From the 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
Department of
Pharmacology, University of Michigan, Ann Arbor, Michigan 48109
Received for publication, July 6, 2000, and in revised form, September 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 A large number of extracellular stimuli act via cell surface
receptors coupled to G-proteins (1). Inactive G-proteins are heterotrimeric proteins consisting of In vitro most purified native or recombinant RGS proteins
can bind G 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 G 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),
GTP 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 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 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 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% 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 G Single Turnover GTP Hydrolysis
Assay--
[ 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
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/ 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/ 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.
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).
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,
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.
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.
Binding of RGS18 to G GAP Activity of RGS18--
The ability of RGS18 to stimulate
GTPase activity of G Inhibition of Gq-mediated Signaling by RGS18--
Since
RGS18 was able to bind the G 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
G 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 G 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 G 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-1 subunit of both Gi and Gq subfamilies.
The results of in vitro GTPase single-turnover assays using
G
i indicated that RGS18 accelerates the intrinsic GTPase
activity of G
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits. Upon binding of a specific ligand to the G-protein-coupled receptor, the
receptor promotes the exchange of GDP to GTP in the
subunit, resulting in dissociation of the
subunit from the
subunits. The
subunits are tightly associated and do not dissociate under physiological conditions. The free
subunit and
subunits then transmit signals through various signal transduction pathways. The
activated
subunit has slow intrinsic GTPase activity. When the
subunit is in the GDP-bound form, it re-associates with the
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 G
i1 as the
GDP-AlF4
-bound form (13). It was shown that RGS binds to
G
i through the switch region, and that site-directed
mutagenesis of the contact residues lead to loss of interaction
(14-16).
q and/or G
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
G
12 and G
13 (23). No RGS that can act on
G
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
G
s protein Gpa2 (24).
i and
G
q in vitro, enhance GTPase activity of
G
i, and attenuate signals from Gq-coupled receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (1250 Ci/mmol), and [
-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 G
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]GTP
S binding.
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).
-actin cDNA.
70 °C for 20 h.
-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.
--
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
subunits of Gi1+2 (AS7 from
PerkinElmer Life Sciences), Gq/11, G12,
G13, and Gs or FLAG (Santa Cruz).
-32P]GTP (1 µM) was allowed
to bind to 50 nM myristylated G
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 G
i1 preloaded with [
-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
[
-32P]Pi release was assessed by liquid
scintillation counting of a 250-µl aliquot of the supernatant in 4 ml
of ScintiVerse.
-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.
-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.
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (44K):
[in a new window]
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.
View larger version (54K):
[in a new window]
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.
View larger version (42K):
[in a new window]
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.
) 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.
View larger version (47K):
[in a new window]
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.
View larger version (42K):
[in a new window]
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."
i and G
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 G
q function (20). RGS5 can bind both
G
i and G
q (9). To determine which
G-protein signaling pathway RGS18 might act on, binding of RGS18 to
endogenous G
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 G
proteins (Fig.
6). In has been shown that RGS binds G
with high affinity when G
is complexed with
GDP-AlF4
, which mimics the transition
state during GTP hydrolysis. As shown in Fig. 6, the RGS proteins bound
G
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 G
protein was seen with the immunoprecipitates prepared from cells transfected with control plasmid
(Fig. 6, pcFLAG). As previously shown (20), RGS2 did interact with G
q but not with G
i, and
RGS4 was able to bind both G
i and G
q.
RGS18 was also able to interact with G
i and G
q. However, RGS18 did not bind G
12,
G
13, or G
s (data not shown).
View larger version (22K):
[in a new window]
Fig. 6.
Interaction between RGS18 and
G 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
G
q, G
i1+2, G
12,
G
13, G
s, and FLAG. Proteins were
visualized with ECL reagents.
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 G
i1. RGS4
dramatically enhanced endogenous GTPase activity of G
i1.
RGS18 also stimulated GTPase activity but not as much as RGS4. RGS4
reduced the calculated t1/2 for
Pi release of G
i1 from 1.04 to 0.19 min and
RGS18 reduced t1/2 to 0.56 min.
View larger version (25K):
[in a new window]
Fig. 7.
Effect of RGS proteins on single-turnover GTP
hydrolysis. HEK293T cells were transfected with FLAG-tagged RGS2
( ), RGS4 (
), 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 G
i1 preloaded with 50 nM
[
-32P]GTP to the beads as described under
"Experimental Procedures." Baseline [
-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.
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 G
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/
-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.
View larger version (17K):
[in a new window]
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 (
), FLAG-RGS2 (
), FLAG-RGS4 (
), or FLAG-RGS18
(
). 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/
-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
-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
i and G
q (Fig. 6). In in
vitro GTPase assays, RGS18 enhanced the intrinsic GTPase activity
of G
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 G
i as well as
G
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.
. The RGS boxes interact with the switch
regions of G
, and these interactions are required for the GAP
activity. Therefore, the specific interaction with G
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 G
i and G
o in vitro,
and attenuated pheromone signaling. The RGS16 core domain was also able
to bind G
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
G
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 G
q.
i and G
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.
, and Epstein-Barr virus-induced
molecule 1 ligand (26, 29, 63, 64). RGS2, which acts on Gq,
showed no effect on chemotaxis. SDF-1
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-1
, bone marrow hematopoiesis was absent, even
though fetal liver hematopoiesis was normal (67). This suggests that
SDF-1
, 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-1
-induced
chemotaxis, it is possible that there might be specificity of RGS to
regulate different chemokine receptors. Since RGS18 can interact with
G
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-1, stromal-derived factor-1
;
TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone;
GTP
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688[CrossRef][Medline] [Order article via Infotrieve] |
2. | Dietzel, C., and Kurjan, J. (1987) Mol. Cell. Biol. 7, 4169-4177[Medline] [Order article via Infotrieve] |
3. | Apanovitch, D. M., Slep, K. C., Sigler, P. B., and Dohlman, H. G. (1998) Biochemistry 37, 4815-4822[CrossRef][Medline] [Order article via Infotrieve] |
4. | De Vries, L., Mousli, M., Wurmser, A., and Farquhar, M. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11916-11920[Abstract] |
5. | Koelle, M. R., and Horvitz, H. R. (1996) Cell 84, 115-125[Medline] [Order article via Infotrieve] |
6. | Hunt, T. W., Fields, T. A., Casey, P. J., and Peralta, E. G. (1996) Nature 383, 175-177[CrossRef][Medline] [Order article via Infotrieve] |
7. | Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Hong, J. X.,
Wilson, G. L.,
Fox, C. H.,
and Kehrl, J. H.
(1993)
J. Immunol.
150,
3895-3904 |
9. | Wu, H. K., Heng, H. H., Shi, X. M., Forsdyke, D. R., Tsui, L. C., Mak, T. W., Minden, M. D., and Siderovski, D. P. (1995) Leukemia 9, 1291-1298[Medline] [Order article via Infotrieve] |
10. |
Chen, C. K.,
Wieland, T.,
and Simon, M. I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12885-12889 |
11. |
Chen, C.,
Zhang, B.,
Han, J.,
and Lin, S. C.
(1997)
J. Biol. Chem.
272,
8679-8685 |
12. |
Faurobert, E.,
and Hurley, J. B.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2945-2950 |
13. | Tesmer, J. J. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261[Medline] [Order article via Infotrieve] |
14. |
Lan, K. L.,
Sarvazyan, N. A.,
Taussig, R.,
Mackenzie, R. G.,
DiBello, P. R.,
Dohlman, H. G.,
and Neubig, R. R.
(1998)
J. Biol. Chem.
273,
12794-12797 |
15. |
Natochin, M.,
McEntaffer, R. L.,
and Artemyev, N. O.
(1998)
J. Biol. Chem.
273,
6731-6735 |
16. |
Natochin, M.,
and Artemyev, N. O.
(1998)
J. Biol. Chem.
273,
4300-4303 |
17. | Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452[Medline] [Order article via Infotrieve] |
18. | Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 38, 172-175 |
19. |
Granderath, S.,
Stollewerk, A.,
Greig, S.,
Goodman, C. S.,
O'Kane, C. J.,
and Klambt, C.
(1999)
Development
126,
1781-1791 |
20. |
Heximer, P. S.,
Watson, N.,
Linder, M. E.,
Blimer, K. J.,
and Hepler, J. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14389-14393 |
21. |
Wylie, F.,
Heimann, K.,
Luan Le, T.,
Brown, D.,
Rabnott, G.,
and Stow, J. L.
(1999)
Am. J. Physiol.
276,
C497-C506 |
22. |
Zhang, Y.,
Neo, S. Y.,
Han, J.,
Yaw, L. P.,
and Lin, S.-C.
(1999)
J. Biol. Chem.
274,
2851-2857 |
23. |
Kozasa, T.,
Jiang, X.,
Hart, V. H.,
and Kehrl, J. H.
(1996)
Science
280,
2109-2111 |
24. |
Versele, M.,
de Winde, J. H.,
and Thevelein, J. M.
(1999)
EMBO J.
18,
5577-5591 |
25. | Heximer, P. S., Cristillo, A. D., and Forsdyke, D. R. (1997) DNA Cell Biol. 16, 589-598[Medline] [Order article via Infotrieve] |
26. |
Denecke, B.,
Meyerdierks, A.,
and Bottger, E. C.
(1999)
J. Biol. Chem.
274,
26860-26868 |
27. | Nomoto, S., Adachi, K., Yang, L. X., Hirata, Y., Muraguchi, S., and Kiuchi, K. (1997) Biochem. Biophys. Res. Commun. 241, 281-287[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Beadling, C.,
Druey, K. M.,
Richter, G.,
Kehrl, J. H.,
and Smith, K. A.
(1999)
J. Immunol.
162,
2677-2682 |
29. |
Reif, K.,
and Cyster, J. G.
(2000)
J. Immunol.
164,
4720-4729 |
30. |
Wang, J.,
Ducret, A.,
Tu, Y.,
Kozasa, T.,
Aebersold, R.,
and Ross, E. M.
(1998)
J. Biol. Chem.
273,
26014-26025 |
31. |
Saitoh, O.,
Kubo, Y.,
Odagiri, M.,
Ichikawa, M.,
Yamagata, K.,
and Sekine, T.
(1999)
J. Biol. Chem.
274,
9899-9904 |
32. | Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529[CrossRef][Medline] [Order article via Infotrieve] |
33. | Thomas, E. A., Danielson, P. E., and Sutcliffe, J. G. (1998) J. Neurosci. Res. 52, 118-124[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Cowan, C. W.,
Fariss, R. N.,
Sokl, I.,
Palczewski, K.,
and Wensel, T. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5351-5356 |
35. | Mumby, S. M., and Linder, M. E. (1994) Methods Enzymol. 237, 254-268[Medline] [Order article via Infotrieve] |
36. | Morrison, S. J., and Weissman, I. (1994) Immunity 1, 661-673[Medline] [Order article via Infotrieve] |
37. |
Spangrude, G. J.,
Brooks, D. M.,
and Tumas, D. B.
(1995)
Blood
85,
1006-16 |
38. | Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York |
39. |
Park, I. K.,
and Soderling, T. R.
(1995)
J. Biol. Chem.
270,
30464-30469 |
40. | Kondo, M., Weissman, I. L., and Akashi, K. (1997) Cell 91, 661-672[Medline] [Order article via Infotrieve] |
41. | Akashi, K., Traver, D., Miyamoto, T., and Weissman, I. L. (2000) Nature 404, 193-197[CrossRef][Medline] [Order article via Infotrieve] |
42. | Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Medline] [Order article via Infotrieve] |
43. |
Thompson, J. B.,
Wade, S., M.,
Harrison, J. K.,
Salafranca, M. N.,
and Neubig, R. R.
(1998)
J. Pharmacol. Exp. Ther.
285,
216-222 |
44. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
45. | Kozak, M. (1984) Nucleic Acids Res. 12, 857-872[Abstract] |
46. | Pelletier, J., and Sonenberg, N. (1985) Cell 40, 515-526[Medline] [Order article via Infotrieve] |
47. | Blum, S., Forsdyke, R. E., and Forsdyke, D. R. (1990) DNA Cell Biol. 9, 589-602[Medline] [Order article via Infotrieve] |
48. | Freter, R. R., Irminger, J. C., Porter, J. A., Jones, D. S., and Stiles, C. D. (1992) Mol. Cell. Biol. 12, 5288-5300[Abstract] |
49. |
Hepler, J. R.,
Berman, D. M.,
Gilman, A. G.,
and Kozasa, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
428-432 |
50. | Chen, W., Shields, T. S., Storks, P. J. S., and Cone, R. D. (1995) Anal. Biochem. 226, 349-354[CrossRef][Medline] [Order article via Infotrieve] |
51. | Shaw, G., and Kamen, R. (1986) Cell 46, 659-667[Medline] [Order article via Infotrieve] |
52. |
Grant, S. L.,
Lassègue, B.,
Griendling, K. K.,
Ushio-Fukai, M.,
Lyons, P. R.,
and Alexander, R. W.
(2000)
Mol. Pharmacol.
57,
460-467 |
53. | Chen, C., and Lin, S. C. (1998) FEBS Lett. 422, 359-362[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Zeng, W.,
Xu, X.,
Popov, S.,
Mukhopadhyay, S.,
Chidian, P.,
Swistok, J.,
Danho, W.,
Yagaolff, K. A.,
Fisher, S. L.,
Ross, E. M.,
Muallem, S.,
and Wilkie, T. M.
(1998)
J. Biol. Chem.
273,
34687-34690 |
55. |
Xu, X.,
Zeng, W.,
Popov, S.,
Berman, D. M.,
Davignon, I., Yu, K.,
Yowe, D.,
Offermanns, S.,
Muallem, S.,
and Wilkie, T. M.
(1999)
J. Biol. Chem.
274,
3549-3556 |
56. |
Chatterjee, T. K.,
and Fisher, R. A.
(2000)
J. Biol. Chem.
275,
24013-24021 |
57. |
DeVries, L.,
Elenko, E.,
McCaffery, M.,
Fischer, T.,
Hubler, L.,
McQuistan, T.,
Watson, N.,
and Farquhar, M. G.
(1998)
Mol. Biol. Cell
9,
1123-1134 |
58. |
Srinivasa, S. P.,
Bernstein, L. S.,
Blumer, K. J.,
and Linder, M. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5584-5589 |
59. |
Chens, C.,
Seow, K. T.,
Guo, K.,
Yaw, L. P.,
and Lin, S. C.
(1999)
J. Biol. Chem.
274,
19799-19806 |
60. | Nigg, E. A. (1997) Nature 386, 779-787[CrossRef][Medline] [Order article via Infotrieve] |
61. |
Fischer, T.,
Elenko, E.,
Wan, L.,
Thomas, G.,
and Farquhar, G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4040-4045 |
62. |
Druey, K. M.,
Ugur, O.,
Caron, J. M.,
Backlund, P. S.,
and Jones, T. L. Z.
(1999)
J. Biol. Chem.
274,
18836-18842 |
63. |
Bowman, E. P.,
Campbell, J. J.,
Druey, K. M.,
Scheschonka, A.,
Kehrl, J. H.,
and Butcher, E. C.
(1998)
J. Biol. Chem.
273,
28040-28048 |
64. |
Moratz, C.,
Kang, V. H.,
Druey, K. M.,
Shi, C. S.,
Schschonka, A.,
Murphy, P. M.,
Kozasa, T.,
and Kehrl, J. H.
(2000)
J. Immunol.
164,
1829-1838 |
65. | Bleul, C. C., Farzan, M., Choe, H., Parolin, C., Clark-Lewis, I., Sodroski, I., and Springer, T. A. (1996) Nature 382, 829-833[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Möhle, R.,
Bautz, F.,
Rafii, S.,
Moore, M. A. S.,
Brugger, W.,
and Kanz, L.
(1998)
Blood
91,
4523-4530 |
67. | Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S. I., Kitamura, Y., Yoshida, N., Kikutani, H., and Kishimoto, T. (1996) Nature 382, 635-638[CrossRef][Medline] [Order article via Infotrieve] |