL-10, IL-8, PAF, PMA, sphingosine-1 RGS1 Abs were prepared by immunizing rabbits with recombinant RGS1, phosphate, or TNF-␣ for 12 h (Fig. 2). PAF, IL-8, and sphin- an N-terminal peptide (MPGMFFSANPK) coupled to keyhole limpet he- gosine-1 phosphate all signal through GPCRs. B cells express PAF mocyanin (KLH), a C-terminal peptide (NDLNANSLK) coupled to KLH, or an internal peptide (DDKMNKRRPK) coupled to KLH. The antiserum receptors, but presumably lack IL-8 receptors. We chose to exam- generated against the N-terminal peptide (referred to as 2247) proved to be ine anti-CD40 and TNF-␣ since both increase NF-␬B in B cells the most efficacious and, since it was raised against a peptide outside of the and the proximal RGS1 promoter contains an NF-␬B site (J. Kehrl, 1832 RGS1 AND Gi␣ SIGNALING

FIGURE 1. Expression of RGS1 in various B cell lines. Cell lysates Downloaded from from various cells lines indicated below were analyzed by immunoblotting for RGS1 expression. In addition, ϳ200 ng of recombinant RGS1 (lane 1) and cell lysates prepared from two of the HS-Sultan RGS1 transfectants (TF no. 8 and TF no. 12, lanes 2 and 3) served as positive controls. On a separate immunoblot (lanes 13–17), lysates (75 ␮g each) from HS-Sultan cells stimulated with PMA (50 ng/ml) for various duration were analyzed for RGS1 expression. The immunoblots were detected by ECL. Represen- http://www.jimmunol.org/ tative of one of three experiments performed. FIGURE 3. The speciﬁcity of RGS1 for G␣ subunits in B cells. A, Ϫ RGS1 binds Gi␣ and Gq␣ in a GDP plus AlF4 -dependent manner. HS- Ϫ unpublished observation). Anti-CD40 stimulation induced low Sultan cell lysates were treated with GDP or GDP plus AlF4 and reacted levels of RGS1 whereas PAF induced a modest increase. Both with His-tagged recombinant RGS1 bound to beads. After washing, the PMA and TNF-␣ were potent inducers of RGS1 expression in bound proteins were eluted and analyzed by immunoblotting with Abs tonsil B cells whereas IL-8 and sphingosine-1 phosphate had es- against various G␣ subunits. A cell lysate was simultaneously analyzed to sentially no effect. Thus, signals through a GPCR like the PAF verify the presence of the G␣ subunit in the lysate. The immunoblot was detected by ECL. B, RGS1 binds G12␣ independent of its nucleotide load- receptor as well as signals via the TNF-receptor can trigger the by guest on September 26, 2021 ing. HS-Sultan cell lysates were treated with GDP (lane 3), GDP plus induction of RGS1 expression. Ϫ ␥ AlF4 (lane 4), or GTP- S(lane 5) and reacted with His-tagged recombinant RGS1 bound to beads. After washing, the bound proteins were RGS1 Binds Gi␣,Gq␣, and G12␣, but not Gs␣ from B eluted and analyzed by immunoblotting for G12␣ as above. The presence of lymphocytes RGS1 in the eluted fractions was veriﬁed by immunoblotting using a mAb

To determine which G␣ subunits RGS1 may regulate in B cells, we reactive with the His-tag (bottom panel). Each experiment was performed 2ϩ twice. reacted recombinant RGS1 immobilized on Ni NTA beads with cell lysates from HS-Sultan cells that had been treated with GDP Ϫ or with GDP with AlF4 . The bound proteins were solubilized and size fractionated by SDS-PAGE, and their identity was determined studied. RGS1 readily extracted Gi␣1/2,Gi␣3, and Gq␣ from the cell Ϫ by immunoblotting with antisera speciﬁc for different G␣ subunits lysate treated with GDP and AlF4 , but not from the lysate treated (Fig. 3A). In each case, an HS-Sultan cell lysate was simulta- with GDP alone. We failed to detect any Gs␣ associated with neously analyzed to verify that it contained the G␣ subunit being RGS1 under either condition. We also examined whether RGS1

could extract G12␣ from the HS-Sultan cell lysates (Fig. 3B). In Ϫ contrast to the results with Gi␣, which exhibited an AlF4 -dependent extraction by RGS1, G12␣ was extracted whether the lysate ␥ Ϫ 2ϩ was GDP, GTP- S, or GDP and AlF4 treated. Since the Ni NTA beads alone did not extract G12␣, the extraction depended upon the presence of RGS1. In contrast to RGS1, recombinant

RGS4 did not extract G12␣ from the HS-Sultan cell lysates (data not shown). Based on these results, we predicted that RGS1 would

act as a GAP for Gi␣ and Gq␣ subunits, but not Gs␣ subunits. Although RGS1 extracted G12␣ from cell lysates, it failed to do so Ϫ in an AlF4 -dependent manner, suggesting that it was unlikely to beaG12␣ GAP. FIGURE 2. Induction of RGS1 in normal tonsil B cells. Puriﬁed tonsil B cells were stimulated for 12 h with media, anti-CD40 (5 ␮g/ml), anti- RGS1 acts as GAP for Gi␣ and Gq␣, but not for Gs␣ or G12␣ CD40 plus IL-10 (50 ng/ml), IL-8 (50 ng/ml), PAF (10Ϫ6 M), PMA (25 ng/ml), sphingosine-1-P (50 ng/ml), or TNF-␣ (50 ng/ml). Cell lysates To directly test the effects of RGS1 on the GTPase activity of were prepared and analyzed for RGS1 expression by immunoblotting with various G␣ subunits, we measured the catalytic activity of puriﬁed the RGS1 anti-peptide Ab no. 2247. This experiment is one of several recombinant G␣ subunits during a single GTPase cycle in the pres- similar experiments that were performed. ence or absence of recombinant RGS1 (Fig. 4). RGS4 served as a The Journal of Immunology 1833

FIGURE 4. GAP activity of RGS1 for speciﬁc G␣ subunits. Different concentrations of recombinant RGS1 or recombinant RGS4 were tested for their ability to accelerate the GTPase activity of Gi␣1,Gq␣ R183C, Gz␣, Go␣,Gs␣, and G12␣. Hydrolysis of GTP was initiated by 32 Downloaded from adding the G␣ subunit loaded with [␥- P]GTP to a buffer containing MgSO4 with the indicated amount of RGS protein. The reaction mixture was incubated, and aliquots were removed at the indicated times and pro- 32 cessed, and the amount of Pi was counted by liquid scintillation spectrometry. HED is buffer without RGS protein. The GAP assays were performed twice with http://www.jimmunol.org/ similar results. by guest on September 26, 2021

positive control since its GAP activity in these assays is well doc- ical to a polarized motile morphology. Although RGS1 failed to umented (19). RGS1 proved nearly as effective Gi␣ GAP as RGS4, act as a G12␣ GAP, the RGS1 G protein binding data indicated that although approximately 3-fold more RGS1 than RGS4 was needed HS-Sultan cells contained significant levels of G12␣ and that RGS1 to achieve a similar level of GAP activity. However, when tested could bind GTP-G12␣ and, thus, potentially act as an effector an- against two other Gi␣ subfamily members, Gz and Go, RGS1 tagonist. To test that possibility, we transiently expressed in COS proved less efficient than did RGS4. To measure the GAP activity cells a GTPase-deficient form of G12␣,G12␣-Q229, which is of RGS1 for Gq␣, we employed a mutant Gq␣,Gq␣R183C. The largely GTP bound. Since the transient expression of G␣12-Q229L analogous mutant in Gi␣,Gi␣R178C, has a markedly reduced GT- in COS cells activates the SAPK pathway (33), we could test the Pase activity, but still responds to RGS proteins (19). The slow effects of RGS1 on SAPK activation by concomitantly transfecting

GTPase activity of Gq␣R183C made it a suitable target for testing an epitope-tagged version of SAPK and assessing its activity in an potential Gq␣ GAPs. Both RGS1 and RGS4 showed good GAP in vitro kinase assay (Fig. 5). Expression of G12␣-Q229L increased activity for Gq␣, although at equal molar concentrations RGS4 was SAPK activity 3- to 10-fold depending upon the experiment, and slightly superior to RGS1 (Fig. 4). As expected, neither RGS4 nor in each of ﬁve experiments the coexpression of RGS1 inhibited the

RGS1 enhanced the intrinsic GTPase activity of Gs␣. Furthermore, activation of SAPK. In contrast, RGS4 at similar expression levels despite the ability of RGS1 to bind G12␣, it lacked GAP activity. had either no effect or actually augmented SAPK activity. Al-

In contrast, p115 RhoGEF, a recently discovered G12␣ GAP (17), though RGS1 inhibited G12␣-Q229L-induced SAPK activation, it signiﬁcantly increased the rate of G12␣ GTP hydrolysis. did not inhibit G13␣-Q226L-induced SAPK activation in COS cells. Since G -Q229L is also a potent activator of serum re- ␣ 12␣ RGS1 inhibits G12 -Q229L signaling in COS cells sponse element (SRE)-dependent transcription (34–36), we could

Although there is no information about the role of G12 proteins in examine whether RGS1 expression impaired the activation of a lymphocytes, they may be involved in chemokine-induced cy- SRE reporter construct. Cotransfection of COS cells with an SRE- toskeletal changes that occur as lymphocytes switch from a spher- responsive reporter construct and G12␣-Q229L resulted in a 27- 1834 RGS1 AND Gi␣ SIGNALING Downloaded from

FIGURE 5. RGS1 inhibits activated G12␣-mediated signal transduction. A, RGS1 impairs G12␣-Q229L-induced SAPK activation. COS cells were ␮ transiently transfected with expression constructs for HA-SAPK, G12␣-Q229L, and varying amounts of FLAG-RGS1 (1, 2, or 4 g) or FLAG-RGS4 (4 ␮g). HA-immunoprecipitates were performed 36 h after the transfection and subjected to an in vitro kinase assay using a GST-N-terminal fragment of c-Jun fusion protein. The amount of kinase activity was quantitated by autoradiography and NIH Image, and expressed as fold induction. The expression of RGS1

and RGS4 was veriﬁed by immunoblotting with an anti-FLAG mAB. This experiment is one of ﬁve performed. B, RGS1 does not impair G13␣-Q226L- http://www.jimmunol.org/ mediated SAPK activation. Similar experiment as in A, except G13␣-Q226L was used. C, RGS1 impairs G12␣-Q229L-induced activation of an SRE reporter. ␤ COS cells were transfected with expression constructs for CMV- Gal, SRE-luc, G12␣-Q229L, or G13␣-Q226L, and varying amounts of RGS1 (the amount of plasmid transfected is indicated below in micrograms). The amount of luciferase and ␤-galactosidase activity in each sample was measured, and the ␤ luciferase activity was normalized to the -galactosidase activity. The data are shown as the fold induction (activated G12␣ or G13␣ vs control). Data are from one of three experiments performed.

fold increase in luciferase activity compared with a control plas- roviral vector served as a control. An immunoblot of cellular ly-

mid (Fig. 5). The concomitant expression of RGS1 resulted in a sates demonstrated RGS1 expression in the RGS1-transduced cell by guest on September 26, 2021 dose-dependent reduction in reporter gene activity. Higher levels lines (an immunoblot of lysates from RGS1-transfected cell lines of RGS1 expression than that shown resulted in further inhibition no. 8 and no. 12 is shown in Fig. 1). We exposed the HS-Sultan of SRE reporter activity. RGS1 failed to significantly impair G13␣- vector control and RGS1-transfected cells to concentrations of Q226L-mediated SRE activation, a result consistent with the data PAF that had triggered the strongest increase in intracellular cal- from the SAPK assay. These results indicate that RGS1 can impair cium. As expected, PAF triggered a sharp increase in the intracel- signal transduction via those receptors that utilize G12␣ as a signal lular calcium levels of the control cells; however, a similar con- transducer. centration of PAF raised the intracellular calcium levels in the RGS1 down-regulates GPCR signaling in B cells RGS1-transfected cells very modestly. Furthermore, the intracellular calcium levels rapidly returned to baseline in the transfected Next, we tested whether RGS1 modulates G protein signaling in B cells (Fig. 6A). Similarly, the control cell line increased its intra- cells. Because of difficulties in manipulating primary B lympho- cellular calcium level following LPA stimulation, whereas the cytes, we used as a model system the B cell line HS-Sultan, since RGS1 transfectants again responded poorly (data not shown). it lacks RGS1 unless stimulated. To identify suitable agonists, we The chemokine SDF-1 triggers directed migration of a number exposed HS-Sultan cells to ligands known to bind heptahelical of cell types, including B lymphocytes, via its specific receptor receptors that activate heterotrimeric G proteins and monitored CXCR4 (4, 40). Although the exposure of HS-Sultan cells to intracellular calcium levels. Of the ligands tested (SDF-1, macro- SDF-1 did not induce a significant calcium flux, a significant per- phage inflammatory protein (MIP)-1␣, RANTES, IL-8, ATP, iso- centage of the HS-Sultan cells responded to SDF-1 in a migratory proterenol, histamine, oxotremorine, sphingosine-1-phosphate, lysophosphatidic acid (LPA), and PAF) only the lipids PAF and LPA assay. To test whether the expression of RGS1 would alter SDF-1 triggered a significant increase in intracellular calcium. The che- signaling, we tested the migratory capacity of the RGS1 transfec- mokine SDF-1 triggered an almost negligible increase in intracel- tants in a transwell chemotaxis assay in the presence of varying lular calcium. Many B lymphocyte cell lines express PAF recep- concentrations of SDF-1 (Fig. 6B). We found that fewer RGS1- tors, and PAF has been shown to increase intracellular calcium transfected cells migrated in response to SDF-1 than did the vector levels and activate mitogen-activated protein kinase (MAPK) (37– control cells at all of the concentrations tested. This was the case 38). Although little is known about the effects of LPA on B lym- with all the RGS1-transfected cell lines that we tested, including phocyte function, LPA increases intracellular calcium levels and two not shown. The levels of CD19 were similar on the various activates MAPK in a variety of other cell types (39). cell lines, and whereas there were some minor variations in the We transduced HS-Sultan cells with a retroviral vector that en- level of CXCR4 expression among the different cell lines, they did codes RGS1 and isolated separate pools of transduced cells by not correlate with responsiveness to SDF-1 in the migratory assay neomycin selection. HS-Sultan cells transduced with an empty ret- (data not shown). The Journal of Immunology 1835

FIGURE 6. RGS1 impairs GPCR signaling in B cells. A, Changes in free intracellular calcium in RGS1-transfected lines (TF7, 8, 12 (dashed lines)) and Downloaded from vector alone transfected control line (solid line) in response to PAF signaling as measured by the change in the ratio of free vs bound intercellular Ca2ϩ. In each stimulation, buffer was added at 30 s, and PAF (10Ϫ5 M) was injected at 60 s as indicated on the x-axis denoting time. The y-axis labeled as ﬂuorescence intensity represents the ratio ﬂuorescence (violet/blue) of the calcium probe indo-1. Shown are representative responses of each line from at least three experiments done in duplicate in each experiment. B, RGS1 impairs SDF-1 directed cell migration. RGS1-transfected lines and the vector- transfected control line were assessed for their ability to migrate in response to increasing concentrations of SDF-1 in a standard chemotaxis assay. The data are shown as the percentage of total input migrating on the y-axis and the concentration of SDF-1 on the x-axis. The graph represents mean and SD of two independent experiments in which each concentration point was done in triplicate for each line. http://www.jimmunol.org/

SDF-1 attracts naive and memory B cells, but not germinal cen- in B cells. RGS1 was a less efﬁcient GAP for two other Gi␣ sub- ter B cells despite their expression of CXCR4 (3). We used T family members, Go␣ and Gz␣, than was RGS4. However, because cell-depleted human tonsil cells in chemotaxis assays to conﬁrm of the slow intrinsic GTPase activity of Go␣ and Gz␣, RGS1 should the migratory responsiveness to SDF-1 of each B lymphocyte sub- be functionally important in tissues where they are coexpressed. set. The cells, which migrated in response to SDF-1, were assessed Furthermore, a previous set of experiments examined using

for their cell surface expression of CD19, CD38, and IgD. slightly different conditions had shown that RGS1 possessed sim- by guest on September 26, 2021 ϩ IgD CD38-negative B cells reside in the mantle zone region and ilar GAP activity for Go␣ and Gi␣1 (20). In contrast to its binding Ϫ are considered to be naive B cells. Germinal center B cells to Gi␣ and Gq␣, which was markedly enhanced by GDP and AlF4 ϩ ϩ Ϫ (CD38 ) can be divided into IgD and IgD fractions, whereas treatment, RGS1 bound G12␣ irrespective of its nucleotide status. Ϫ Ϫ memory B cells are IgD /CD38 . The majority of B cells in each This suggested that the binding of RGS1 would not stabilize the fraction expressed the chemokine receptor CXCR4 (Fig. 7A); how- “switch regions” in G12␣ nor enhance the rate at which G12␣ hy- ever, in a standard chemotaxis assay, only the memory cells drolyzes GTP. In fact, when tested in vitro, RGS1 failed to act as (IgDϪ/CD38Ϫ B cells) and the naive B cells (IgDϩ/CD38Ϫ)mi- aG12␣ GAP. The binding of RGS1 to GTP-G12␣ suggested to us grated in response to SDF-1. In contrast, the two other populations that RGS1 could bind to GTPase-deficient G12␣ mutants, and we were refractory to chemokine (Fig. 7B). In preliminary experiments, found that immobilized recombinant RGS1 readily extracted the germinal center B cells are refractive in migrational responses to other GTPase-deficient G12␣-Q229L from transiently transfected cell ly- B cell chemoattractants (data not shown). To examine whether RGS1 sates (Kirk Druey, unpublished observation). This contrasts with expression correlates with nonresponsiveness to SDF-1, we used the inability of RGS proteins to interact with a comparable Gi␣ FACS to isolate three populations of CD19-positive lymphocytes mutant (41). Finally, RGS1 exerted no activity on Gs␣; however, based on their differential expression of IgD and CD38. We tested the RGS1 like RGS4 can indirectly regulate Gs␣ signaling by inhibit- germinal center B cell fractions (IgDϪ, CD38ϩ), the memory B cell Ϫ Ϫ ϩ ing Gi␣, thus allowing unopposed Gs␣ signaling. fraction (IgD , CD38 ), and the naive B cell fraction (IgD , A surprising result in this study is that, not only did RGS1 bind CD38Ϫ) for their expression of RGS1 by immunoblotting. We found G12␣, but also its expression impaired the activation of down- that the naive and memory B cells expressed low levels of RGS1 stream signaling by GTP-G12␣. The direct downstream effectors of whereas the germinal center B cells contained significant levels of GTP-bound G12␣ and G13␣ are poorly characterized although a RGS1 as assessed by immunoblotting, equivalent to what we ob- recent study showed that activated G13␣ directly stimulates the served following PMA activation (Fig. 7C). Thus the refractoriness of guanine nucleotide exchange activity of p115 RhoGEF, indicating germinal center B cells to SDF-1-triggered chemotaxis may be be- that it is a G13␣ effector (42). However, activated G12␣ failed to cause they express RGS1. have a similar effect, suggesting that these ␣ subunits may possess both overlapping and distinct effectors. Consistent with that pos- Discussion ϩ ϩ sibility, G12␣ and G13␣ differentially activate Na /H exchanger

The studies of RGS1 G␣ binding speciﬁcities and GAP activity for isoforms (43). We also provide evidence that G12␣ and G13␣-trig- different G␣ subunits provides insight into the types of signaling gered signaling pathways differ. G12␣, but not G13␣, signaling is pathways that RGS1 might regulate. Although a slightly less ef- impaired by RGS1. Precisely how RGS1 inhibits G12␣-mediated

ﬁcient GAP for Gi␣1 and Gq␣ than RGS4, RGS1 potentially reg- signaling requires clariﬁcation. We have no evidence that the GAP ulates ligand-receptors pairs that couple through either Gi␣ or Gq␣ activity of RGS1 is important for its inhibition of G12␣ signaling. 1836 RGS1 AND Gi␣ SIGNALING

FIGURE 7. Correlation of SDF-1 migratory refractoriness and RGS1 expression. A, CXCR4 expression by human tonsil B cell subsets. Human tonsil B cells were phenotyped into subsets based on differential expression of IgD and CD38 into naive, memory, and germinal center B cells. The expression level of CXCR4 receptor was assessed on each B cell subset, with the MFI of CXCR4 staining by the majority of each subset given. B, SDF-1-induced migratory responsiveness of human tonsil B cell subsets. Human tonsillar B cells were used in a chemotaxis assay to determine the migratory responsiveness to an optimal concentration (50 ng/ml) of SDF-1. Input and transmigrated populations were analyzed and phenotyped by ﬂow cytometry. Thus, the percentage of each subset population migrating

could be determined. The data are expressed as the Downloaded from percentage of total for each subset; thus, the percentage of naive cells migrating is indicative of the percentage of total naive cells in the population, not the percentage of total or percentage of B cells. C, RGS1 expression in human tonsil B cell subsets. Human tonsillar B cells were sorted into naive,

memory and germinal center B cells. Lysates were http://www.jimmunol.org/ made from each of the subpopulations, and 30 ␮gof each lysate was used to fractionate by SDS-PAGE, was transferred to nitrocellulose membrane, and was used for immunoblotting with anti-RGS1 Ab. Shown is a representative blot from three blots using lysates from three independent sorts. by guest on September 26, 2021

However, since all the GAP assays in this study were done in the LPA and inhibit their effects on B cell function. We previously

absence of receptors, it remains possible that RGS1 is a G12␣ GAP showed that the transient expression of RGS1, RGS3, or RGS4 in the presence of the appropriate receptor. A precedent for such a inhibited the migration of a pre-B cell line transfected with CXCR1

possibility is that the G␣ speciﬁcity of RGS2 was revealed only in and CCR2 to IL-8 or MCP1 (50). In that study, the inhibitory effect

the presence of a receptor (28). Nevertheless, based on the G12␣ of RGS3 substantially exceeded that of either RGS1 or RGS4. binding data, we would predict that RGS1 inhibits G12␣ signaling Furthermore, in HEK 293 cells transfected with CXCR1, RGS3,

pathways by behaving as a G12␣ effector antagonist. and RGS4 both proved superior to RGS1 in inhibiting IL-8-in- We analyzed the consequence of RGS1 expression on signal duced MAPK activation (16). In this study, RGS1 potently inhib- transduction initiated by two chemoattractants, LPA and PAF. The ited the migration of a B cell line triggered by the chemokine presence of RGS1 impaired the increase in intracellular calcium SDF-1 via its cognate receptor CXCR4. In contrast, when we that occurs following exposure to PAF or LPA. Both PAF and tested the migratory response to SDF-1 of HS-Sultan cell lines LPA are potent phospholipid agonists produced by a variety of cell permanently transfected with RGS3, SDF-1 attracted the RGS3- types. Although there is little information on the effects of LPA on transfected cells just like it did the vector control cells (C. Moratz, B-lymphocyte function, PAF has been extensively studied. The unpublished observation). This is in contrast to previous experi- binding of PAF to its receptor stimulates c-Fos and c-Jun tran- ments in which RGS3 inhibited chemotaxis in transient transfec- scription; increases phospholipid turnover; results in the tyrosine tions; however, these current data are the results of RGS3 stable phosphorylation of several proteins including p53/56lyn and p59fyn; overexpression in a B cell line in response to signaling by an ␬ and increases NF- B binding activity in nuclear extracts (44–46). endogenous receptor. Thus, similar to the findings with Gq␣-cou- Functionally, PAF augments the proliferation of B cell lines, stim- pled receptors where receptor-RGS protein specificity has been ulates TNF-␣ production, and increases Ig secretion (47, 48). Both noted (51, 52), RGS proteins may differ in their abilities to inhibit PAF and LPA increase intracellular calcium by activating phos- signaling through specific chemokine receptors. pholipase C-␤, which eventually results in calcium mobilization. Both SDF-1 and CXCR4 are necessary for normal B cell de- Since pertussis toxin only partially inhibits the PAF-induced cal- velopment (53–55). SDF-1-induced migratory response depends

cium ﬂux in B cells, PAF likely signals through Gq␣ in addition to upon the release of Gi-associated G␤␥ subunits (53, 56, 57). RGS1 Gi␣ to mobilize calcium (38, 49). Thus, the effect of RGS1 of does not bind G␤␥ (20); therefore, it is unlikely to directly inﬂu- ϩ2 PAF-induced Ca mobilization is consistent with its GAP activ- ence the interaction of G␤␥ with its downstream effectors. How-

ity for Gi␣ and Gq␣. ever, since RGS1 is a GAP for Gi␣, its presence will reduce the The induction of RGS1 in B lymphocytes would be predicted to duration that Gi␣ remains bound to GTP in the cell. Since the impair the recruitment of B cells to inﬂammatory sites by PAF and GDP-bound form of Gi␣ has a high afﬁnity for G␤␥, it will rapidly The Journal of Immunology 1837

recombine with free G␤␥, thereby reducing the amount of G␤␥ 3. Bleul, C. C., J. L. Schultze, and T. A. Springer. 1998. B lymphocyte chemotaxis available to interact with effectors. An apt illustration of the ability regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement. J. Exp. Med. 187:753. of RGS proteins to indirectly inhibit G␤␥ signaling was provided 4. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti, and T. A. Springer. by experiments in yeast with Sst2p and RGS4 (16). The response 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived fac- ␤␥ tor 1 (SDF-1). J. Exp. Med. 184:1101. to pheromone is mediated by the release of from the yeast G␣ 5. Spangrude, G. J., F. Sacchi, H. R. Hill, D. E. Van Epps, and R. A. Daynes. 1985. subunit. Although both Sst2p and RGS4 inhibit responses trig- Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin. J. Immu- gered by pheromone exposure, they fail to inhibit when the signal nol. 135:4135. 6. Chaffin, K. E., and R. M. Perlmutter. 1991. A pertussis toxin-sensitive process transduction pathway is initiated directly by G␤␥. Thus, the inhi- controls thymocyte emigration. Eur. J. Immunol. 21:2565. bition of HS-Sultan cells to migrate in response to SDF-1 by RGS1 7. Cyster, J. G., and C. C. Goodnow. 1995. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J. Exp. Med. 182:581. is likely related to its G ␣ GAP activity. i 8. Vicente-Manzanares, M., M. C. Montoya, M. Mellado, J. M. Frade, What role might RGS1 have in germinal center B cells? Al- M. A. del Pozo, M. Nieto, M. O. de Landazuri, A. C. Martinez, and though its elevated expression of germinal center B cells suggests F. Sanchez-Madrid. 1998. The chemokine SDF-1␣ triggers a chemotactic re- a role in regulating signaling through a GPCR or receptors, no such sponse and induces cell polarization in human B lymphocytes. Eur. J. Immunol. 28:2197. receptors are known to participate in the B cell proliferation or the 9. Hamm, H. E., and A. Gilchrist. 1996. Heterotrimeric G proteins. Curr. Opin. Cell selection processes that occurs in the germinal center. One possi- Biol. 8:189. 10. Bohm, S. K., E. F. Grady, and N. W. Bunnett. 1997. Regulatory mechanisms that bility is that high levels of RGS1 inhibit signaling through a che- modulate signalling by G-protein-coupled receptors. Biochem. J. 322:1. mokine receptor that regulates egress from the germinal center. 11. Katz, M. E., and F. McCormick. 1997. Signal transduction from multiple Ras RGS1 levels may remain high in germinal center B cells until they effectors. Curr. Opin. Genet. Dev. 7:75. 12. Sprang, S. R., and D. E. Coleman. 1998. Invasion of the nucleotide snatchers: are ready to exit the germinal center. At that point, their RGS1 structural insights into the mechanism of G protein GEFs. Cell 95:155. Downloaded from levels decline, they become chemokine responsive, and they es- 13. Kehrl, J. H. 1998. Heterotrimeric G protein signaling: roles in immune function cape from the germinal center. A candidate for such a chemokine and fine-tuning by RGS proteins. Immunity 8:1. 14. Berman, D. M., and A. G. Gilman. 1998. Mammalian RGS proteins: barbarians is SDF-1. The majority of the B cells that reside in germinal center at the gate. J. Biol. Chem. 273:1269. are not attracted by SDF-1 despite their expression of CXCR4 15. Koelle, M. R., and H. R. Horvitz. 1996. EGL-10 regulates G protein signaling in receptors. Germinal center B cells are less responsive to B cell the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 84:115. chemoattractants in general; thus it is possible that the migrational 16. Druey, K. M., K. J. Blumer, V. H. Kang, and J. H. Kehrl. 1996. Inhibition of http://www.jimmunol.org/ unresponsiveness reflects an overall lack of motility rather than an G-protein-mediated MAP kinase activation by a new mammalian gene family. Nature 379:742. effect of RGS1 expression. It may also indicate that RGS1 may 17. Kozasa, T., X. Jiang, M. J. Hart, P. M. Sternweis, W. D. Singer, A. G. Gilman, regulate more than just CXCR4 signaling or that other RGSs are G. Bollag, and P. C. Sternweis. 1998. p115 RhoGEF, a GTPase activating protein involved in regulating B cell responses. However, we know that for G␣12 and G␣13. Science 280:2109. 18. De Vries, L., M. Mousli, A. Wurmser, and M. G. Farquhar. 1995. 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