|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Tsukuba Life Science Center, The Institute of
Physical and Chemical Research, Japan, and the Department of
Pharmacology, University of Illinois at Chicago.
The regulator of G-protein signaling (RGS) negatively regulates the
The guanosine triphosphate (GTP)-binding protein
(G-protein) signaling pathway is one of the most important signaling
cascades used to relay extracellular signals and sensory stimuli to
eukaryotic cells.1 Heterotrimeric G proteins, which couple
heptahelical receptors to effectors in the signal transduction pathway,
are composed of Recently, a new family of proteins, the regulator of the
G-protein-signaling (RGS) family, has been identified.5-7
Genetic screenings for negative regulators for the pheromone response pathway in yeast identified a protein, Sst2.8 Further
analyses revealed that Sst2 interacted directly with the G-protein Outside the RGS domain, members of the family are structurally diverse
and can contain additional motifs that could mediate subcellular
targeting or assembly of signaling complexes or both. Additionally,
certain RGS family members exhibit highly restricted patterns of
expression, implying cell-specific functions in embryogenesis and cell
differentiation. An Axin family, containing Axin and Axil, was
identified from mammals, and the proteins in this family containing the
RGS domain were shown to regulate an early step in embryonic axis
formation by RNA injection into Xenopus embryos.11,12 Moreover, the loco gene was identified in Drosophila, and
its mutants revealed a severe glial cell differentiation defect.
The loco encodes 2 RGS domain proteins, and these
were found to show a significant similarity to rat RGS12. The
interaction and the coexpression of LOCO and G Production of blood cells is regulated by the interplay of various
cytokines and bone marrow stromal cells.17-21 Platelets are originally derived from pluripotent hematopoietic stem cells. The
stem cells differentiate into committed megakaryocyte progenitors, and
finally the matured megakaryocytes release a number of platelets. Past
studies on megakaryocyte differentiation and platelet production have
been hampered because of the rarity of megakaryocytes in bone marrow;
the delay of identification of megakaryocyte-specific cytokine,
thrombopoietin (TPO); and the lack of a useful platelet-inducible megakaryocytic cell line. Since TPO was identified in 1994, it has been
reported to regulate the proliferation and maturation of
megakaryocytes22-27 and to actually stimulate
polyploidization of primary immature megakaryocytes.28-31
The availability of TPO and its capacity to induce the proliferation
and differentiation of megakaryocyte progenitor cells have resulted in
the expansion of megakaryocyte numbers in vitro. This culture system
has allowed us to investigate the molecular and cellular mechanisms of
megakaryocyte maturation and platelet formation.
During the isolation of TPO-inducible transcripts in primary
megakaryocytes, a complementary (cDNA) encoding a novel member of the
RGS family, termed RGS18, was incidentally isolated. We found that
RGS18 was expressed predominantly in megakaryocytes and that it had GAP
activity for G Preparation of megakaryocytes and cell culture
Isolation of RGS18 cDNA
Northern blot analysis A mouse multiple-tissue blot filter was purchased from OriGene (Rockville, MD). We separated 5 µg polyA+ mRNA of mouse hematopoietic cell lines on a 1% agarose gel and then transferred it to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, England). The filters were hybridized with the digoxigenin (DIG)-labeled RGS18 cDNA probe (nucleotides 350 to 750) at 42°C for 16 hours in DIG Easy Hyb solution (Roche Diagnostics, Indianapolis, IN). After washing at 68°C for 30 minutes in 1 × SSC containing 0.2% SDS, the hybridized bands were detected by chemiluminescent detection by means of CDP-Star substrate (Roche Diagnostics).Reverse transcriptase-polymerase chain reaction analysis Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed by means of Multiple Tissue cDNA Panels (Clontech), following the manufacturer's protocol. We used 1 ng cDNA in 50 µL PCR mixture. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), was used as endogenous mRNA standard. Annealing temperature was 50°C for RGS18 and 60°C for G3PDH. The following RGS18 primers were used: 5'-TACAGAGGCCTGACTTCCAT-3' and 5'-TTCCATGAGCTGGTACACTC-3', and the following G3PDH primers were used: 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-C ATGTAGGCCATGAGGTCGTCCACCAC-3'.Preparation of antibody The peptide (CESKEKTFFKLMHGS) of RGS18 (amino acids 14 to 28) was synthesized (Sawady, Tokyo, Japan), and conjugated to multiple antigen peptide. The conjugated peptide (100 µg) was injected into 8-week-old female WKY/NCrj rats (Charles River Japan, Yokohama, Japan) 3 times at 2-week intervals. Polyclonal RGS18 antiserum was purified by GST-RGS18 affinity chromatography.Indirect immunofluorescence microscopic analysis Smear samples of bone marrow cells and purified megakaryocytes were fixed with 100% methanol for 10 seconds at room temperature and then washed with phosphate-buffered saline (PBS) for 10 minutes at room temperature. For the sections of mouse spleen, adult BL6 mice (SLC, Shizuoka, Japan) were perfused with 4% paraformaldehyde in 100 mM PBS (pH 7.4) for 20 minutes. The removed spleens were fixed with 4% paraformaldehyde in 100 mM PBS for 20 minutes and washed with PBS 5 times. After that, the spleens were cryoprotected overnight in PBS containing 30% sucrose and embedded in O.C.T. compound (Miles, Elkhart, IN) and then cut on a freezing, sliding microtome at 6 µm. Various lineages of hematopoietic progenitors were purified from mouse bone marrow by FACS Vantage (BD Pharmingen, San Diego, CA), by means of anti-CD45R/B220 (RA3-6B2), anti-CD3e (145-2C11), anti-Ly-6G/Gr-1 (RB6-8C5), anti-CD11b/Mac-1 (M1/70), and anti-TER-119 (TER-119) antibodies (BD Pharmingen).Coverslips of fixed suspension cells and fixed mouse spleen sections were blocked for 10 minutes in blocking buffer (PBS containing 5% FCS), and then incubated in blocking buffer containing anti-RGS18 rat antibody or fluorescein isothiocyanate (FITC)-labeled anti-RGS18 antibody for 2 hours at 37°C and overnight at room temperature, respectively. After rinsing with PBS 5 times, blocking buffer containing FITC-conjugated F(ab')2 fragment donkey or Cy3-conjugated F(ab')2 fragment goat antirat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was applied for 1 hour at 37°C. The coverslips were washed with PBS 5 times, and mounted in 90% glycerol/PBS containing 0.1% p-phenylenediamine and 0.2 µg/mL 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI). To assess the specificity of the antibody, parallel series of cells and sections were incubated in blocking buffer without primary antibody or in blocking buffer with rat preimmune antiserum of primary antibody. The coverslips were observed under a fluorescence microscope (Olympus BX60-34-FLBD1, Olympus, Tokyo, Japan) at a final magnification of 600 × or 1500 ×. In vitro binding assay The RGS18 cDNA (708 base pairs [bp]) in pGEX4T-1 was expressed in Escherichia coli BL21. The binding of endogenous G proteins with GST-RGS18 was performed as follows: The purified megakaryocytes were starved in S-Clone containing 1% BSA for 6 hours. FD-TPO cells were starved in RPMI 1640 containing 0.4% FCS, 0.125 mg/mL transferrin, and 0.01% BSA without TPO for 7 hours. The starved cells were restimulated with or without 50 ng/mL SDF-1 (R&D Systems, Minneapolis, MN) and/or 1 U/mL mouse recombinant TPO for up to 1 hour.
Cells were then lysed in lysis buffer A (50 mM Hepes, pH 7.5, 300 mM
NaCl, 1 mM dithiothreitol (DTT), 6 mM MgCl2, 1% Triton X-100, 2 mM Pefabloc, 10 ng/mL leupeptin, and 10 ng/mL aprotinin). Cell lysates were activated with GDP (30 µM) or GDP plus
30 µM AlCl3 and 100 mM NaF for 30 minutes at 30°C. We
then incubated 1 mg of the cell lysates with GST-RGS18 (40 µg) bound to glutathione Sepharose 4B (Amersham Pharmacia Biotech) (25 µL) for 2 hours at 4°C. After washing 3 times with lysis buffer A containing 0.025% C12E10 (Sigma, St Louis, MO)
instead of Triton X-100, the bound proteins were eluted with Laemmli
buffer and boiled for 10 minutes. Samples were fractionated by 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to ECL membrane (Amersham Pharmacia Biotech). The membrane was blocked with 5% milk in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20 (TBS-T), and incubated with anti-Gi-3 (C-20) or anti-Gq/11 (C-19) antibody (Santa Cruz Biotechnology, CA)
for 2 hours. After washing 3 times with TBS-T, the membrane was
incubated with antirabbit immunoglobulin G-conjugated horseradish peroxidase antibody, and the antibody complexes were visualized by an ECL system (Amersham Pharmacia Biotech).
G s and Gi were expressed in and purified from E
coli.35 G12 and Gq were expressed in Sf9 cells
and purified as described.36 Gi, Gs, and G12 (50 pmol) were loaded with 5 to 20 µM [ -32P]GTP
(approximately 5000 Ci/mmol) at 20°C (for Gs) or 30°C (for Gi
and G12) for 20 to 30 minutes in the presence of 5 mM EDTA. Samples
were then gel-filtered at 4°C through a Sephadex G-50 spin
column (Amersham Pharmacia Biotech) equilibrated with buffer B (25 mM
Hepes, pH 8.0, 1 mM DTT, 5 mM EDTA, 0.05%
C12E10) to remove free
[-32P]GTP and
[32P]O loaded with [-32P]GTP to
buffer B containing 8 mM MgSO4 and 1 mM GTP with the indicated amount of RGS proteins. The reaction mixture was incubated on
ice (for Gi and Gs) or 15°C (for G12). Aliquots (50 µL) were removed at the indicated times and mixed with 750 µL of 5% (wt/vol) activated charcoal in 50 mM NaH2PO4. The
mixture was centrifuged at 2000 rpm for 10 minutes, and 400 µL
supernatant containing [32P]Oq was assayed with the use
of mutant GqR183C.37 The slow GTPase activity of
GqR183C made it possible to load [-32P]GTP on Gq
without accelerating GDP-GTP exchange by agonist-bound receptor.
GqR183C was loaded with 10 µM [-32P]GTP in the
presence of 50 mM Hepes, pH 7.4, 0.1 mg/mL BSA, 1 mM DTT, 1 mM EDTA,
0.9 mM MgSO4, 30 mM
(NH4)2SO4, 4% glycerol, and 5.5 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) at 20°C for 2 hours. The reaction mixture
was gel-filtered through a Sephadex G50 spin column equilibrated with
50 mM Hepes, pH 7.4, 1 mM DTT, 1 mM EDTA, 0.9 mM MgSO4, 0.1 mg/mL BSA, and 1 mM CHAPS. GTPase assays were initiated by addition of
1 mM GTP and the indicated amount of RGS proteins followed by
incubation at 20°C. Aliquots (50 µL) were removed and processed as
described above.
Isolation of a novel RGS To study the megakaryocyte-specific differentiation/maturation process, we isolated the genes whose transcription is induced by the stimulation of megakaryocyte growth and differentiation factor TPO. Subtraction of mRNAs of primary mouse bone marrow megakaryocytes before and after TPO stimulation led to the isolation of a number of novel cDNA fragments encoding factors related to the megakaryocyte-specific differentiation/maturation events. We focused on a novel mouse RGS, termed RGS18, although this gene expression turned out not to be up-regulated by TPO stimulation (data not shown). Figure 1 shows the nucleotide sequence of the isolated RGS18 cDNA (1937 bp) and the amino acid sequence of the encoded polypeptide. The cDNA contained an open reading frame, which encoded a highly basic polypeptide of 235 amino acids with a calculated molecular mass of 27 500 d. By a BLAST search of the NCBI database, mouse RGS18 cDNA was found to be highly homologous to a human RGS13 (GenBank AAF80227), which was different from the previously reported human RGS13 (GenBank AF030107).38 To date, at least 26 different RGS proteins have been described in mammals and designated from RGS1 to RGS17. Therefore, we used the term RGS18 for this novel mouse RGS (accession number, AB042807) and human RGS13 (GenBank AAF80227). Alignment of the deduced amino acid sequences of mouse and human RGS18 (previously described as RGS13 for the human) shows that they have 83% identity and 91% similarity in amino acid sequences. Like other members of the RGS family, RGS18 contain a highly conserved RGS domain, which mediates G-protein interactions,39,40 and several potential phosphorylation sites for protein kinase A, protein kinase C, and casein kinase II.
RGS18 in subfamily B To examine the relationship of RGS18 to other RGS family members, a phylogenetic tree was constructed by using the amino acid sequences of RGS domains of all known mammalian RGS family members (Figure 2A). It was proposed that the mammalian RGS family consists of at least 6 distinct subfamilies.38 As shown in Figure 2A, RGS18 turned out to be a member of subfamily B. Figure 2B shows an alignment of RGS domains of RGS18 with those of other subfamily B members. The RGS domain of mouse RGS18 has 85% identity with and 89% similarity to that of human RGS18 (previously described as RGS13) and has a significant homology (between 45% and 60% identity and between 65% and 75% similarity) to that of other subfamily B members. The essential amino acid residues, especially N152 (single-letter amino acid code), constituted with RGS domains are conserved in RGS18. The S152 is a characteristic residue that exists only in subfamilies B, C, and D, and the N residue at this position is believed to be crucial for the GAP activity and might be involved in stabilization of the transition state of G subunits.41 However, S127
(single-letter amino acid code), which has heretofore been viewed as a
subfamily B-specific residue, was not found in either mouse or human
RGS18 or in mouse RGS1, indicating that it is no longer a unique
residue in this subfamily.
Figure 2C shows the phylogenetic tree of RGS subfamily B, which was constructed with the use of full-length amino acid sequences of all subfamily B members. When outside sequences of RGS domains were included in this assay, RGS18 turned out to be closest to RGS5, which was not clear when we compared RGS18 with other RGS members within RGS domains. RGS18 is expressed in spleen and hematopoietic cells Expression of RGS18 in various mouse tissues was examined by Northern blot analyses. As shown in Figure 3A, a single hybridized band of about 2 kilobases (kb) was clearly detected in spleen (Figure 3A, lane 9) and a very faint band was detectable in lung (Figure 3A, lane 5), suggesting that RGS18 is expressed predominantly in hematopoietic cells.
In order to detect rare RGS18 mRNA in various tissues, RT-PCR at 2 different cycles was performed in whole mouse embryos at different developmental stages as well as in several mouse tissues (Figure 3B). G3PDH was used as an internal control (Figure 3B, lower panel). It was found that RGS18 expression began in day-11 embryos, reached the maximum at day 15, and decreased thereafter (Figure 3C, lanes 9-12), suggesting that RGS18 was expressed in embryonic hematopoietic cells in fetal liver. It was also confirmed that RGS18 was predominantly expressed in spleen (Figure 3B, lane 3), weakly expressed in lung (Figure 3B, lane 4), but not expressed in other tissues examined (Figure 3B. lanes 1, 2, 5-9). We next analyzed RGS18 expression in various mouse hematopoietic cell lines (Figure 3C); erythroid cells SKT6; TPO-dependent hematopoietic progenitor cells FD-TPO, which has megakaryocytic characters27; IL-3-dependent pro-B cells Ba/F3; myeloid cells WEHI-3B; and IL-2-dependent killer T cells CTLL-2. RGS18 transcripts were found most abundantly in FD-TPO cells (Figure 3C, lane 4) and less abundantly in Ba/F3 cells and WEHI-3B cells (Figure 3C, lanes 1 and 3). The transcripts were weakly seen in SKT6 cells (Figure 3C, lane 5), but no transcript was detected in CTLL-2 cells (Figure 3C, lane 2). These results indicate that RGS18 is expressed predominantly in hematopoietic cells. RGS18 expression in megakaryocytes The expression of RGS18 in the protein level of hematopoietic cells was examined by preparing anti-RGS18 rat antiserum by injecting a peptide (CESKEKTFFKLMHGS) encoding outside of the RGS domain; this avoided cross-reaction with other RGS family members, and the antibody was purified by RGS18 affinity chromatography. This antibody clearly detected mouse RGS18 of 27.5 kd in ES cell-derived megakaryocytes in immunoblot analysis as well as immunoprecipitation (data not shown).The immunohistochemical staining of the cells isolated from adult mouse
bone marrow (Figure 4A) and spleen
(Figure 4B) was performed with the purified anti-GST18-specific
antibody. The megakaryocytes with a large cytoplasmic region, which was
stained with anti-RGS18 antibody in red, as well as with large
polyploid chromosomes stained with DAPI in blue, were clearly observed
(Figure 4A-B). However, RGS18 was not detectable in other hematopoietic cells such as erythroid progenitors in bone marrow (Figure 4A) or
spleen (Figure 4B), which were seen as a number of small cells stained
only with DAPI in blue.
We further performed the immunofluorescence stainings of the other
lineages of the hematopoietic progenitor cells in mouse bone marrows.
We isolated Hoechst RGS18 is localized in cytoplasm of megakaryocytes Subcellular localization of RGS18 in primary megakaryocytes was determined by indirect immunofluorescence microscopic analysis (Figure 5). We prepared 2 kinds of primary megakaryocytes: those from mouse bone marrow (Figure 5A-B) and those isolated from in vitro culture of ES cells on OP9 cells with TPO (Figure 5C-D). The endogenous RGS18 were clearly stained in green and found to be localized in cytoplasm but not in nucleus of either of these primary megakaryocytes (Figures 5A-C). The polyploid chromosomes were stained with DAPI in blue in both megakaryocytes (Figure 5B,D). A number of other hematopoietic cells in bone marrow were stained only with DAPI (Figure 5B). These results clearly demonstrated that RGS18 is localized predominantly in cytoplasm of megakaryocytes.
It was also found that the platelets isolated from peripheral blood (Figure 5E) as well as the megakaryocytes during proplatelet formation (PPF) (Figure 5F) were clearly stained with purified FITC-labeled anti-RGS18-specific rat antibody. Thus, RGS18 may also perform its function in platelets. RGS18 acts as GAP for G subunit of G proteins is divided into 4 subfamilies,
Gi, Gq, Gs, and G12,4 and each RGS directly
interacts with a subset of G subunits. To directly test the effects
of RGS18 on the GTPase activity of various G subunits, we measured
the catalytic activity of purified recombinant G subunits during a
single GTPase cycle in the presence or absence of recombinant RGS18
(Figure 6). RGS4 or p115RhoGEF was used
as a positive control for GAP activity for Gi and Gq, or for
G12, respectively.10,42 RGS18 was found to be a nearly
as effective Gi GAP as RGS4 (Figure 6A), although at equal molar
concentrations RGS4 was slightly superior. To measure the GAP activity
of RGS18 for Gq, we employed a mutant Gq, GqR183C, since slow
GTPase activity of GqR183C made it a suitable target for testing
potential Gq GAPs. RGS18 showed good GAP activity for Gq exactly
as did RGS4 (Figure 6B). RGS18 did not show GAP activity for Gs or
for G12 (Figure 6C-D). These results indicate that RGS18 acts as GAP
for Gi and Gq but not for Gs or G12.
Specific binding of RGS18 to G , which is an inactive form,
but does bind with high affinity to the
G-GDP-AlF4 complex, which mimics
the transition states of GTP hydrolysis and is the active
form.5 Because we found that RGS18 acts as GAP for Gi
and Gq but not for Gs or G12, we performed in vitro binding
assay of RGS18 to Gi and Gq as follows: The primary megakaryocytes prepared from ES cells (Figure
7A) and FD-TPO cells (Figure 7B) were
pretreated with excess GDP to inactivate G, or with GDP and
AlF4 to activate G, and the cell lysates
were incubated with recombinant GST-RGS18-bound beads. The bound
proteins were separated by SDS-PAGE, and immunoblotted with antibodies
specific to Gi-3 or Gq/11. Anti-Gi-3 rabbit antibody (C-10)
specifically reacts with Gi-1, Gi-2, and Gi-3, but does not
cross-react with other G subunits. Anti-Gq/11 rabbit antibody
(C-19) specifically reacts with Gq and G11, but does not react
with other G subunits.
None of G SDF-1 affects the binding of RGS18 to G binding to RGS18, we performed in
vitro binding assays before and after SDF-1 stimulation with or
without TPO in ES cell-derived megakaryocytes (Figure
8). The bindings of RGS18 to Gi or
Gq with or without AlF4 treatment are
shown as controls (Figure 8A-B, lanes 9-10). RGS18 weakly and
constitutively interacted with Gi without any stimulation in
megakaryocytes (Figure 8A, lane 2), and TPO alone did not enhance the
binding of RGS18 to Gi within 15 or 60 minutes after
stimulation (Figure 8A, lanes 3-4). Surprisingly, however, stimulation
of megakaryocytes with SDF-1 clearly enhanced the binding of RGS18 to Gi within 15 minutes or 60 minutes after stimulation (Figure 8A,
lanes 5-6), although the level of enhancement was low compared with
that with AlF4 treatment (Figure 8A, lane
10). The addition of TPO did not affect the enhancement of RGS18
binding to Gi caused by SDF-1 (Figure 8A, lanes 7-8). In contrast,
little interaction of RGS18 with Gq was detected with or without
stimulation (Figure 8B, lanes 2-8). These results suggest that SDF-1
binding to its receptor CXCR4 induces modification such as
depalmitoylation and/or translocation of Gi but not of Gq, which
in turn enhances the affinity and/or capacity of Gi binding to
RGS18. Therefore, the SDF-1 receptor-mediated signaling cascade, as
well as the TPO signaling pathway, may play an important role in
megakaryocyte growth and differentiation.
We report the isolation of an additional novel member of the RGS
subfamily B. The existence of a large family of RGS proteins prompted
questions about functional differences among members of this family.
Our findings suggested that RGS18 expressed in megakaryocytes may act
on a specific chemokine receptor-mediated signal. A variety of RGS
family members may be required for the specific function of G proteins
in response to various and specific stimuli in various tissues. RGS, as
well as G proteins, is in general ubiquitously expressed in a variety
of tissues. RGS9 is, however, specifically expressed in
brain,14-16 and we found that RGS18 is specifically
expressed in hematopoietic cells. Therefore, the expression of some RGS
family members is restricted to specific tissues. Our findings are the
first observation that the binding of endogenous RGS to specific G Immunofluorescent microscopic analysis clearly showed that RGS18 is a
cytosolic protein. However, the subcellular localization of other
endogenous RGS family members varies. It was recently reported that
RGS2 and RGS10 accumulated in the nucleus, that RGS4 and RGS16
accumulated in the cytoplasm, and that RGSZ localized to the Golgi
complex, when these RGSs were forced to be expressed in COS-7
cells.43 The study of intracellular distribution of RGS3
showed that it was diffusely localized in cytoplasm, and agonist
stimulation induced its translocation from the cytosol to the plasma
membrane when it was forced to be expressed in a human mesangial cell
line.44 RGS1 was reported to be localized to the plasma
membrane.45 G SDF-1 is a member of the CXC family of chemokines constitutively secreted from the bone marrow stromal cells and several other cell types48 and was initially characterized as a pre-B-cell-stimulating factor and as a highly efficient chemotactic factor for T cells and monocytes.49 The biological effects of SDF-1 are mediated through its receptor CXCR4, which is expressed on leukocytes and hematopoietic stem cells.50,51 Unlike most chemokines and chemokine receptors, SDF-1 is the only known ligand for CXCR4, and CXCR4 is the only known receptor for SDF-1.52-55 Genetic elimination of SDF-1 causes perinatal lethality, owing to severe abnormalities in cardiac development, B-cell lymphopoiesis, and bone marrow myelopoie |
i and G
Top
Abstract
Introduction
, and 
