Structure, chromosomal localization and expression of the mouse regulator of G-protein signaling10 gene (mRGS10)

Gene 297 (2002) 39–49 www.elsevier.com/locate/gene

Structure, chromosomal localization and expression of the mouse regulator of G-protein signaling10 gene (mRGS10) Corinne Haller a, Simon Fillatreau b, Reinhard Hoffmann a, Fabien Agene`s a,c,* a

Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland Institute for Cell, Animal and Population Biology (ICAPB), University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK c Institut Pasteur, Unite´ Biologie des Populations Lymphocytaires, URA CNRS 1965, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France b

Received 1 February 2002; received in revised form 31 July 2002; accepted 9 August 2002 Received by E. Boncinelli

Abstract Regulator of G-protein signaling (RGS) proteins negatively regulate signaling pathways involving seven transmembrane receptors and heterotrimeric G proteins. The purpose of this study was to determine the chromosomal localization, structure and expression profile of the gene coding for mouse regulator of G-protein signaling10 (mRGS10). Fluorescence in situ hybridization analysis indicated that mRGS10 maps to band F3–F4 of the mouse chromosome 7. Sequence analysis revealed that the RGS10 gene encompasses six exons spanning more than 40 kb of genomic DNA. The RGS domain is encoded by exons 3–6; alternative splicing of the first exons allows the generation of two isoforms in the mouse system which differ in their N-terminal portion. Thus, mRGS10 encodes two intracellular proteins of 167 and 181 amino-acids which are highly homologous to the human and rat polypeptides. The deduced amino-acid sequences of mouse RGS10 show 92% sequence identity to their orthologues from human. The mRGS10 gene is expressed predominantly in brain and testis but it is also found in heart, lung, bone marrow, lymph node and spleen. Differential display between mature B lymphocytes and marginal zone B cells, as well as reverse transcription–polymerase chain reaction and Northern blot, showed that mRGS10 is differentially transcribed during B-cell differentiation. Finally, mRGS10 protein was detected in plasma cells of secondary lymphoid organs by immunofluorescence. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Mouse regulator of G-protein signaling10; mRGS10; Survival; Migration; B Lymphocyte

1. Introduction During the last decade, chemokines have emerged as the most important regulators of leukocyte trafficking (reviewed in Luther and Cyster, 2001; Moser and Loetscher, 2001; Thelen, 2001). Several chemokines are responsible for Blymphocyte homing in secondary lymphoid organs. Mature B cells are attracted into lymphoid follicles by BCA1 (also called BLC, CXCL13) which binds to the chemokine recepAbbreviations: BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization; GAP, GTPase activating protein; GEF, guanine-nucleotide exchange factors; GDP/GTP, guanosine diphosphate and guanosine triphosphate; GPCR, G-protein-coupled receptor; HEK 293, human embryonic kidney 293 cells; LA, in vivo lymph node activated B cells; LN, lymph node cells; mRGS10l, Mouse regulator of G-protein signaling10 long isoform; mRGS10vs, Mouse regulator of G-protein signaling10 very-short isoform; PKA, cAMP-dependent protein kinase A; RGS, regulator of G-protein signaling; RT–PCR, reverse transcription– polymerase chain reaction; SA, splenic activated B cells/marginal zone B cells; SN, splenic naı¨ve B cells/mature B cells * Corresponding author. Tel.: 133-1-4568-8544; fax: 133-1-4568-8921. E-mail address: [email protected] (F. Agene`s).

tor CXCR5 (formerly BLR1) (Legler et al., 1998). Naı¨ve T cells and B lymphocytes expressing CCR7 are attracted by SLC (CCL21) and ELC (MIP3b, CCL19) in the T zones of lymphoid organs (Gunn et al., 1998; Ngo et al., 1998). SDF1 (CXCL12) attracts B lymphocytes expressing CXCR4 to secondary lymphoid organs (Casamayor-Palleja et al., 2001). Chemokine receptors are heptahelical transmembrane molecules, members of the very large G-protein-coupled receptor family (GPCRs). Intracellular signals induced by chemokine receptors are mediated by heterotrimeric Gprotein constituted of a (39–44 kDa), b (36 kDa) and g (8 kDa) subunits. Upon ligand binding, GPCRs stimulate G-a subunit to release guanosine diphosphate (GDP) and to bind guanosine triphosphate (GTP). Stimulation of chemotaxis by chemokines requires the functional coupling of the receptor to G-a(i) because migration is completely abolished by treatment of the cells with pertussis toxin (PTX). In the GTP-bound form, G-a dissociates from the G-b–g dimer, each of which independently binds and activates downstream effectors. It was shown that only b–g subunits released from G-a(i) coupled receptors, but not those

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00883-1

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released from G-a(s) or G-a(q) coupled receptors, can mediate cell migration (Neptune et al., 1999). Signaling is terminated when G-a subunit hydrolyses GTP, returning to the GDP-bound state. This GDP-bound G-a protein reassembles with the b–g dimer to form the inactive heterotrimeric G-protein. RGS proteins are recognized as key modulators of the signals generated by the binding of hormones, peptides or other ligands to G-protein-coupled receptors. These proteins negatively regulate intracellular signaling events mediated by G proteins by increasing their intrinsic GTPase activity; reaction responsible for their deactivation. Regulator of Gprotein signaling proteins are a family of highly diverse, multifunctional signaling proteins found in eukaryotic species ranging from yeast to mammals. The RGS family has grown rapidly since its discovery, 5 years ago. To date, at least 80 different RGS proteins have been identified. Mammalian RGS proteins (RGS 1–18, RGS19 previously known as RGS-GAIP, RGS20 formerly called RGSZ), Mammalian G-protein-coupled receptor kinases (GRK), animal axin/conductin, Caenorhabditis elegans Egl-10, Drosophila Loco and Saccharomyces cerevisiae SST2 are known to contain an RGS domain (Dietzel and Kurjan, 1987; De Vries et al., 1995; Druey et al., 1996; Koelle and Horvitz, 1996; Dohlman et al., 1995). This conserved, characteristic RGS domain (120 amino acid residues) is responsible for their physical interaction with G-a protein subunits. It also confers the GAP (GTPase activating protein) activity of the proteins by stabilizing the GTP to GDP transition state of the G-a subunit (Hunt et al., 1996). Outside the RGS domain, the sequences show great diversity, with disparate domain structure. Recent sequences analysis has suggested that mammalian RGS proteins may be divided into six subfamilies (Zheng et al., 1999). Lymphoid cells have been found to express various RGS molecules (reviewed in Kehrl, 1998). Human RGS1 (BL34) has been cloned from lymphocytes (Hong et al., 1993) and RGS3 has been identified from a human tonsil B-cell cDNA library (Druey et al., 1996). RGS2, RGS14 and RGS16 have also been shown to be expressed in lymphoid cell types (Siderovski et al., 1994; Snow et al., 1997; Beadling et al., 1999; Cho et al., 2000). Following antigen challenge in vivo, B lymphocytes rapidly up-regulate RGS1 and RGS2 transcripts; whereas RGS3 and RGS14 are down-modulated. Modulation of RGS1 and RGS3 expression attenuate B-cell migration induced by the lymphoid chemokines BCA1, ELC and SDF1 (Moratz et al., 2000; Reif and Cyster, 2000). It is likely that the expression of many RGS proteins is differentially regulated during B-cell development, affecting cell subpopulation migration capacities. In this paper, we present evidence that RGS10 is differentially expressed during mouse B-cell development in vivo. We describe the genomic locus encoding for the gene and introduce the various isoforms of the RGS10 transcripts. The protein sequences are also analysed with regard to their putative function.

2. Materials and methods 2.1. Cell transfers and flow cytometry Eight-week-old unmanipulated C57BL/6 mice, obtained from Iffa-Credo (Lyon, France), were used as a source of splenic mature B cells (SN) and splenic marginal zone B cells (SA). In order to obtain in vivo activated LN cells (Agenes and Freitas, 1999), 8-week-old B6 Rag2 2/2Ly5.2 mice, obtained from the Centre de De´ veloppement des Techniques Avance´ es (CDTA, Orleans, France), were exposed to a dose of 300 rad given with a cesium source (Shinkai et al., 1992). Suspensions of pooled mesenteric and inguinal lymph nodes (LN) collected free of blood were prepared from B6.Ly5.1 mice bred in the Basel Institute for Immunology (Basel, Switzerland). Following irradiation, the host mice received intravenously 5–30 £ 106 LN cells. Animals were kept in SPF conditions and spleen cells were recovered 8 months to 1 year after injection into the recipients. For flow cytometry, the following monoclonal antibodies were used: anti-Ly5.1 (A20), anti-B220 (RA3-6B2), antiIgM (R6-60.2), anti-IgD (54.1 clone, a kind gift of Katrin Hafen, Basel Institute for Immunology) and anti-CD21 (7G6). Cell surface staining was performed with the appropriate combinations of FITC, PE, CyChrome and biotin labeled antibodies, followed by streptavidin Phar-red (Becton Dickinson, Franklin Lakes, NJ). Dead cells were excluded from the analysis by light-scatter and/or propidium iodide. The phenotype of the sorted B-cell populations were the following: Lymph node cells (LN) ¼ B220 1 IgM 1 PI 2, lymph node activated B cells (LA) ¼ B220 1 IgM 1 PI 2, splenic mature B cells (SN) ¼ B220 1 IgM low IgD high CD21 int., splenic marginal zone B cells (SA) ¼ B220 1 IgM high IgD low CD21 high (Agenes and Freitas, 1999; Loder et al., 1999). Cell sorting was performed on a Moflo (Cytomation, Fort Collins, CO). Flow cytometry analysis on a FACScalibur (Becton Dickinson) confirmed that purity of the isolated B cells was . 98%. 2.2. Detection of the mRGS10 gene, cloning and sequencing Differential display analysis between SA vs. SN and LA vs. LN was accomplished using ‘RNA map kits’ from GenHunter (Nashville, TN). The mRNA differential display technology works by systematic amplification of the 3 0 terminal portions of mRNAs and resolution of those fragments on a DNA sequencing gel (Liang and Pardee, 1992). Differentially expressed gene products were cloned and sequenced on a ABI Prism DNA Sequencer (Applied Biosystems, Foster city, CA). We performed BLAST searches of the NCBI mouse expressed sequence tag (EST) (Altschul et al., 1997). The full length cDNA of the most interesting genes were obtained by ‘Marathon cDNA amplification’ (Clontech, Palo Alto, CA). Total cDNAs were then cloned using the ‘TOPO TA cloning kit’ (Invitrogen, Carlsbad, CA).

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In order to perform genomic studies, a Bacterial Artificial Chromosome (BAC) was obtained from Incyte Genomics after screening of a 129/SvJ library with the mRGS10 cDNA probe. BAC sequencing reactions were performed on a LICOR DNA sequencer (MWG-Biotech, Germany).

TCT GGA AGC-3 0 ) and hRGS10vs (5 0 -CAG GTG GAC ACC AGA GCA TGG A-3 0 and 5 0 -TCA TGT GTT ATA AAT TCT GGA AGC-3 0 ).

2.3. Fluorescence in situ hybridization

Plasmids encoding the long or the very-short isoform of mRGS10 (mRGS10l or mRGS10vs) were transfected into human embryonic kidney 293 cells with lipofectamine (Gibco/Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, cell pellets were lysed with 2£ SDS buffer and sonicated. Proteins were run on 10% SDS–polyacrylamide gels and subsequently transferred to nitrocellulose membranes (BioRad, Hercules, CA). Membranes were blocked; rabbit anti-RGS10 sera were added, followed by anti-rabbit IgG HRP (Promega, Madison, WI). The membranes were then extensively washed and stained with an ECL solution containing ‘luminol’ (Fluka, Buchs, Switzerland). Membranes were finally exposed to Biomax MR film (Kodak, Rochester, NY). Rabbit anti-RGS10 sera were raised against several peptides of mRGS10 proteins: N-terminal peptide of the long isoform of mRGS10 (‘PNTL’: M F T R A V S R L S R K R P P S D I H D G D G S S), C-terminal peptide (‘PCT’: K R T E E E E E E P P D A Q T A A K R A S R I Y N T) and internal peptide (‘PI’: F R E F L K K E F S E E N V L F W L A C E D F K K) shared by the two mRGS10 isoforms.

A probe was generated by labeling of mouse BAC 26812 with digoxigenin dUTP by nick translation. This labeled probe was combined with sheared mouse DNA and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblast cells in a solution containing 50% formamide, 10% dextran sulfate and 2£ salinesodium-citrate buffer (SSC). Specific hybridization signals were detected by incubating the hybridized slides with fluoresceinated anti-digoxigenin antibodies followed by counterstaining with DAPI (4 0 -6-diamidino-2-phenylindole). 2.4. Northern blot analysis and semi-quantitative RT–PCR Cells were sorted as previously described and total RNA was extracted with RNAzolB from AMS Biotechnology (Oxon, GB). For Northern blots, 10–15 mg of RNA from mouse tissues, purified cells or cell line was subjected to gel electrophoresis and transferred to a Genescreen membrane (NEN, Boston). Similar experiments were performed using ‘Clontech multiple tissue Northern blots’ (Clontech). Blots were probed overnight with random-primed 32P-labeled mouse RGS10 DNA probes at 42 8C. Membranes were then washed according to the manufacturer’s instructions and exposed for a 2 h. DNA fragment of 720 bp used as a probe for the mRGS10 gene was obtained by PCR using the following primers (720 bp): 5 0 -AAC TTC TCA GGT GGA CAC CAG AGC ATG GAA C-3 0 and 5 0 -TAC TTT TGT TTT TAA TGG AAG ATT AGC ATC A-3 0 . Membranes were re-probed with a mouse b-actin probe from Clontech to control for loading and RNA integrity. For semi-quantitative RT–PCR, cDNA synthesis and PCR were performed with the Superscript One-Step RT–PCR system (Life Technologies, Gaithersburg, MD) using dilutions starting at 100 ng of RNA. The following primers were used for distinguishing mRGS10 isoforms: mRGS10l (5 0 -GCC GAC TGA GCA GGA AGC GGC C-3 0 and 5 0 -CTG TAG CTG TCA TAC TTC ATG A-3 0 ) and mRGS10vs (5 0 -AAC ACA TCA GCT CTG AGA AGG CAA CGG GC-3 0 and 5 0 -CTG TAG CTG TCA TAC TTC ATG A-3 0 ). For comparison and quantification, we titrated the hypoxanthine phosphoribosyltransferase (HPRT) gene product (177 bp) with the following primers: 5 0 -GTA ATG ATC AGT CAA CGG GGG AC-3 0 and 5 0 -CCA GCA AGC TTG CAA CCT TAA CCA-3 0 . The following primers were used for distinguishing hRGS10 isoforms: hRGS10l (5 0 -ATG TTC AAC CGC GCC GTG AGC C-3 0 and 5 0 -TCA TGT GTT ATA AAT TCT GGA AGC T-3 0 ), hRGS10s (5 0 -TTA AAA TGA CAG TGT CTG TTG GC-3 0 and 5 0 -TCA TGT GTT ATA AAT

2.5. Western blot and rabbit anti-RGS10 sera

2.6. Immunohistology Spleens were harvested, embedded in Cryo-M-Bed embedding compound (Bright Instrument Co. Ltd., Huntingdon, UK) and frozen at 280 8C. Frozen sections (5 mm thick) were fixed on cold acetone and dried extensively. The sections were stained with goat anti-mouse IgM conjugated to Texas Red (Southern Biotechnology Associates, Birmingham, AL) to visualize B cells and the rabbit polyclonal serum anti-mRGS10 PI. FITC-conjugated anti rabbit IgG (SAPU, Carluke, UK) was added 2 h later. Blocking of the non-specific binding was performed using anti-Fc receptor antibody 2.4G2. Slides were viewed on an Olympus BX50 microscope under reflected light fluorescence. Images were captured using a Hamamatsu digital camera and Openlab image analysis software (Improvision, Coventry, UK). 3. Results and discussion We have previously shown that after cell transfer, B lymphocytes persisting in immunodeficient hosts have a phenotype related to splenic marginal zone B cells; whereas lymph node cells originally injected resemble mature B cells found in the spleen (Agenes and Freitas, 1999). Our studies aimed at identifying genes selectively associated with this long lived B-cell population. For this purpose,

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we followed two approaches. In the first approach, we generated ‘long-lived/activated B cells’ by injecting lymph node B cells (LN) in immunodeficient host. After 1 year, we compared the gene expression profile of this in vivo lymph node derived ‘long-lived/activated B cells’ (LA) and the cells present in the original inoculum (LN) by differential display (Loder et al., 1999). mRGS10 gene was identified as a gene differentially expressed by ‘activated/long-lived B cells’. In the second approach, we compared the gene expression profile of marginal zone B cells (splenic activated B cells ¼ SA) and mature B lymphocytes (splenic naı¨ve B cells ¼ SN) purified by cell sorting. mRGS10 was found to be selectively present in marginal zone B cells. We therefore focused our attention on RGS10. We determined its chromosomal localization, the structure of the gene and the transcripts. We analysed the putative protein sequences. Finally, we describe the tissue expression of the messenger and the protein. Chromosomal localization of RGS10 was determined by fluorescence in situ hybridization (FISH). The initial FISH experiment resulted in specific labeling of the distal region of a medium sized chromosome, which seemed to be chromosome 7 on the basis of DAPI staining. This localization was confirmed by a second experiment in which a probe specific for the centromeric region of chromosome 7 was co-hybridized with BAC 26812 DNA. This experiment resulted in the specific labeling of the distal region and the centromere of chromosome 7 (Fig. 1). Measurements of ten specifically labeled chromosomes 7 demonstrated that mRGS10 is located at a position which is 93% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome 7; an area that corresponds to band 7F3–F4 (,68 cM). Proteins with a broad tissue distribution are thought to posses a general function, whereas those with a narrow tissue expression seem to be more specialized. Tissue expression of mRGS10 was assessed by Northern blot with a ,700 bp PCR product probe amplified from the 3 0 end of mRGS10 cDNA. The length of the messenger RNA encoded by the mRGS10 gene is 950 bp. These experiments demonstrate prominent expression of mRGS10 in brain and testis, but the mRNA is also present in heart, lung, bone marrow, lymph node and spleen. The mouse RGS10 gene is not expressed in liver, muscle and kidney as it is in humans (Fig. 2 and data not shown). Genomic DNA encoding the mRGS10 gene and cDNA were cloned and studied in order to determine the protein(s) potentially generated. In order to obtain a BAC genomic murine clone encoding the RGS10 gene, a BAC ES 129/ SvJ library was screened by hybridization with a cDNA probe. Sequence analysis of BAC 26812 reveal that the RGS10 gene encompasses six exons spanning more than 40 kb of genomic DNA (Fig. 3). We were able to clone two different cDNA encoded by the mRGS10 gene. As observed on Northern blot analysis, the size of these two messengers is 950 bp (Fig. 2). They differ in the 5 0 end of the

Fig. 1. Chromosomal localization of mRGS10 by fluorescence in situ hybridization. Normal metaphase chromosomes derived from mouse embryo fibroblast cells were hybridized with digoxigenin labeled probe, incubated with fluoresceinated antidigoxigenin antibodies and counterstained with DAPI. Red arrow, mRSG10 probe; white arrow, centromeric chromosome 7 probe.

transcripts and contain open reading frames of 546 and 504 bp, respectively (Fig. 3). Thus, mRGS10 potentially encodes two intracellular proteins of 181 (mRGS10l) and 167 amino acids (mRGS10vs), which are highly homologous to the human and rat polypeptides. The deduced amino acid sequences of mouse RGS10 show 92% sequence identity to their human orthologues (Hunt et al., 1996 and Fig. 4). As can be seen, these two isoforms are generated by alternative splicing of the first exons. Using a yeast 2-hybrid system with a mutationally activated form of Rat G-a(i) as the bait, Hunt et al. isolated from HeLa cells a cDNA encoding hRGS10 (Hunt et al., 1996). The isoform originally identified by this group (173 amino acids, denominated from now on hRGS10s) is encoded by a mRNA expressed at least in brain, spleen and testis (data not shown). We generated, by homology between mouse and human genomic sequences, primers that would amplify by RT–PCR the mouse RGS10s mRNA if existing. We were never able to obtain any PCR product of a mouse orthologue for hRGS10s

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Fig. 2. Expression pattern in mouse tissues of mRGS10. Northern blot analysis of total RNA showing levels of mRGS10 in the indicated cells and tissues. As a control for RNA loading, the blots were hybridized with an actin probe (two possible isoforms at 1.7 and 2 kb).

in the organs in which it has been found in the human tissues (data not shown). Moreover, from our sequences of the BAC containing the mRGS10 genomic DNA, we discovered that the ‘ATG’ codon used for initiation of the translation of hRGS10s is replaced by a ‘GTG’ codon on the mouse genomic sequence. Thus, even if a messenger encoding the orthologue of hRGS10s was transcribed in the mouse system, it could not generate a protein of 173 amino acids as there is no initiating codon. In order to determine the protein size of mRGS10l and mRGS10vs, we generated rabbit anti-sera and transfected HEK 293 cell line with the DNA encoding these two isoforms. Western blot analysis showed that transfected HEK 293 cells expressed proteins of 21 kDa (mRGS10l) and 19.5 kDa (mRGS10vs) (Fig. 5). Like all members of the RGS family, the predicted 167and 181-amino-acid mRGS10 proteins contain a 120amino-acid core domain that is strongly conserved with the yeast SST2 protein. The RGS domain is a four-element fingerprint that provides a signature for regulator of Gprotein signaling proteins. The RGS domain of mRGS10

is encoded by exons 3–6 (Fig. 6). The four subdomains can be either nearly continuous, as it is the case for mRGS10, or widely dispersed within non-conserved sequence (Burchett, 2000). Resolution of the crystal structure of the RGS4 protein complexed with a stable transition state mimic of G-a-GTP has revealed that the RGS domain is formed by nine a-helices that fold into two small subdomains. These subdomains contact the G-a surface at three different sites (Tesmer et al., 1997). Due to their relatively small size, mRGS10 proteins do not seem to contain other large domains as often seen for other members of the family. These additional domains, like GGL (G-protein GammaLike), DEP (from Dishevelled, EGL-10 and Pleckstrin) or PH (Pleckstrin Homology) link the RGS proteins to other signaling networks, where they constitute effector type molecules (Burchett, 2000). Polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) serve as proteolytic signals. Thus, the PEST motif found in the C-terminus end of mRGS10 (157 RTEEEEEEPPDAQTAAK 173 of mRGS10l) is likely to

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Fig. 3. Structure of the mRNA encoded by the RGS10 gene. The mRGS10 gene is composed of six exons. Intron lengths were evaluated by PCR. mRGS10 mRNAs all contain the last four exons and splicing of exons 1–3 generate transcripts encoding for mRGS10l (exons 1, 3, 4, 5, 6); whereas the messengers containing exons 2, 3, 4, 5, 6 encode the mRGS10vs protein. The human RGS10 gene is composed of seven exons.

induce rapid destruction of the protein, as it has been shown for other RGS members (Rechsteiner and Rogers, 1996; Kim et al., 1999; De Vries et al., 2000). Palmitylation is a reversible, frequent modification of eukaryotic signaling proteins. Like other RGS proteins, the hRGS10s protein can be palmitylated on the conserved cysteine 66C, modulating its GAP activity (Tu et al., 1997, 1999; Resh, 1999). Thus, the cysteine present in the RGS box of mRGS10 may

be a target for auto or cellular palmitylation (74C of mRSG10l). hRGS10s protein activity is also regulated by cAMP-dependent protein kinase A (PKA) phosphorylation on serine 158S. Interestingly, the mouse orthologues also possess the putative ‘KRAS motif’ in their C-terminus end, known to be required for phosphorylation by PKA (Burgon et al., 2001). Our initial goal was to identify genes involved in B-

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Fig. 4. Comparison of murine and human RGS10 polypeptides. (A) Alignment of murine (mRGS10l, mRGS10vs) and human (hRGS10l, hRGS10s, hRGS10vs) RGS10 proteins. Differences in amino acids between mouse and human proteins are shown as open boxes. The sequences data are available from NCBI web site under accession numbers BF685740 (hRGS10l), S71812 (hRGS10s), NM002925 (hRGS10vs), AK009283 (mRGS10l). (B) Percentage of identity between RGS10 polypeptides present in mouse and human (long (l) and very-short (vs) isoforms).

lymphocyte differentiation and survival; therefore, we present in Fig. 7 the initial observation of the differential expression of mRGS10 in the cell populations described

above (LN vs. LA/SN vs. SA). We studied extensively, using a panel of methods, mRGS10 expression during Bcell differentiation. We found that mRGS10 expression is

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Fig. 5. mRGS10l and mRGS10vs protein size. Proteins expressed by HEK 293 cells transfected with mRGS10l and mRGS10vs DNA were subjected to Western blot analysis in which various rabbit anti-RGS10 sera were used as first-step antibodies. Rabbits were immunized with peptides from the N-terminal part of mRGS10l (PNTL), C-terminal part (PCT) and an internal sequence (PI) shared by the two mRGS10 isoforms (see Section 2.5 for sequences). Columns from left to right: migration of proteins from HEK 293 cells transfected with mRGS10l (left), mRGS10vs (middle) or control DNA (right). Lines from top to bottom: rabbit anti-sera raised against the N-terminal peptide of mRGS10l (top line), the C-terminal peptide (middle line) or an internal peptide (lower line) were added on the blots. Note that the anti-PNTL serum recognizes specifically the mRGS10l protein; whereas anti-PCT and anti-PI sera recognize both mRGS10l and mRGS10vs. The deduced protein size of mRGS10l and mRGS10vs are, respectively, 21 and 19.5 kDa.

up-regulated only at late stage of B-cell differentiation. By Northern blot we only found mRGS10 mRNA in marginal zone B cells. We did not detect any mRGS10 transcripts in bone marrow pre-B cells, splenic mature B cells, nor in

lymphoma cell lines WEHI-3 and A20 (Fig. 2). The recent ‘GeneChip Arrays’ technology is a powerful method to study gene expression profile in various cell populations (Affymetrix, Santa Clara, CA). Hoffmann et al. performed

Fig. 6. Schematic representation of the four elements of the RGS box. RGS domain of mRGS10 is encoded by the last 4 exons: motif1 (22 amino acids; red box) is encoded by exon 3 and 4, motif 2 (19 amino acids; yellow box) by exon 4, motif 3 (24 amino acids; pink box) by exon 5 and motif 4 (20 amino acids; blue box) by exons 5 and 6.

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Fig. 7. Regulation of mRGS10 expression during B-cell differentiation. B lymphocytes were purified by cell sorting according to the following phenotypes: splenic mature B cells from C57BL/6 mice (SN) ¼ B220 1 IgM low IgD high CD21 int.; splenic marginal zone B cells from C57BL/6 mice (SA) ¼ B220 1 IgM high IgD low CD21 high; lymph node cells from unmanipulated animals (LN) ¼ B220 1 IgM 1 PI 2; in vivo lymph node derived activated B cells (LA) ¼ B220 1 IgM 1 PI 2. mRNA were used for the gene differential display experiment and semi-quantitative RT–PCR. (A) Gene differential display. Systematic amplification of the 3 0 end of mRNAs from the various cell populations presented above were performed. PCR products were resolved on a DNA sequencing gel. Triplicates of each cell population are shown, the arrow indicates the position of the mRGS10 transcript that is differentially expressed, in contrast to the gene in the upper part of the figure. (B) Semi-quantitative RT–PCR. cDNA and PCR were performed with Superscript One-Step RT–PCR system using dilutions starting at 100 ng of RNA. Results obtained for the housekeeping gene HPRT, mRGS10l and mRGS10vs are shown for the purified cell populations presented above. The expression of mRGS10l is increased at least 20 times in SA and LA compared to SN and LN, respectively. This result confirms Northern blot analysis, in which the mRGS10 transcript was detected in SA and not in SN. We did not detect any mRGS10vs mRNA in these B-cell subpopulations, in contrast to splenic T cells.

an extensive study of gene expression for B- and T-cell development (Hoffmann et al., 2002). B-Cell differentiation in the BM, from preBI cells to mature IgM low B cells, and Tcell development in the thymus, from DN1 to single positive cells, was studied; no expression of mRGS10 was detected in these subpopulations. However, in the human system, we detected an up-regulation of hRGS10 in tonsillar germinal center IgD 2 CD38 1 B cells (data not shown) (Liu and Arpin, 1997). As the mRGS10 gene encode two transcripts, we determined by RT–PCR which isoform was up-regulated in our original differential display experiment. Semi-quantitative RT–PCR showed that the expression of the long isoform of mRGS10 is increased in marginal zone B cells (SA) compared to mature B cells (SN); as well as in ‘longlived/activated B cells’ (LA) compared to ‘lymph node B cells’ (LN) (Fig. 7). In order to define which B-cell populations express RGS10 protein in vivo, we performed immunohistological analysis. Interestingly, RGS10 protein could be detected in some plasma cells located in the red pulp (Fig. 8). Only a fraction of the plasma cells express RGS10 protein. The biological significance of this finding is not clear at present. It is known that the life span of the plasma cells present in the red pulp is heterogeneous (Ho et al., 1986). As RGS10 has been cloned as a gene differentially expressed by long-

lived B cells, it is possible that it is preferentially expressed by long-lived plasma cells. We are currently investigating this possibility. Only very few marginal zone B cells were positive for RGS10 (data not shown), suggesting a tight post-transcriptional regulation of the expression of this protein. RGS10 and chemokine receptors act on the same G-a(i) proteins, as GAP or GEF respectively (GEF: Guaninenucleotide exchange factors) (Bargatze et al., 1993; Cyster and Goodnow, 1995; Hunt et al., 1996; De Vries and Gist Farquhar, 1999). It is possible that the migration of cells expressing RGS10 is dependent on the level of expression of this protein and the inhibitory signals driven by this molecule.

4. Conclusion In this paper, we describe the cloning of the mouse regulator of G-protein signaling10. We identified two isoforms, a long (mRGS10l) and a very short (mRGS10vs). We localized the mRGS10 gene to chromosome 7 (F3–F4 band) and detected expression of mRGS10 in brain, testis, heart, lung, bone marrow, lymph node and spleen. We can state that the original isoform of hRGS10 described by Hunt et al.

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Kohler for cell sorting and Drs. Freitas and Scotet for reviewing the manuscript. F.A. would like to dedicate this paper to Corinne De´ mollie`re. The Basel Institute for Immunology was founded and supported by F. Hoffmann-La Roche Ltd., CH-4070 Basel. References

Fig. 8. Expression of the mRGS10 protein in secondary lymphoid organs. Immunohistological stain showing the expression of RGS10 protein by a subset of plasma cells (magnification £40Þ. The colors have been artificially generated using Openlab software. The IgM staining (Texas Red) is shown in blue. The RGS10 staining (FITC) is shown in red. Co-expression of RGS10 and IgM results in a pink color.

(hRGS10s; 173 amino acids) has no homologue in the mouse system (Hunt et al., 1996). mRGS10l (181 amino acids) and mRGS10vs (167 amino acids) proteins differ only in their N-terminal end and they both possess the 120-amino-acid RGS domain. They are highly homologous to the human and rat polypeptides. The RGS10 gene is differentially expressed during B-cell differentiation. Interestingly, the RGS10 protein is selectively expressed by a subset of plasma cells. We recently generated several lines of transgenic mice and are currently working on an mRGS10-deficient mouse. With the help of these mouse models, we hope to clarify the involvement of mRGS10 in cell migration, cell survival and lymphocyte activation; as well as determine if this gene is involved in neurological disorders as previously described for other RGS-deficient mice (Oliveira-Dos-Santos et al., 2000).

Acknowledgements We would like to thank Tracy Hayden and Hubertus

Agenes, F., Freitas, A.A., 1999. Transfer of small resting B cells into immunodeficient hosts results in the selection of a self-renewing activated B cell population. J. Exp. Med. 189, 319–330. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bargatze, R.F., Butcher, E.C., 1993. Rapid G protein-regulated activation event involved in lymphocyte binding to high endothelial venules. J. Exp. Med. 178, 367–372. Beadling, C., Druey, K.M., Richter, G., Kehrl, J.H., Smith, K.A., 1999. Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes. J. Immunol. 162, 2677–2682. Burchett, S.A., 2000. Regulators of G protein signaling: a bestiary of modular protein binding domains. J. Neurochem. 75, 1335–1351. Burgon, P.G., Lee, W.L., Nixon, A.B., Peralta, E.G., Casey, P.J., 2001. Phosphorylation and nuclear translocation of a regulator of g protein signaling (rgs10). J. Biol. Chem. 276, 32828–32834. Casamayor-Palleja, M., Mondiere, P., Amara, A., Bella, C., Dieu-Nosjean, M.C., Caux, C., Defrance, T., 2001. Expression of macrophage inflammatory protein-3alpha, stromal cell-derived factor-1, and B-cell-attracting chemokine-1 identifies the tonsil crypt as an attractive site for B cells. Blood 97, 3992–3994. Cho, H., Kozasa, T., Takekoshi, K., De Gunzburg, J., Kehrl, J.H., 2000. RGS14, a GTPase-activating protein for Gi alpha, attenuates Gi alphaand G13alpha-mediated signaling pathways. Mol. Pharmacol. 58, 569– 576. Cyster, J.G., Goodnow, C.C., 1995. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J. Exp. Med. 182, 581–586. De Vries, L., Gist Farquhar, M., 1999. RGS proteins: more than just GAPs for heterotrimeric G proteins. Trends Cell Biol. 9, 138–144. De Vries, L., Mousli, M., Wurmser, A., Farquhar, M.G., 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, 11916–11920. De Vries, L., Zheng, B., Fischer, T., Elenko, E., Farquhar, M.G., 2000. The regulator of G protein signaling family. Annu. Rev. Pharmacol. Toxicol. 40, 235–271. Dietzel, C., Kurjan, J., 1987. Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. Mol. Cell. Biol. 7, 4169–4177. Dohlman, H.G., Apaniesk, D., Chen, Y., Song, J., Nusskern, D., 1995. Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 3635–3643. Druey, K.M., Blumer, K.J., Kang, V.H., Kehrl, J.H., 1996. Inhibition of Gprotein-mediated MAP kinase activation by a new mammalian gene family. Nature 379, 742–746. Gunn, M.D., Tangemann, K., Tam, C., Cyster, J.G., Rosen, S.D., Williams, L.T., 1998. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. Natl. Acad. Sci. USA 95, 258–263. Ho, F., Lortan, J.E., MacLennan, I.C., Khan, M., 1986. Distinct short-lived and long-lived antibody-producing cell populations. Eur. J. Immunol. 16, 1297–1301.

C. Haller et al. / Gene 297 (2002) 39–49 Hoffmann, R., Seidl, T., Neeb, M., Rolink, A., Melchers, F., 2002. Changes in gene expression profiles in developing B cells of murine bone marrow. Genome Res. 12, 98–111. Hong, J.X., Wilson, G.L., Fox, C.H., Kehrl, J.H., 1993. Isolation and characterization of a novel B cell activation gene. J. Immunol. 150, 3895– 3904. Hunt, T.W., Fields, T.A., Casey, P.J., Peralta, E.G., 1996. RGS10 is a selective activator of G alpha i GTPase activity. Nature 383, 175–177. Kehrl, J.H., 1998. Heterotrimeric G protein signaling: roles in immune function and fine- tuning by RGS proteins. Immunity 8, 1–10. Kim, E., Arnould, T., Sellin, L., Benzing, T., Comella, N., Kocher, O., Tsiokas, L., Sukhatme, V.P., Walz, G., 1999. Interaction between RGS7 and polycystin. Proc. Natl. Acad. Sci. USA 96, 6371–6376. Koelle, M.R., Horvitz, H.R., 1996. EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell 84, 115–125. Legler, D.F., Loetscher, M., Roos, R.S., Clark-Lewis, I., Baggiolini, M., Moser, B., 1998. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5. J. Exp. Med. 187, 655–660. Liang, P., Pardee, A.B., 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967– 971. Liu, Y.J., Arpin, C., 1997. Germinal center development. Immunol. Rev. 156, 111–126. Loder, F., Mutschler, B., Ray, R.J., Paige, C.J., Sideras, P., Torres, R., Lamers, M.C., Carsetti, R., 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190, 75–89. Luther, S.A., Cyster, J.G., 2001. Chemokines as regulators of T cell differentiation. Nat. Immunol. 2, 102–107. Moratz, C., Kang, V.H., Druey, K.M., Shi, C.S., Scheschonka, A., Murphy, P.M., Kozasa, T., Kehrl, J.H., 2000. Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes. J. Immunol. 164, 1829–1838. Moser, B., Loetscher, P., 2001. Lymphocyte traffic control by chemokines. Nat. Immunol. 2, 123–128. Neptune, E.R., Iiri, T., Bourne, H.R., 1999. Galpha i is not required for chemotaxis mediated by Gi-coupled receptors. J. Biol. Chem. 274, 2824–2828. Ngo, V.N., Tang, H.L., Cyster, J.G., 1998. Epstein–Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in

49

lymphoid tissues and strongly attracts naive T cells and activated B cells. J. Exp. Med. 188, 181–191. Oliveira-Dos-Santos, A.J., Matsumoto, G., Snow, B.E., Bai, D., Houston, F.P., Whishaw, I.Q., Mariathasan, S., Sasaki, T., Wakeham, A., Ohashi, P.S., Roder, J.C., Barnes, C.A., Siderovski, D.P., Penninger, J.M., 2000. Regulation of T cell activation, anxiety, and male aggression by RGS2. Proc. Natl. Acad. Sci. USA 97, 12272–12277. Rechsteiner, M., Rogers, S.W., 1996. PEST sequences and regulation by proteolysis. Trends Biochem. Sci. 21, 267–271. Reif, K., Cyster, J.G., 2000. RGS molecule expression in murine B lymphocytes and ability to down- regulate chemotaxis to lymphoid chemokines. J. Immunol. 164, 4720–4729. Resh, M.D., 1999. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451, 1–16. Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A.M., et al., 1992. RAG-2deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867. Siderovski, D.P., Heximer, S.P., Forsdyke, D.R., 1994. A human gene encoding a putative basic helix-loop-helix phosphoprotein whose mRNA increases rapidly in cycloheximide-treated blood mononuclear cells. DNA Cell Biol. 13, 125–147. Snow, B.E., Antonio, L., Suggs, S., Gutstein, H.B., Siderovski, D.P., 1997. Molecular cloning and expression analysis of rat Rgs12 and Rgs14. Biochem. Biophys. Res. Commun. 233, 770–777. Tesmer, J.J., Berman, D.M., Gilman, A.G., Sprang, S.R., 1997. Structure of RGS4 bound to AlF4-activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261. Thelen, M., 2001. Dancing to the tune of chemokines. Nat. Immunol. 2, 129–134. Tu, Y., Wang, J., Ross, E.M., 1997. Inhibition of brain Gz GAP and other RGS proteins by palmitoylation of G protein alpha subunits. Science 278, 1132–1135. Tu, Y., Popov, S., Slaughter, C., Ross, E.M., 1999. Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10. J. Biol. Chem. 274, 38260–38267. Zheng, B., De Vries, L., Gist Farquhar, M., 1999. Divergence of RGS proteins: evidence for the existence of six mammalian RGS subfamilies. Trends Biochem. Sci. 24, 411–414.