Molecular Cloning and Characterization of a Novel Regulator of G-protein Signaling from Mouse Hematopoietic Stem Cells*

In-Kyung ParkDagger , Christopher A. Klug§, Kaijun Li§, Libuse Jerabek§, Linheng Li||**, Masakatsu NanamoriDagger Dagger , Richard R. NeubigDagger Dagger , Leroy Hood||§§, Irving L. Weissman§, and Michael F. ClarkeDagger ¶¶

From the Dagger  Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, Ann Arbor, Michigan 48109, the § Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, the || Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195, and the Dagger Dagger  Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, July 6, 2000, and in revised form, September 21, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH2 and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and short-term hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the alpha  subunit of both Gi and Gq subfamilies. The results of in vitro GTPase single-turnover assays using Galpha i indicated that RGS18 accelerates the intrinsic GTPase activity of Galpha i. Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on Gq-mediated signaling pathways in vivo.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A large number of extracellular stimuli act via cell surface receptors coupled to G-proteins (1). Inactive G-proteins are heterotrimeric proteins consisting of alpha , beta , and gamma  subunits. Upon binding of a specific ligand to the G-protein-coupled receptor, the receptor promotes the exchange of GDP to GTP in the alpha  subunit, resulting in dissociation of the alpha  subunit from the beta gamma subunits. The beta gamma subunits are tightly associated and do not dissociate under physiological conditions. The free alpha  subunit and beta gamma subunits then transmit signals through various signal transduction pathways. The activated alpha  subunit has slow intrinsic GTPase activity. When the alpha  subunit is in the GDP-bound form, it re-associates with the beta gamma subunits, leading to an inactive form. The duration of the G-protein signal depends on the rate of GTP hydrolysis and the rate of subunit re-association. For small GTP-binding proteins such as ras, there are GAP1 proteins (GTPase activating protein), which increase the GTP hydrolysis rate. Recently, functional homologs of the ras-GAP have been identified for the heterotrimeric G-protein. These are called RGS (regulator of G-protein signaling) proteins. The first RGS identified, Sst2 (supersensitivity to pheromone) in yeast, is a negative regulator of pheromone signaling (2). Later, the SST2 gene product was shown to function as a GAP for Gpa1, a molecule involved in pheromone desensitization (3). So far ~20 RGS have been identified (4-12), and more could be anticipated. All RGS proteins have a highly conserved domain consisting of 120 amino acid residues, the RGS box, with varying lengths of NH2 and COOH termini. RGS4 can be expressed in bacteria, and it has been co-crystallized with Galpha i1 as the GDP-AlF4--bound form (13). It was shown that RGS binds to Galpha i through the switch region, and that site-directed mutagenesis of the contact residues lead to loss of interaction (14-16).

In vitro most purified native or recombinant RGS proteins can bind Galpha q and/or Galpha i via the RGS box (4, 6, 17-19). Overexpression by transient transfection of a RGS into mammalian cells can attenuate signaling from Gi and/or Gq-linked receptors (20-22). The very recently discovered RGS protein, p115RhoGEF, can act as a GTPase activator for Galpha 12 and Galpha 13 (23). No RGS that can act on Galpha s in mammals has been found so far. However, in yeast, Rgs2 was shown to function as a negative regulator of glucose-induced cAMP signaling through direct GTPase activation of the Galpha s protein Gpa2 (24).

There seems to be tissue specific distribution of RGS. For example, RGS1 is predominantly expressed in B-lymphocytes (8) and monocytes (25, 26), and RGS4 is expressed in neural tissue (27). RGS1, RGS2, RGS3, RGS4, and RGS16 are present in lymphocytes (28, 29). RGSZ1 (30), RGS7 (31), RGS8 (32), and RGS9 (33) are abundant in brain, and RGS9 in rods (34). RGS3 seems to be ubiquitous.

In this paper, we describe cloning of a novel RGS from a long-term hematopoietic stem cell cDNA library. The new RGS, designated as RGS18, is highly expressed in long-term as well as short-term hematopoietic stem cells, and less in more committed hematopoietic populations. RGS18 can bind both Galpha i and Galpha q in vitro, enhance GTPase activity of Galpha i, and attenuate signals from Gq-coupled receptors.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Cell Culture-- Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Jurkat human leukemic T lymphocyte (clone E6-1), chronic myelogenous leukemia K-562, acute lymphoblastic leukemia MOLT-3, Burkitt lymphoma Ramos (RA1), and histocytic lymphoma U-937 cells were obtained from American Type Culture Collections and maintained in RPMI 1640 supplemented with 10% fetal bovine serum.

Materials-- Dulbecco's modified Eagle's medium, glutamine, penicillin, streptomycin, and Trizol were obtained from Life Technologies, Inc., and RPMI 1640 was from BioWhittaker. Fetal bovine serum was from HyClone. Leupeptin and glycogen were purchased from Roche Molecular Biochemicals, pepstatin A, benzamidine, TLCK, phenylmethylsulfonyl fluoride, anti-FLAG M2-agarose, and Saralasin were from Sigma. [125I]Angiotensin II (2200 Ci/mmol), GTPgamma S (1250 Ci/mmol), and [gamma -32P]GTP (30 Ci/mmol) were from PerkinElmer Life Sciences. myo-[3H]inositol (88 Ci/mmol) was from Amersham Pharmacia Biotech. Dowex AG 1-X8 anion exchange resin was from Bio-Rad. ScintiVerse was from Fisher. GR/B filter was from Whatman. C57 Bl/Ka-Thy 1.1 strain of mice was bred in the animal facility at Stanford University. Myristylated Galpha i1 was expressed in Escherichia coli (JM109) and purified to homogeneity by anion exchange and hydrophobic interaction chromatography as described (35). Specific activity was 16 nmol/mg of protein as determined by [35S]GTPgamma S binding.

Sorting of Long-term Hematopoietic Stem Cells (HSCs)-- Bone marrow cells were obtained by flushing the tibias and femurs of the C57 Bl/Ka-Thy1.1 strain of mice. Cells were stained with a mixture of rat monoclonal antibodies to various cell surface proteins of mature blood cell lineage: 6B2 (anti-B220), M1/70 (anti-Mac-1), 8C5 (anti-Gr-1), Ter-119 (anti-erythrocyte-specific antigen), KT31.1 (anti-CD3), 53-7.3 (anti-CD5), GK1.5 (anti-CD4), and 53-6.7 (anti-CD8), washed and then incubated with goat anti-rat antibody conjugated with phycoerythrin. After washing, nonspecific sites were blocked with 1 mg of normal rat serum or rat IgG/ml of phosphate-buffered saline. Cells were again washed and then incubated with biotinylated E13-161-7 (anti-Sca-1), fluorescein isothiocyante-conjugated 19XE5 (anti-Thy-1.1), and APC-conjugated 2B8 (anti-c-kit). Washed cells were then incubated with streptavidin-magnetic beads (Miltenyi Biotec) for 10 min and then with avidin-Texas Red (Caltag) for an additional 10 min. Sca-1+ cells were enriched with a mini-MACS column (Miltenyi Biotec). The Sca-1+Lin-Thy-1.1loc-kit+ long-term hematopoietic stem cells were sorted on a Vantage fluorescence-activated cell sorter (Becton Dickinson) and then resorted to ensure a high degree of purity as described previously (36). The long-term hematopoietic stem cells were >98% pure using this approach as demonstrated by repopulation assay (37).

Construction of a cDNA Library from Long-term HSCs of C57 Bl/Ka-Thy1.1 Mice-- Twenty-eight thousand twice-sorted long-term HSCs were resuspended in 100 µl of Trizol reagent containing 20 µg of glycogen. Total RNA was isolated as described by manufacturer's instructions except the sample was re-extracted with 100 µl of Trizol. Total RNA was precipitated with isopropyl alcohol followed by ethanol and then resuspended in water. cDNAs were synthesized using CapFinder cDNA synthesis kit (CLONTECH) with modifications. There were 7.5 million clones in the original library. To test the quality of the library, plasmid DNA from 150 random clones were isolated and sequenced using ABI 3700 sequencer.

Northern Analysis-- Total RNA was isolated from various mouse tissues and human cell lines using Trizol reagent according to the manufacture's instructions. Poly(A)+ RNA was then isolated from total RNA using oligo(dT) paramagnetic beads (Dynal). Two micrograms of poly(A)+ RNA per tissue were separated on a 1% agarose/formaldehyde gel, and transferred to Hybond-XL (Amersham Pharmacia Biotech) or Zeta-Probe (Bio-Rad). The membrane was blocked with salmon sperm DNA at 0.1 mg/ml in ExpressHyb buffer (CLONTECH) for 1 h and then hybridized with 32P-labeled NotI-EcoRI fragment of RGS18 for 1 h at 68 °C. The membrane was washed twice with 2 × SSC, 0.1% SDS at room temperature and then with 0.1 × SSC, 0.1% SDS at 50 °C, and exposed to film for 2 to 4 days. Human Multiple Tissue Northern blot and Human Immune System Multiple Tissue Northern blot II were obtained from CLONTECH, and hybridized with 32P-labeled 0.5-kb EcoRI fragment of human expressed sequence tag clone za69c05. The probes were stripped and the membranes were reprobed with mouse beta -actin cDNA.

Southern Analysis-- Genomic DNA was isolated from mouse spleen according to Maniatis et al. (38). Fifteen micrograms of genomic DNA was digested with various restriction endonucleases, separated on a 0.7% agarose gel, and then transferred onto Hybond-N+. The membrane was blocked with 100 µg/ml salmon sperm DNA and hybridized with the 32P-labeled 0.5-kb NotI-EcoRI fragment of RGS18 cDNA at 55 °C overnight at 2 × 106 cpm/ml of hybridization buffer (10 × Denhardt's, 6 × SSC, 0.1% SDS). The membrane was washed twice with 2 × SSC, 0.1% SDS at room temperature and then with 0.2 × SSC, 0.1% SDS at 65 °C, and exposed to XAR-5 film (Kodak) at -70 °C for 20 h.

RGS18 Antibody Production and Western Blotting of Mouse Tissue Extracts-- Rabbit polyclonal sera were raised against bacterially expressed RGS18 containing the first 202 amino acids, and purified using Protein A column. Various tissues of a BA mouse were homogenized in Buffer B containing 0.2% beta -mercaptoethanol with 10 strokes in Potter-Elvehjem tissue grinder, and centrifuged for 10 min in a microcentrifuge at 4 °C. Thirty micrograms of protein were separated on a 12% SDS-polyacrylamide gel, and Western blot was performed as described (39) using anti-RGS18 antibody at 5 µg/ml.

RT-PCR Analysis of RGS18 in Hematopoietic Progenitor Cells-- Long-term and short-term HSCs, common lymphoid progenitors, common myelocyte progenitors, granulocyte macrophage progenitors, and megakaryocyte erythroid progenitors were isolated as described previously (36, 40, 41). Five thousand cells of each of the above populations were double-sorted on a Vantage fluorescence-activated cell sorter. To ensure the correct populations were isolated to sufficient purity, day 12 spleen colony assays were performed on 100 long-term and short-term HSCs from the above sort. Similarly, common myelocyte progenitors, granulocyte macrophage progenitors, and megakaryocyte erythroid progenitors were functionally assayed in methocellulose cultures according to Ref. 41. Total RNA was prepared from these fluorescence-activated cell sorter-purified cells using the Qiagen RNeasy miniprep kit. RNA was treated with DNase I to eliminate residual DNA contamination prior to reverse transcription reaction. cDNA was obtained using Superscript II (Life Technologies, Inc.) according the manufacture's recommendations. PCR was performed using 32P-labeled primers and KlenTaq-1 (CLONTECH). Hypoxanthine-guanine phosphoribosyltransferase control PCR was performed to normalize the amount of cDNAs to be used in the PCR reaction. Generally, 50 cells worth of cDNA was used to long term and short term-HSCs, 30 cells worth of cDNA for common myelocyte progenitors and granulocyte macrophage progenitors, and 20 cells worth for megakaryocyte erythroid progenitors. After 28 cycles of PCR, one-fifth of the products were run on polyacrylamide gels. Gels were dried and exposed to x-ray films or a PhosphorImager for data acquisition and analyses. RT-PCR of RGS18 from 2 µg of total RNA isolated from thymus was negative (data not shown).

Binding of RGS to Galpha -- Three and a half million 293T cells were transfected with 10 µg of various RGS constructs by the calcium-phosphate method (42). Twenty-four hours after transfection, medium was changed, and cells were further grown for another 24 h. Cells were rinsed once with cold phosphate-buffered saline, and resuspended in Buffer A containing 50 mM HEPES, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 5 µg of pepstatin A, TLCK, and TPCK, and 10 µg of leupeptin and soybean trypsin inhibitor per ml. After 15 min on ice, cell lysates were centrifuged in a microcentrifuge for 15 min at 4 °C. The RGS proteins were immunoprecipitated from the supernatant with 20 µl of anti-FLAG M2 affinity gel for 1 h at 4 °C. The immunoprecipitates were washed three times with Buffer A. Jurkat cell extracts (50 million cells per point) were prepared as described (28) using Buffer B (Buffer A plus 1 mM MgCl2 and 0.3 M NaCl). The extracts were treated with 30 µM GDP alone or 30 µM GDP, 30 µM AlCl3, and 0.1 M NaF for 30 min at 30 °C, and then incubated with immunoprecipitated RGS proteins for 1 h at 4 °C. Bound proteins were washed once with Buffer B without Triton X-100, eluted by boiling in 30 µl of SDS sample buffer, and separated on a 12% SDS-polyacrylamide gel. Proteins were transferred electrophoretically to nitrocellulose membrane (Schleicher & Schuell). Western blot was performed using polyclonal antibodies against the alpha  subunits of Gi1+2 (AS7 from PerkinElmer Life Sciences), Gq/11, G12, G13, and Gs or FLAG (Santa Cruz).

Single Turnover GTP Hydrolysis Assay-- [gamma -32P]GTP (1 µM) was allowed to bind to 50 nM myristylated Galpha i1 for 15 min at 30 °C in Buffer E consisting of 50 mM HEPES, pH 8, 5 mM EDTA, 100 mM NaCl, 0.1% Lubrol, and 1 mM dithiothreitol. After lowering the temperature to 4 °C, single turnover GTP hydrolysis was initiated by mixing equal volumes of Galpha i1 preloaded with [gamma -32P]GTP and Buffer E plus 30 mM MgSO4, 400 µM unlabeled GTP, and FLAG-tagged RGS proteins bound to M2-agarose beads. The hydrolysis reaction was terminated by adding 1 ml of 15% (w/v) charcoal solution containing 50 mM NaH2PO4, pH 2.3, and placing samples on ice at the indicated time points. The charcoal was removed by centrifugation for 20 min at 4,000 × g, and [gamma -32P]Pi release was assessed by liquid scintillation counting of a 250-µl aliquot of the supernatant in 4 ml of ScintiVerse.

Receptor Binding Assay-- Angiotensin binding by whole cells was determined as described previously (43). Briefly, cells were harvested, washed, and resuspended in Buffer C (Opti-MEM, 0.1% bovine serum albumin, and 0.1 mg/ml bacitracin). The binding reaction was initiated by adding [125I]angiotensin II at a final concentration of 100 nM into each cell suspension, and incubating at 37 °C for 1 h. Unbound radioligands were filtered through a GF/B filter and the filters were washed three times with Buffer C. Cell-bound radioligands on filter was quantitated by gamma -counting. Nonspecific binding (less than 5% of the total) was determined by adding 1 µM unlabeled Saralasin. Protein assay was performed on each sample according to Bradford (44). Total specific binding of angiotensin II was normalized to protein content.

Measurement of Inositol Phosphate Release-- Inositol phosphate measurements were carried out as described (43). Seven hours after transfection, cells were incubated with myo-[3H]inositol (10 µCi/ml) in Dulbecco's modified Eagle's medium for 24 h at 37 °C. Cells were harvested in phosphate-buffered saline containing 0.02% EDTA, washed twice with ice-cold Buffer D (142 mM NaCl, 30 mM HEPES, pH 7.4, 5.6 mM KCl, 3.6 mM NaHCO3, 2.2 mM CaCl2, 1 mM MgCl2, and 1 mg/ml D-glucose), and then resuspended in ice-cold Buffer D containing 60 mM LiCl. The reaction was initiated by mixing 0.25 ml of pre-warmed cell suspension with 0.25 ml of varying concentration of angiotensin II at 37 °C. After 30 min, 0.5 ml of 20% trichloroacetic acid was added and the samples were centrifuged at 4100 × g for 20 min. The supernatant was extracted five times with ethyl ether, neutralized with sodium bicarbonate, and adsorbed to 0.5-ml Dowex AG1-X8 formate resin (50:50 slurry). Resin was washed five times with 2.5 ml of unlabeled 5 mM myo-inositol and inositol phosphates were eluted with 1 ml of 1.2 M ammonium formate, 0.1 M formic acid mixture. The eluates were counted by liquid scintillation counting in 10 ml of ScintiVerse. Released [3H]inositol phosphates were normalized to the amount of [3H]inositol incorporated into cellular lipids. The pellet after centrifugation was resuspended in 0.5 ml of H2O and 1.5 ml of chloroform/methanol, and vortexed vigorously. An additional 0.5 ml of H2O and 1.5 ml of chloroform were added, and a 200 µl-aliquot of the organic phase was counted by liquid scintillation spectrophotometer in 10 ml of ScintiVerse to determine lipid associated radioactivity.

Transcriptional Activation Assay-- HEK293T cells (3.5 × 106) were transfected with 2 µg of pCMV-M1, 2 µg of pCRE/beta -gal, and 8 µg of control or FLAG-tagged RGS proteins. After 24 h, cells were serum starved for additional 24 h. Cells were stimulated with 1 mM carbachol for 6 h. Cell extracts were prepared and luciferase activity was measured using a luciferase assay kit (Promega) according to the manufacture's instruction.

Plasmids-- FLAG-tagged RGS18 was generated by PCR using two primers (5'-CGGGTCATGAGATATGTCACTGGTTTTCTTCTC-3' and T3 primer) and RGS18 cloned in pBlueScript. The PCR reaction consists of 1 cycle of 2 min at 94 °C, 30 cycles of 30 s at 94 °C/30 s at 60 °C/1 min at 72 °C and 1 cycle of 10 min at 72 °C. The PCR product was cleaned using Qiaspin mini-prep kit (Qiagen). After PCR, the DNA was digested with XbaI and ApaI, and ligated to pcFLAG. Human RGS2 was FLAG-tagged at the C terminus by PCR using primers (5'-TTCAGGATCCAAGAGAGATACCACCATGCAAAGTGCTATGTTCTTG-3' and 5'-CTTCTCGAGTGTAGCATGAGGCTCTGTGGTG-3'). The PCR product was digested with BamHI and XhoI and ligated to pcFLAG. FLAG-tagged rat RGS4 was provided by Dr. Robert McKenzie (Parke Davis, Ann Arbor, MI). The angiotensin receptor 1a cDNA in pCDM8 has been described previously (43). pCRE/beta -gal was provided by Dr. Roger Cone (Oregon Health Sciences University, Portland, OR), and pCMV-M1 was by Dr. J. Silvio Gutkind (National Health Institute, Bethesda, MD).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of a Novel Regulator of G-Protein Signaling from Mouse Hematopoietic Stem Cell-- The hematopoietic cells are constantly replenished by a self-replicating common precursor called the HSC. A large body of data on the biology of these cells has been accumulated. However, due to their rarity (less than 0.01% of the bone marrow cells; Ref 36) and the inability to grow these cells in vitro, there is little information regarding the molecular mechanisms that regulate stem cell functions. To better understand long-term self-renewing hematopoietic stem cells on the molecular level, a cDNA library was constructed from small numbers of highly purified long-term self-renewing hematopoietic stem cells. Approximately 150 clones were randomly chosen for sequencing to evaluate the quality of this library. The results of DNA sequencing indicated that the library contained ~50% previously unknown genes that are not present in the expressed sequence tag or GenBankTM data base (data not shown). One of the unknown clones showed limited homology to RGS (regulator of G-protein signaling), and this clone was further analyzed. The novel RGS will be referred to as RGS18. Complete sequencing and translation of the cDNA clone indicated that the clone contained the entire coding sequence (Fig. 1). The first ATG codon in the sequence is at nucleotide position 187, and conforms to the consensus sequence of Kozak (45). The base composition of the entire 1399 base pairs is 65.9% A + T. In the 3'-untranslated region, a single polyadenylation signal sequence, AATAAA, is present at nucleotide position 1122, and three ATTTA or ATTTTA sequence motifs (46) are indicated (Fig. 1). In addition, a TTTTGAT sequence motif followed by an AT-rich sequence is present in the 3'-untranslated region. This motif is present in immediate early genes and suggested to play a role in transcriptional activation (47, 48). Translation of cDNA showed that RGS18 has 234 amino acids containing a central RGS box. A data base search using NCBI BLAST generated many nonredundant clones. The homology lies mostly within the RGS box (data not shown). Among the clones, RGS2 and RGS5 are most closely related to RGS18 (Fig. 2A). RGS2 has 51% identity and 67% homology, and RGS5, 49% identity and 66% homology.



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Fig. 1.   Sequence analysis of mouse RGS18. A, schematic diagram of mouse and human RGS18 cDNA structure. The black bars indicate the coding sequences. B, BglII; E, EcoRI; H, HindIII; N, NcoI; P, PstI; X, XbaI. B, cDNA and protein sequences (GenBankTM accession number AF302685). Boldfaced letters in the cDNA sequence indicate the polyadenylation signal. Underlined letters indicate mRNA destabilization signal.



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Fig. 2.   Amino acid sequence comparison between mouse RGS18 and other RGS proteins. A, homology among mouse RGS2, RGS5, and RGS18. Blacked areas indicate identity and shaded areas homology. GenBankTM accession numbers for RGS2 and RGS5 are AF215668 and NM_009063, respectively. B, homology between mouse and human RGS. Shaded areas indicate sequence differences. Blacked areas indicate putative phosphorylation sites. CK-II, casein kinase-II; PK-C, protein kinase C; PK-A, cAMP-dependent protein kinase A.

By searching the expressed sequence tag data base, we have found a human fetal lung expressed sequence tag clone (GenBankTM accession number N98410), showing 85% identity spanning from nucleotide position 203 to 501. IMAGE clone 297800, from which the sequence was derived, was obtained and completely sequenced. In the coding sequence, the human clone has 86% identity at the nucleotide level and 82% identity and 90% homology at the protein level (Fig. 2B), strongly suggesting that the human clone is a RGS18 ortholog. The human ortholog had a longer 3'-untranslated region than its mouse counterpart (Fig. 1), and there are two polyadenylation signal sequences and three ATTTA or ATTTTA sequences in the 3'-untranslated region. RGS18 proteins from both species contained putative phosphorylation sites for casein kinase II, protein kinase C, and protein kinase A (Fig. 2B)

Expression of RGS18 mRNA in Tissues and Cells-- Poly(A)+ RNAs isolated from different mouse tissues were analyzed by Northern using the RGS18 cDNA. The NotI-EcoRI fragment containing the 5'-untranslated region and the partial coding region detected a 2.4-kb transcript (Fig. 3A). The highest level of RGS18 expression was observed in bone marrow followed by spleen, fetal liver, and then lung. RGS18 was undetectable in brain, thymus, liver, kidney, and skeletal muscle. A very faint signal was seen in heart. The expression pattern of human RGS18 was also analyzed. In tissues, human RGS18 is highest in peripheral leukocytes followed by bone marrow, spleen, and fetal liver (Fig. 3B). Thymus, as well as lymph nodes, did not express human RGS18, similar to the mouse RGS18 expression pattern. No signal was detected in other tissues tested. In cultured cell lines, RGS18 was expressed only in the monocytic line U937, but not in Molt3 (acute lymphoblastic T-cell leukemic line), K562 (chronic myelogenous leukemic line), and Ramos (B-lymphocytes).



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Fig. 3.   Tissue-specific expression of RGS18. A, Northern analysis of mouse RGS18. Two micrograms of poly(A)+ RNA isolated from various tissues were separated on a 1% agarose-formaldehyde gel, and then transferred to Hybond-XL membrane. The membrane was hybridized with mouse RGS18 cDNA as described under "Experimental Procedures." B, Northern analysis of human RGS18. Human Multiple Tissue Northern blot and Human Immune System Multiple Tissue Northern blot II were obtained from CLONTECH and hybridized with human RGS18 cDNA. Note a similar tissue expression pattern between the two species. C, Western analysis of mouse RGS18. Mouse tissue extracts were separated on a 12% SDS-polyacrylamide gel, transferred onto nitrocellulose membrane, and incubated with rabbit antibody raised against mouse RGS18. - and + indicate antibody has been preincubated in the absense or presence of with 30-fold excess antigen protein, respectively. RGS18 was visualized with ECL reagent.

A rabbit antibody against recombinant RGS18 containing the first 202 amino acids was generated and used to test RGS18 expression in mouse tissue extracts (Fig. 3C). Anti-RGS18 recognized a specific protein with an apparent molecular mass of 26 kDa on a SDS-polyacrylamide gel (Fig. 3C, -) but not when the antibody was preincubated with the recombinant RGS18 polypeptide (Fig. 3C, +). As predicted from the Northern blot, RGS18 was most highly expressed in the bone marrow.

To confirm expression of RGS18 in long-term self-renewing hematopoietic stem cells, cells at various stages of hematopoiesis were purified from bone marrow by fluorescence-activated cell sorter, and RT-PCR was performed (Fig. 4). Compared with hypoxanthine-guanine phosphoribosyltransferase control, RGS18 signal was highest in long-term and short-term HSCs, and the level was lower in common lymphoid progenitors, common myeloid progenitors, granulocyte macrophage progenitors, and megakaryocyte erythroid progenitors. This indicates that RGS18 is expressed more in the primitive cells, and is down-regulated as cells differentiate to more committed linages.



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Fig. 4.   RT-PCR of mouse RGS18 from early hematopoietic progenitors. Cells from early stages of hematopoiesis were isolated from mice bone marrow, and RT-PCR was performed as described under "Experimental Procedures." LT-HSC, long-term self-renewing hematopoietic stem cells; ST-HSC, short-term hematopoietic stem cells; CLP, common lymphoid progenitors; CML, common myeloid progenitors; GMP, granulocyte macrophage progenitors; MEP, megakaryocyte erythroid progenitors.

Southern Analysis of RGS18-- Mouse genomic DNA was digested with BamHI, EcoRI, or HindIII, and transferred to the membrane, and hybridized with the 0.5 kb of 5' end of RGS18 cDNA (Fig. 5). BamHI, EcoRI, and HindIII generated single bands of 9, 2.8, and 6 kb, respectively, suggesting that there is a single copy for RGS18.



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Fig. 5.   Southern analysis of RGS18. Mouse genomic DNA (50 µg) was digested with indicated restriction endonucleases and separated on a 0.7% agarose gel. DNA was transferred onto the Hybond-XL membrane and probed with the RGS18 cDNA fragment as described under "Experimental Procedures."

Binding of RGS18 to Galpha i and Galpha q from Jurkat T Leukemic Cell Extracts-- From sequence comparison, RGS18 showed the most homology to RGS2 and RGS5. RGS2 has been shown to selectively bind and inhibit Galpha q function (20). RGS5 can bind both Galpha i and Galpha q (9). To determine which G-protein signaling pathway RGS18 might act on, binding of RGS18 to endogenous Galpha protein was analyzed. HEK293T cells were transfected with the plasmids carrying FLAG-tagged RGS2, RGS4, and RGS18 cDNA. RGS proteins were immunoprecipitated with anti-FLAG M2 antibody coupled to agarose beads, and incubated with Jurkat cell extracts to facilitate binding to endogenous Galpha proteins (Fig. 6). In has been shown that RGS binds Galpha with high affinity when Galpha is complexed with GDP-AlF4-, which mimics the transition state during GTP hydrolysis. As shown in Fig. 6, the RGS proteins bound Galpha only in the transition state (Fig. 6, + AlF4-). No binding was observed in the GDP-bound state (Fig. 6- AlF4-). The amount of different RGS proteins used in the reaction was similar (Fig. 6, FLAG). No bound Galpha protein was seen with the immunoprecipitates prepared from cells transfected with control plasmid (Fig. 6, pcFLAG). As previously shown (20), RGS2 did interact with Galpha q but not with Galpha i, and RGS4 was able to bind both Galpha i and Galpha q. RGS18 was also able to interact with Galpha i and Galpha q. However, RGS18 did not bind Galpha 12, Galpha 13, or Galpha s (data not shown).



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Fig. 6.   Interaction between RGS18 and Galpha proteins. RGS proteins were immunoprecipitated from transfected HEK293T cells using anti FLAG-M2-agarose beads and incubated with Jurkat cell extracts in the presence or absence of AlF4- formation. The bound proteins were separated on a 12% SDS-polyacrylamide gel and transferred for Western blot using antibodies against Galpha q, Galpha i1+2, Galpha 12, Galpha 13, Galpha s, and FLAG. Proteins were visualized with ECL reagents.

GAP Activity of RGS18-- The ability of RGS18 to stimulate GTPase activity of Galpha i1 was compared with other RGS proteins. To obtain large amounts of RGS18 protein, His-tagged RGS18 was expressed in bacteria. However, recombinant protein was insoluble. Therefore, HEK293T cells were transfected with FLAG-tagged RGS plasmids, and RGS proteins were immunoprecipitated as described before. To normalize the amount of RGS proteins in the assays, the immunoprecipitates were resolved on a SDS-polyacrylamide gel and stained with Coomassie Blue. RGS proteins were scanned with a densitometer, and the RGS was normalized with FLAG-agarose beads. Immunoprecipitates prepared from HEK293T cells transfected with pcFLAG showed no stimulation of GTPase activity (Fig. 7, Vector). As shown before, RGS2 showed no GTPase activity toward Galpha i1. RGS4 dramatically enhanced endogenous GTPase activity of Galpha i1. RGS18 also stimulated GTPase activity but not as much as RGS4. RGS4 reduced the calculated t1/2 for Pi release of Galpha i1 from 1.04 to 0.19 min and RGS18 reduced t1/2 to 0.56 min.



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Fig. 7.   Effect of RGS proteins on single-turnover GTP hydrolysis. HEK293T cells were transfected with FLAG-tagged RGS2 (black-diamond ), RGS4 (black-triangle), RGS18 (), or empty vectors (), and RGS proteins were purified by immunoprecipitation using anti-FLAG M2-agarose beads. Single-turnover GTP hydrolysis was initiated by adding Galpha i1 preloaded with 50 nM [gamma -32P]GTP to the beads as described under "Experimental Procedures." Baseline [gamma -32P]GTP hydrolysis measured at t = 0 was ~40% of total, and was subtracted. Data are presented as percentage of total G-protein-dependent GTP hydrolysis measured at 20 min, and are mean ± S.E. for four independent experiments, each performed in duplicate.

Inhibition of Gq-mediated Signaling by RGS18-- Since RGS18 was able to bind the Galpha q subunit, biological assays were used to determine whether this interaction has functional significance. If RGS18 can modulate a signal from Gq-coupled receptors, it will be indicative of a functional interaction with Galpha q. HEK293T cells were co-transfected with angiotensin 1a receptor plasmid and a FLAG-RGS or control plasmid. Angiotensin 1a receptor has shown to be coupled to the Gq signaling pathway and activation of phospholipase C, which generates inositol 3-phosphate (49). Transfected cells were labeled with myo-[3H]inositol and stimulated with angiotensin II peptide. Both RGS2 and RGS4 inhibited [3H]inositol phosphates release (Fig. 8A). RGS18 was also able to attenuate Gq signaling mediated by angiotensin II. There was no difference in the amount of [125I]angiotensin binding to the cells transfected with the RGS constructs or the empty vector (data not shown). Next, we tested whether RGS18 could attenuate Gq-mediated transcriptional activity. HEK293T cells were transfected with RGS or control plasmid, and M1 muscarinic receptor and pCRE/beta -gal. It has been shown that activation of M1 muscarinic receptor, which couples Gq protein, resulted in transcriptional activation through binding of cAMP responsive element-binding protein to cAMP responsive element (50). Carbachol treatment of cells transfected with control plasmid showed ~20-fold activation of transcription of the reporter gene (Fig. 8B). All RGS constructs inhibited transcriptional activation. RGS2 inhibited activation by 75%, RGS4 by 71%, and RGS18 by 77.5% of the control. These results indicate that RGS18 can modulate signals from Gq-coupled receptors.



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Fig. 8.   Effects of RGS on Gq-mediated signal pathways. A, RGS blocks angiotensin II-stimulated inositol phosphate release. HEK293T cells were transiently transfected with either pcFLAG vector alone () or AT1R and pcFLAG (black-square), FLAG-RGS2 (), FLAG-RGS4 (black-triangle), or FLAG-RGS18 (black-diamond ). Cells were labeled with myo-[3H]inositol, and then incubated in the presence of 30 mM LiCl for 30 min at 37 °C with the indicated amount of angiotensin II. Inositol phosphates were measured as described under "Experimental Procedures." Data are expressed as percentage of maximum inositol phosphate release by AT1R plus vector-transfected cells, and are means ± S.E. from four independent experiments, each performed in duplicate. 125I-Angiotensin II binding to each transfected cell was 2.2 ± 1.7% for pcFLAG vector alone, 100% for AT1R and pcFLAG, 74 ± 10% for AT1R and FLAG-RGS2, 94 ± 8% for AT1R and FLAG-RGS4, and 98 ± 16% for AT1R and FLAG-RGS18. Statistical analysis by ANOVA with Bonferroni corrected post-tests showed that p > 0.05 for differences between receptor alone and coexpression of RGS18 proteins. B, RGS inhibits carbachol-induced transcription of reporter gene. HEK293T cells were transfected with pCRE/beta -gal, pCMV-M1, and various FLAG-RGS plasmids. Twenty-four h after transfection, cells were starved for serum for 24 h, and then stimulated with 1 mM carbachol for 6 h. Cell extracts were prepared and beta -galactosidase activity was measured as described under "Experimental Procedures." Data are expressed as means ± S.E. from four separate experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this paper, we report the cloning of a novel RGS from mouse long-term self-renewing hematopoietic stem cells. The sequence surrounding the third ATG located at nucleotide position 187 was in agreement with the Kozak's consensus sequence for eukaryotic initiation codons (45). The 702-nucleotide open reading frame encodes a polypeptide of 234 residues. This new RGS protein was designated as RGS18. The entire RGS18 cDNA is A + T-rich, and the 3'-untranslated region contains three ATTTA motifs. These features have been linked to mRNA stability (46) and translational control (51). The presence of these structures suggests that expression of RGS18 could be highly regulated. Both mouse and human RGS18 were expressed as a 2.4-kb transcript as determined by Northern hybridization, and showed a hematopoietic tissue-specific expression pattern, with the highest levels in peripheral leukocytes and bone marrow followed by fetal liver and spleen (Fig. 4). There was no RGS18 message detected in thymus and lymph nodes. In cultured cells, only monocytic U937 but not B, T, and myelocyte-derived cell lines expressed RGS18. RT-PCR of RGS18 from cells at the early stages of hematopoiesis indicated that RGS18 is highly expressed in both long-term and short-term HSCs, and less so in cells with more committed lineages (Fig. 4). RGS18 protein can bind Galpha i and Galpha q (Fig. 6). In in vitro GTPase assays, RGS18 enhanced the intrinsic GTPase activity of Galpha i1 to a lower extent compared with RGS4 (Fig. 7). RGS2, which acts only on Gq, was not able to stimulate the GTPase activity. Furthermore, RGS18 inhibited the inositol phosphates production mediated by angiotensin 1a receptor and transcriptional activation mediated by M1 muscarinic receptor in HEK293T cells (Fig. 8). Even though the RGS18 sequence is more homologous to RGS2 than RGS4, it clearly interacts with Galpha i as well as Galpha q. Effects of RGS18 on the Gi pathway are contradictory. Therefore, it is necessary to study gain of function and/or loss of function mice to verify the role of RGS18 in Gi pathway in vivo.

There are over 20 RGS genes cloned so far, but their in vivo regulation is not well understood. There are several ways in which cells can regulate RGS functions. First, many RGS proteins are expressed in tissue and cell-type specific manners. For example, some RGS proteins are abundant in lymphocytes and monocytes (8, 25, 26), brain (30-33), and rods (34). Furthermore, the level of RGS can be modulated under certain conditions. For example, in antigen-activated B cells, RGS1 and RGS2 are up-regulated and RGS3 and RGS14 are down-regulated (29). RGS1 and RGS2 are also up-regulated in phorbol ester-stimulated B cells and ConA- and cyclohexamide-treated human blood mononuclear cells (25). In vascular smooth muscle, RGS2 message was rapidly increased upon angiotensin stimulation (52). RGS16 expression is induced in human T cells by IL-2 and the induction was diminished by cAMP. RGS2 expression, however, was reciprocated (28). RGS18 also showed a tissue and cell-type specific expression pattern. It is expressed highly in long-term and short-term HSCs, and its level is decreased as these cells are more committed to differentiated pathways. In mature cells, RGS18 appears to be most highly expressed in peripheral blood leukocytes of myelomonocytic lineage.

Another way to regulate the RGS activity is by regulating specific interaction between the Galpha . The RGS boxes interact with the switch regions of Galpha , and these interactions are required for the GAP activity. Therefore, the specific interaction with Galpha would be determined by divergent sequences outside of the RGS box. The first evidence that the RGS box alone might not be enough to function normally in vivo comes from the Sst2 complementation assay in yeast (53). The full-length RGS16 protein could bind and function as a GAP for Galpha i and Galpha o in vitro, and attenuated pheromone signaling. The RGS16 core domain was also able to bind Galpha and enhance GTPase activity in vitro; however, the mutants lacking the NH2-terminal region were unable to attenuate pheromone signaling (53). Further evidence for the requirement of the non-RGS box is that deletion of the NH2-terminal domain of RGS4 diminished its biological potency by 10,000-fold (54). It has been demonstrated that different RGS can differentially inhibit Ca2+ mobilization induced by carbachol, bombesin, and cholecytokinin, whose receptors are coupled to Gq. The pattern of inhibition did not change regardless of Galpha q gene deletion (55), and deletion of the NH2-terminal region of RGS4 abolished receptor selectivity by carbachol and cholecytokinin (54). These results indicate that the specificity of RGS functions depend on their interaction with the G-protein-coupled receptor complex rather than a specific Galpha q.

RGS proteins show differential subcellular distribution. Using confocal microscopy, Chatterjee and Fisher (56) have shown that RGS2 and RGS10 are present in the nucleus, that RGS4 and RGS16 are in the cytoplasm, and that RGSZ is localized to the trans-Golgi network. RGS-GAIP was also shown to be associated with Golgi membranes (57). The deletion of the NH2-terminal 15 residues of RGS4 and RGS16 resulted in nuclear accumulation of the RGS proteins. The deleted sequence contained a nuclear export signal, and mutations of the conserved leucine residues also resulted in nuclear accumulation. These data seem inconsistent with the report that RGS4 (58) and RGS16 (59) are associated with the membrane. Chen et al. (59) showed that amino acid residues 7 to 32 of RGS16 are required for RGS16 membrane association. Mutation of the second leucine led to the loss of RGS16 biological activity and membrane association. They proposed that the NH2-terminal domain contained an amphipathic structure that was responsible for membrane binding. Furthermore, Srinivasa et al. (58) showed that the first 33 amino acid residues are required for the membrane binding of RGS4. Since the NH2-terminal sequences of RGS4, RGS5, and RGS16 are conserved, they might share the same type of regulation. Indirect immunofluorescent staining of FLAG-tagged RGS18 showed that RGS18 is localized exclusively in the cytoplasm (data not shown). Furthermore, there is a possible nuclear export signal (NES, L5XXFXXL, Ref. 60) in RGS18. Studies using the NH2-terminal deletion and point mutants of RGS18 would verify this sequence functions as a NES.

RGS proteins undergo post-translational modification. It has been shown that RGS-GAIP is phosphorylated at serine 24 by casein kinase II on clathrin-coated vesicles, and that the phosphorylated form is associated with the membrane (61). RGS18 has two putative casein kinase II sites, one protein kinase C site, and one protein kinase A site outside of the RGS box. It is thus possible that phosphorylation of RGS at these residues might regulate intracellular localization and/or functions of RGS18. Recently, it has been demonstrated that two cysteine residues at positions 2 and 12 of RGS16 are palmitoylated in vivo (62). Mutation of either of these residues decreased RGS16 activity in both Galpha i and Galpha q signaling pathways induced by isopreterenol/somatostatin and carbachol, respectively. Cysteine mutation did not significantly affect the cellular localization of RGS16 and in vitro GAP activity, suggesting that reversible palmitoylation of the protein might be important for biological activity of RGS16. This would also be true for RGS4 and RGS5 whose sequences are conserved at the NH2 terminus. In RGS18 there is no amphipathic structure at the NH2 terminus, and no possible palmitoylation site, suggesting a different mode of regulation for RGS18.

The fact that RGS18 is highly expressed in HSC and mature myelomonocyte compartment and that many other RGS proteins are lymphoid specific suggest that RGS proteins may have functions in regulation of hematolymphoid systems. For example, lymphocyte migration during inflammatory response is induced by a number of chemokines, whose receptors are coupled to Gi. RGS1, RGS3, RGS4, and RGS14, which are expressed by the lymphoid system, can inhibit chemotaxis induced by various chemokines including proinflammatory factors, stromal cell-derived factor-1alpha , and Epstein-Barr virus-induced molecule 1 ligand (26, 29, 63, 64). RGS2, which acts on Gq, showed no effect on chemotaxis. SDF-1alpha is the ligand for CXCR4 (65), which is expressed by various cells including HSCs (66). During the fetal development, hematopoiesis occurs in the fetal liver. As the fetus develops, hematopoiesis moves to the bone marrow. In adult, bone marrow is the primary site for hematopoiesis. It has been shown that in mice lacking SDF-1alpha , bone marrow hematopoiesis was absent, even though fetal liver hematopoiesis was normal (67). This suggests that SDF-1alpha , expressed by bone marrow stromal cells, is responsible for migration of HSCs from fetal liver to bone marrow. Even though many reports show that multiple RGS proteins could modulate SDF-1alpha -induced chemotaxis, it is possible that there might be specificity of RGS to regulate different chemokine receptors. Since RGS18 can interact with Galpha i, it would be of interest to test the possible role of RGS18 in inflammatory response and HSC migration.


    FOOTNOTES

* This work was supported by Grant DK53074-04 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF302685.

Present address: Dept. of Microbiology, Div. of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL 35294.

** Present address: Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110.

§§ Present address: Institute for Systems Biology, Seattle, WA 98105.

¶¶ To whom correspondence should be addressed: Dept. of Internal Medicine, Div. of Hematology and Oncology, University of Michigan, CCGC 4431, 1500 E. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-647-3428; Fax: 734-647-9654; E-mail: mclarke@umich.edu.

Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M005947200


    ABBREVIATIONS

The abbreviations used are: GAP, GTPase activating protein; HSC, hematopoietic stem cell; RGS, regulator of G-protein signaling; SDF-1alpha , stromal-derived factor-1alpha ; TLCK, Nalpha -p-tosyl-L-lysine chloromethyl ketone; GTPgamma S, [35S]guanosine 5-3-O-(thio)triphosphate; HEK, human embryonic kidney; RT-PCR, reverse transcriptase-polymerase chain reaction; kb, kilobase pair(s); TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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