(Received for publication, March 14, 1997, and in revised form, May 28, 1997)
From the Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084
We report the cDNA cloning and characterization of a novel GTP-binding protein, termed Rem (for Rad and Gem-related), that was identified as a product of polymerase chain reaction amplification using oligonucleotide primers derived from conserved regions of the Rad, Gem, and Kir Ras subfamily. Alignment of the full-length open reading frame of mouse Rem revealed the encoded protein to be 47% identical to the Rad, Gem, and Kir proteins. The distinct structural features of the Rad, Gem, and Kir subfamily are maintained including a series of nonconservative amino acid substitutions at positions important for GTPase activity and a unique sequence motif thought to direct membrane association. Recombinant Rem binds GTP in a specific and saturable manner. Ribonuclease protection analysis found Rem to be expressed at comparatively high levels in cardiac muscle and at moderate levels in lung, skeletal muscle, and kidney. The administration of lipopolysaccharide to mice, a potent activator of the inflammatory and immune systems, results in the general repression of Rem mRNA levels in a dose- and time-dependent manner. Thus, Rem is the first Ras-related gene whose mRNA levels have been shown to be regulated by repression.
The Ras family of low molecular weight GTP-binding proteins has been implicated in a wide range of cellular processes, including cell growth and differentiation, intracellular vesicular trafficking, nucleocytoplasmic transport, and cytoskeletonal reorganization. To date, six subfamilies have been identified: Ras, Rho, Rab, Ran, ARF, and a newly described family composed of the Rad, Gem, and Kir proteins (1, 2). These subfamilies are defined largely by primary sequence relationships but also by their regulation of common cellular functions. All GTPases of the Ras superfamily contain five well conserved amino acid motifs involved in guanine nucleotide binding and hydrolysis (1, 3). These primary sequence motifs have been evolutionarily conserved and define a conserved structure whose importance has been confirmed through extensive mutational analysis (4). Therefore, the sequence of all GTPases share approximately 20-30% amino acid identity, whereas the sequence identity is considerably higher within subfamilies (5). In addition, most family members share conserved COOH-terminal cysteine rich motifs needed for covalent modification by isoprenoid lipids (6). Prenylation is the initial step in the attachment of these proteins to the cytoplasmic leaflets of a variety of cellular organelles (7) and has been shown to be required for normal membrane localization and biological activity (8).
The Ras-related GTPases are thought to act as binary switching molecules, alternating between an active GTP-bound and an inactive GDP-bound structural state (4). They respond to external signals by exchanging GTP for constitutively bound GDP, thereby triggering intracellular signaling cascades. The signal is terminated when the protein hydrolyzes its bound GTP to GDP in a reaction that is stimulated by guanosine triphosphatase (GTPase)-activating proteins (GAPs)1 (9). Progression through this GTPase cycle is regulated by additional regulatory proteins, including factors that stimulate guanine nucleotide exchange. These regulatory proteins may themselves possess intrinsic effector activity (9) and associate with specific GTPases largely through interactions with their G2 effector domains. A variety of G2 regions allow the Ras-related GTPases to interact with a wide range of cellular effector molecules (4). Thus, small GTPases are extremely versatile and found to regulate an array of cellular processes (2, 9).
The Rad, Gem, and Kir proteins are the first members of a new class of
Ras-like GTPases (10-12). Rad shares 61% identity at the amino acid
level with Gem (11) and Kir (12), whereas the coding sequences of the
Gem and Kir genes are 98% identical, differing significantly only in
the 5-untranslated sequences (12). The RGK proteins share structural
features that are distinct from other Ras-related proteins. These
include several nonconservative amino acid substitutions within regions
known to be involved in guanine nucleotide binding and hydrolysis
including unique G2 effector and G3 domains, extended NH2
and COOH termini and a conserved COOH-terminal motif thought to mediate
membrane association but lacking classical CAAX motifs
needed to direct protein isoprenylation. In addition, the members of
this Ras subfamily are subject to transcriptional regulation. Rad was
found to be overexpressed in muscle of type II diabetes patients (10),
and Gem expression is induced in mitogen-stimulated T-cells (11),
whereas Kir expression is induced by oncogenic kinases (12). Although
the cellular function of these proteins remains to be established, Rad
has been shown to associate with skeletal muscle
-tropomyosin and the cytoskeleton of muscle cells (13) and to inhibit insulin-stimulated glucose uptake in a variety of cultured cell lines (14). This suggests
a role for Rad in skeletal muscle function and cytoskeletal organization. The deregulated expression of Gem prevents proliferation of normal and transformed 3T3 cells, suggesting that Gem is involved in
regulating signaling pathways that influence cell growth (11). Finally,
the cellular levels of Kir are dramatically increased in pre-B cells
transformed by a select set of abl tyrosine kinase oncogenes
(12). The correlation between Kir expression and the tumorigenic and
metastatic potential of BCL/ABL and v-ABL transformed cells suggests
that Kir may participate in the processes of invasion or metastasis.
When expressed in yeast, Kir leads to the formation of pseudohyphae, a
developmental transition normally induced by nitrogen starvation (15).
Genetic analysis suggests that Kir acts upstream of the
STE20 kinase and results in the activation of a
mitogen-activated protein kinase cascade (15). These results are
consistent with a model in which Kir, and perhaps other members of the
RGK family, may regulate cellular signaling cascades by controlling the
activity of mitogen-activated protein kinases that have yet to be
determined.
In this report, we describe the initial characterization of a novel Ras-related GTP-binding protein, Rem, first identified using a degenerate PCR strategy. On the basis of structural criteria, Rem is a new member of the Rad, Gem, and Kir subfamily of Ras-related proteins. Rem mRNA is expressed predominantly in skeletal and cardiac muscle, lung, and kidney, and the bacterially expressed protein is shown to bind GTP in a specific and saturable manner. Because other members of the RGK family are transcriptionally regulated, we examined whether a similar method of regulation controlled the expression of Rem. Surprisingly, we find that in mice treated with lipopolysaccharide, a potent activator of cells of the immune and inflammatory systems, the levels of Rem mRNA are repressed in a dose- and time-dependent manner. Thus, Rem is the first Ras-related GTP-binding protein whose mRNA levels are regulated by repression.
Standard molecular biology techniques were used (16). cDNA clones were subcloned to plasmid pBluescript II vectors (Stratagene) and sequenced by the dideoxy chain termination method (16) using the M13 universal primer or specific internal primers. Nick-translated probes were synthesized using a labeling kit (Life Technologies, Inc.).
Reverse Transcriptase Polymerase Chain Reaction Identification and Cloning of RemThe strategy used to generate the PCR-derived
fragment of Rem from conserved amino acid sequences found within the
Rad and Gem/Kir small GTP-binding proteins is outlined in Fig. 1. First strand cDNA from mouse testis and brain total RNA was a kind gift from Dr. Kevin Sarge of this department. The first strand cDNA was
used for PCR with the degenerate PCR primers
AT(A/C/T)AT(A/C/T)(C/T)TNGTNGGNAA(C/T)AA and GT(T/C)TC(A/T/G)
AT(A/G)AA(C/T)TT(G/A)CA(A/G)TC(AG)AA. The resultant 105-base PCR
product was blunt ended with T4 polymerase, and 5-phosphate was added
with polynucleotide kinase (17). The PCR product was then subcloned
into the EcoRV site of pBluescript KS+
(Stratagene, La Jolla, CA). Sixty individual clones were sequenced using a SequenaseTM kit (U. S. Biochemical Corp.). Two PCR products, when sequenced, were found to contain a unique cDNA fragment that was used to design a Rem-specific oligonucleotide (Rem-3; see Fig. 1).
The Rem-3 oligonucleotide was then used to screen an oligo(dT)-primed
Uni-ZAP XR adult mouse testis cDNA library (the kind gift of Dr.
Debra J. Wolgemuth, Center for Reproductive Sciences, Columbia
University College of Physicians and Surgeons, New York, NY).
Approximately 30,000 plaques were transferred in duplicate to filters
that were then probed with 1 × 106cpm/ml of the end
labeled oligonucleotide, TTCTACCGAGACTTCCCGGC. Filters were hybridized
at 42 °C in hybridization solution containing 6 × SSC, 1 × Denhardt's solution, 100 µg/ml yeast tRNA, and 0.05% sodium
pyrophosphate (18). The filters were washed for 2 h with 6 × SSC and 0.05% sodium pyrophosphate at room temperature with three
changes of wash buffer. Three positives were identified, and the
largest (Rem
10-1, 1.1-kilobase insert) was plaque purified, rescued
using phage mediated in vivo excision (according to
manufacturer's protocol, Stratagene), and sequenced. To obtain a
larger cDNA clone, the mouse testis library was rescreened with
1 × 106 cpm/ml of the nick-translated
EcoRI/XhoI fragment of pRem 10-1. Duplicate
filters were hybridized at 42 °C in a 5 × SSPE solution (18)
containing 50% formamide. The filters were washed in 0.2 × SSC
and 0.1% SDS for 1 h at 60 °C with three changes of wash buffer. Of five positives, the clone Rem
6-2 was plaque purified and
rescued as a pBS plasmid because of its ability to hybridize to
5
-directed Rem-specific oligonucleotides.
To obtain a full-length cDNA clone, a bacteriophage cDNA
library was constructed from mouse kidney. Poly(A)+ RNA was
isolated using the Straight A'sTM mRNA isolation system (Novagen,
Madison, WI) and used to construct a random primed cDNA library
(Directional RH Random Primer cDNA Library Construction System,
Novagen). Approximately 30,000 plaques were transferred in duplicate to
filters that were probed under high stringency with 1 × 106 cpm/ml of the nick-translated
EcoRI/XhoI fragment of pRem 6-2. Of the four
positive clones identified, pRem
5, which contained the largest and
most 5
-extended insert (0.9 kilobase), was rescued and characterized
by DNA sequencing. To construct a plasmid that contained the full Rem
coding region, nucleotides 1-502 of pRem 5 were ligated to nucleotides
56-1050 of pRem 6-2 using a unique NheI site. The resulting
plasmid, pRemWT, was characterized by restriction mapping and
sequencing.
Mouse total RNA was isolated using
a STAT-60 kit (Tel-Test B, Friendswood, TX) according to the
manufacturer's protocol. The PstI fragment of pRem 6-2 was
subcloned into pBluescript KS+ (Stratagene, La Jolla, CA)
to create the plasmid pRem PstI. Plasmids containing an
89-bp fragment of the ribosomal protein L32 (19) or a 303-bp fragment
of mouse TNF- cDNA subcloned in the vector pGEM-4 were a gift of
Dr. Daniel Noonan of this department. Antisense radiolabeled riboprobes
were prepared using linearized templates and a MaxiscriptTM (Ambion,
Austin, TX) kit according to the manufacturer's protocol. RNase
protection assays were performed according to the method of Hobbs (19)
with minor modifications. Briefly 20 µg of total RNA was dissolved in
8 µl of hybridization buffer (80% formamide, 0.3 M NaCl,
1 mM EDTA, 40 mM Pipes, pH 6.7). For LPS
experiments, two protection assays were performed for each tissue. The
first contained a mixture of Rem and L32 riboprobes, whereas the second
contained the TNF-
and L32 riboprobe mix. 1 µl (500,000 cpm) of
each probe (in hybridization buffer) was added to each sample. Samples
were overlaid with mineral oil, incubated for 1 min at 90 °C, and
subsequently incubated for 12-16 h at 56 °C. Single-stranded RNA
was digested as described by Hobbs (19). The reaction was stopped by
the addition of an equivalent volume (110 µl) of 4 M
guanidine thiocyanate, 0.5% sodium N-lauryl-sarcosine, 25 mM sodium citrate, pH 7.0, 0.1 M
-mercaptethanol. Yeast tRNA (25 µg) was added as carrier, and
total RNA was precipitated by addition of isopropanol (225 µl)
followed by centrifugation (20). The pellet was dissolved in 10 µl of
loading buffer (80% formamide, 10 mM EDTA, 1 mg/ml
bromphenol blue, 1 mg/ml xylene cyanol) and electrophoresed in a 5%
acrylamide/8 M urea sequencing gel. The gel was dried and
exposed to X-OMAT AR film (Eastman Kodak Co.) for the indicated time.
The gel was quantitated using a Molecular Dynamics PhosphorImager SF
(model 455A). Simultaneous measurement of the rpL32 transcripts, which
encode the L32 ribosomal protein (19), served as an internal control
for housekeeping gene levels.
The tissues of adult female C57BL6/C3H mice were kindly provided by Dr. Mark Kindy of this department. Lipopolysaccharide stimulation was achieved by intraperitoneal injection (10 µg/animal) to paired animals for each time point. Lipopolysaccharide was from Escherichia coli 0111:B4 (DIFCO). Control animals were not injected. At the indicated times the animals were treated with metafane and euthanized by cervical dislocation, and tissues were harvested immediately and quick frozen on dry ice. RNA was isolated and used for ribonuclease protection analysis as described above. These studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Recombinant Protein ProductionRecombinant Rem was
expressed as a glutathione S-transferase (GST)-fusion
protein. A Rem PCR product containing a 5 BamHI restriction
site was generated using oligonucleotides 5
-CGCGGATCCATGACTCTTAACACGCA and 3
-TTCTACCGAGACTTCCCGGC, sequenced to verify the cDNA, and subcloned to BamHI/NheI-digested pRemWT to create
pRem express. The BamHI/XhoI fragment of pRem
express was subsequently cloned in-frame to pGEX-KG (21) to create pRem
GEX. GST-Rem was produced in BL21DE3 cells upon
isopropyl-
-D-thiogalactopyranoside addition, purified on
glutathione agarose beads (Sigma), and removed from GST by thrombin
cleavage as described (22). Thrombin cleavage was deemed to be >95%
efficient as judged by Coomassie Blue staining after SDS-polyacrylamide
gel electrophoresis analysis and Western blotting with anti-GST
antibodies. A bacteria protein co-purified with Rem and was resistant
to extensive high salt washes of the glutathione-agarose beads (see
Fig. 4C). Thrombin was removed by incubation with
benzamidene-Sepharose 6B (Pharmacia Biotech Inc.) for 1 h at
4 °C with mixing. The protein was then dialyzed against 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
dithiothreitol, and 5% glycerol and stored in multiple aliquots at
70 °C.
GTP Binding Assays
GTP binding to Rem was determined with
the rapid filtration assay (23). Rem (1 µg) or bovine serum albumin
(1 µg) was incubated in binding buffer (20 mM Tris, pH
7.5, 50 mM NaCl, 0.1% Triton X-100, 1 mM
dithiothreitol, 40 µg/ml bovine serum albumin, and 1 µM
GTPS (0.45 µCi/sample)) with the indicated concentration of
Mg2+ or EDTA at 22 °C. At the indicated times, aliquots
of 100 µl were withdrawn in duplicate, and the reaction was stopped
by addition of 400 µl of ice-cold wash buffer (20 mM
Tris, pH 7.5, 50 mM NaCl, and the indicated
Mg2+ concentration) and immediately filtered through BA 85 nitrocellulose filters (Schleicher & Schuell) followed by washing with
12 ml of ice-cold wash buffer. The radioactivity remaining on the
filters was determined by scintillation counting. Nonspecific
background was determined by performing the binding assay in the
absence of added protein. To assess the specificity of GTP binding,
cold ribonucleotides were added to a final concentration of 20 µM, and the binding reaction was allowed to proceed for
1 h prior to rapid filtration analysis.
We sought a
comprehensive method to identify additional members of the RGK family
of low molecular weight GTP-binding proteins. A PCR-based strategy
using degenerate oligonucleotide primers designed to exploit amino acid
differences between the guanine nucleotide-binding domains of the RGK
family and other Ras-related GTP-binding proteins was used to isolate
novel RGK-related cDNA fragments from mouse testis cDNA (Fig.
1 and see "Experimental Procedures").
The 5 oligonucleotide was designed to partially overlap the G4 guanine
nucleotide-binding site motif IILVGNK, whereas the 3
oligonucleotide
recognized a portion of the G5 guanine nucleotide-binding region and a
flanking region of amino acids highly conserved within the RGK family
FDCKFIET. PCR generated a pool of cDNAs of the approximate
predicted size (105 bp), which were subcloned and sequenced. Sequencing
of 60 individual clones revealed that approximately 90% of the
products were identical to the previously characterized Rad, Gem, or
Kir genes (10-12). Two of the remaining clones encoded a previously
unknown sequence that was homologous to but clearly different from RGK
genes (Fig. 1). To emphasize this relationship we designated this clone
Rem, for Rad and Gem-related.
A Rem-specific oligonucleotide probe was designed (Rem-3) and used to
screen a mouse testis cDNA library, from which several clones were
isolated (Fig. 1; see "Experimental Procedures"). The largest of
these cDNA clones was Rem 6-2, which extended from a
polyadenylation tract to nucleotide 441 (nucleotide positions refer to
the final sequence of the cDNA that can be retrieved from
GenBankTM U91601). Preliminary ribonuclease protection assays using a
portion of the
Rem 6-2 cDNA indicated that Rem was abundantly
expressed in kidney (data not shown). Based on the high levels of
expression in kidney, a random-primed mouse kidney cDNA library was
constructed and screened with a probe generated from the
Rem 6-2 clone to obtain the 5
end of the Rem cDNA (see "Experimental
Procedures"). Four additional cDNA clones were isolated, one of
which (
Rem 5) extended from nucleotides 1 to 881. These overlapping
cDNA clones (
Rem 6-2 and
Rem 5) were spliced together to form
a full-length cDNA. The 1.5-kilobase cDNA sequence included a
portion of the 5
-untranslated region, an 891-nucleotide (219-1109)
open reading frame with a putative initiator methionine in a region
that matched the Kozak sequence motif (24), and a large 3
-untranslated
region followed by a polyadenylate tail. No other methionines were
observed 5
to the putative start codon in any other reading frame. In
addition, 5
to the Kozak sequence and 3
to the in-frame stop codon,
stop codons were observed in all three reading frames. Analysis of the
open reading frame revealed significant identity at the nucleotide level with members of the Ras superfamily.
The deduced amino acid sequence of Rem is
shown in Fig. 2. This cDNA predicts a
protein of 297 amino acids with a calculated molecular size of 32,893 Da. A data base search was performed to determine the degree of
homology of Rem with the RGK family and to identify additional
G-proteins with structural similarities. The Rem protein contains a
core sequence (amino acids 84-246) that is highly related to members
of the Ras superfamily of small GTP-binding proteins. The highest
degree of similarity was with mouse and human Gem, Kir, and Rad
(46.7-47.2% sequence identity), but there was also a high degree of
homology with additional Ras-like GTPases, including 22.8, 25.9, and
22.3% identity to dictyostelium RasA (25), human Rap-2A (26), and
Rap-2B (27). To assure ourselves that Rem was a new member of the RGK
Ras subfamily, we performed a general comparative protein analysis
among the different Ras-like subfamilies (data not shown). On the basis of this comparison, it is evident that Rem is a novel member of the RGK
Ras-related subfamily. Fig. 2 depicts the alignment of Rem with a
select subset of these proteins. The greatest similarity exists in
regions that correspond to the guanine nucleotide-binding domains
conserved in all Ras family members. The Rem protein exhibits all five
of the domains (G1-G5) that have been shown to take part in both
guanine nucleotide binding and the catalytic functions of the Ras
protein superfamily (4). Although both the NH2- and
COOH-terminal extensions past the Ras core region are divergent, the
COOH-terminal 10 amino acids of Rem are highly conserved in Rad, Gem,
and Kir proteins (Fig. 2). This region does not contain a typical
CAAX, XXCC, or CXC prenylation site
(where A is an aliphatic amino acid and X is any amino acid)
present in almost all Ras family members, although it does contain a
conserved cysteine residue at position 7 from the COOH terminus. This
conserved COOH-terminal motif may therefore represent a novel lipid
modification site or direct the association of these proteins with
membranes by interaction with an anchoring protein.
Tissue Distribution of Rem mRNA
Ribonuclease protection
analysis revealed that Rem was expressed at detectable levels in every
mouse organ examined (Fig. 3). The
highest basal levels were detected in cardiac and skeletal muscle with
slightly lower levels of Rem mRNA in lung and kidney. Low levels of
mRNA were identified in spleen and brain with barely detectable
levels in several additional tissues. The significance of the abundance
and distribution of Rem message in these tissues is unclear. The tissue
distribution of Rem contrasts with that of both Rad and Gem; Gem
mRNA is most abundant in kidney, lung, and spleen, whereas Rad is
expressed in cardiac and skeletal muscle and lung (10, 11).
Rem Is a GTP-binding Protein
The close homology between Rem
and Ras in the regions associated with the binding of GTP suggested
that Rem was a GTP-binding protein. To determine whether the Rem
cDNA coded for a protein possessing GTP-binding activity,
recombinant Rem was expressed and affinity purified (see
"Experimental Procedures"). The recombinant protein migrated with
an apparent molecular mass of 39 kDa, which is in close agreement with
the calculated molecular mass of the Rem cDNA of 33 kDa. Binding of
GTP was then assayed with [35S]GTPS, and bound GTP was
separated by rapid filtration on nitrocellulose. As seen in Fig.
4A, Rem binds GTP
S rapidly
and in a Mg2+-dependent manner upon incubation
under standard nucleotide exchange conditions. Nucleotide binding was
saturable and completely blocked by excess (20-fold) unlabeled GTP and
GTP
S, whereas pyrimidine nucleotides do not compete to any extent
(Fig. 4B). ATP also showed slight inhibition of GTP binding
to Rem at high concentrations. These GTP-binding properties closely
resemble those of recombinant Rad and indicate that Rem specifically
associates with guanine nucleotide (28).
The mRNA
levels of both Gem and Kir are transcriptionally induced, respectively,
in mitogen-stimulated T-cells and abl tyrosine kinase
transformed B-cells (11, 12). To determine whether Rem was regulated in
a similar fashion, we characterized the level of Rem mRNA expressed
in a variety of mouse tissues after exposure to the potent immune and
inflammatory system stimulant, LPS (29, 30). Surprisingly,
intraperitoneal injection of LPS caused a potent and transient decrease
in the levels of Rem mRNA in cardiac muscle, skeletal muscle,
kidney, spleen, and thymus (data not shown). To further characterize
the lipopolysaccharide-stimulated repression of Rem mRNA levels in
mouse tissues, its time course was determined in cardiac muscle (see
"Experimental Procedures"). As is illustrated in Fig.
5, Rem mRNA was repressed 8.5-fold
below basal levels upon LPS treatment (10 µg/animal), with repression first detected within 2 h and maximal repression achieved within 4 h of LPS treatment (Fig. 5B). Rem repression was
sustained to 8 h and returned to basal levels by 20 h. The
degree of repression and its duration were both dose- and mouse
strain-dependent (data not shown). During this same time
course, the inflammation marker TNF-, a cytokine that is known to be
markedly stimulated by LPS treatment (30), was analyzed (Fig.
5A). The time course of TNF-
expression somewhat preceded
that of Rem with maximal expression at 2 h. In addition, TNF-
stimulation was transient with return to basal levels of mRNA by
8 h after LPS stimulation. Because LPS is known to stimulate cells
of the monocytic lineage as well as lymphocytes, we examined the level
of Rem mRNA in primary T cells, B cells, and macrophages as well as
in the macrophage cell lines RAW 264.7 and P388D1 (data not shown).
However, mRNA was not detected in these cells, suggesting that Rem
is not expressed in cells of the immune system.
We report here the identification of a 33-kDa GTP-binding protein, Rem, a novel member of the Ras family of GTP-binding proteins whose mRNA levels are rapidly and markedly decreased by LPS in a variety of mouse tissues. The predicted amino acid sequence of Rem is most highly homologous to the Rad, Gem, and Kir gene products, and by virtue of this similarity we include Rem as the newest member of the RGK subfamily of Ras-related small G-proteins. Rem is an interesting gene both by virtue of its rapid repression by LPS and because the amino acid sequence indicates several novel features that distinguish it from the majority of Ras-related proteins.
Analysis of the Rem cDNA sequence indicates that it encodes a protein of 297 amino acids with a deduced molecular mass of 32,893 Da. The regions of highest homology between Rem and Ras family members correspond to the five regions of Ras that constitute the core consensus involved in GTP binding (G1-G5; Fig. 2) (4), whereas the flanking NH2- and COOH-terminal sequences are unrelated to Ras. These extended regions account for the larger molecular weight of Rem when compared with the majority of members of the Ras family. The guanine nucleotide consensus sequences GX4GK(T/S) (G1 motif), NKXD (G4 motif), and EXSA (G5 motif) are present and well conserved in Rem (Fig. 2). However, Rem, along with other proteins in the RGK subfamily, contains a number of divergent amino acids at highly conserved positions that are known to be critical for normal GTP hydrolysis. Positions 89, 135, and 137 in Rem, which correspond to amino acids 12, 59, and 61 in Ras, are substituted relative to most of the previously characterized small GTPases. These three positions when changed to a variety of amino acids confer oncogenicity by rendering Ras defective for GTP hydrolysis or insensitive to GAP stimulation, thereby causing Ras proteins to be maintained in a GTP-bound active state (3, 31, 32). However, whereas mutagenesis studies have suggested that amino acid substitutions within this conserved catalytic region impair GTPase activity, it appears that some deviation from the consensus structure can be tolerated. Indeed, recent studies have found that Rad exhibits detectable GTP hydrolysis that is stimulated by a cellular GAP activity (28). Studies to characterize the biochemical properties of recombinant Rem are ongoing.
Rem contains an unusual G3 (DXXG) region, which participates
in the binding of both Mg2+ and GTP in Ras GTPases. The
sequence, DTWE (residues 133-136), contains the invariant aspartic
acid residue but lacks the highly conserved glycine thought to play a
critical role in the conformational change Ras-related GTPases undergo
in response to differences in the bound guanosine nucleotide (GTP or
GDP) (3, 4). These structural changes suggest that Rem may use a
different mechanism to bind and hydrolyze the -phosphate of GTP.
Nevertheless, we are able to demonstrate that Rem binds GTP in a
specific and saturable manner. Structural studies will be needed to
determine whether these amino acid substitutions result in alterations
to the active site.
The sequence of the G2 domain in Rem is unique and diverges significantly from the same domain in Rad, Gem, Kir, and other Ras-related proteins. This region is termed the effector domain and mutational analysis has shown it to be responsible for GAP binding by Ras-related proteins (9). This difference is significant because the G2 domains of GTPases play a central role in defining effector protein interactions (4) and are often perfectly conserved within a specific subfamily but not among different subfamilies. Thus, the Rem, Rad, Gem, and Kir proteins constitute the first Ras subfamily to lack a conserved effector domain and therefore may not be regulated by known GAP-like enzymes.
The COOH terminus of Rem lacks one of the typical CAAX, XXCC, or CXC prenylation motifs found on most Ras-like proteins and required for the attachment of Ras to the plasma membrane (6, 7). Although Rem, Rad, Gem, and Kir do not contain classic prenylation motifs, they all contain a highly conserved COOH-terminal motif. This region contains a conserved cysteine residue seven amino acids from the COOH terminus that may serve as a site for lipid modification. The COOH-terminal domain also contains an extended and highly conserved polybasic region. Because polybasic domains have been implicated in providing the binding energy necessary for membrane association of Ras-related proteins (34), it seems likely that this region may also be involved in membrane association. Indeed, the conserved COOH-terminal sequence of Gem has been reported to be required for plasma membrane localization (11). In addition, when the Kir protein is expressed in yeast, an intact COOH terminus is essential to induce pseudohyphal formation (15). However, the mechanism by which the COOH terminus could direct membrane localization is unclear. Whether the process relies on direct membrane binding or interaction with an anchoring protein remains to be determined.
The most surprising finding of the current study was the rapid and transient suppression of Rem mRNA levels in a variety of mouse tissues upon LPS treatment. In cardiac muscle, decreases in Rem mRNA are detected within 2 h of LPS injection, reach their lowest levels after 4 h, and return to basal levels by 20 h. Although several previously characterized Ras-like genes (35-37) including the Rad, Gem, and Kir genes are known to be regulated by transcriptional induction, to our knowledge, Rem is the first Ras-related GTP-binding protein to be shown to undergo suppression of mRNA levels in response to stimulation. It is not known whether this regulation is at the transcriptional level or due to differences in mRNA stability. We are presently searching for a cell line in which to examine these issues.
LPS treatment is known to act primarily by stimulating monocytes,
neutrophils, and endothelial cells (38). This results in the rapid
production of a variety of cytokines, primarily the production of
TNF- and interleukin-1 by activated macrophages (29, 30). However,
additional cytokines as well as a variety of diverse and potent
cellular regulators including arachidonic acid derivatives and nitric
oxide are also triggered by LPS treatment (38). The modulation of Rem
mRNA by LPS and the previous finding that Gem expression was
regulated in immune cell populations by a variety of mitogenic stimuli
led to our analysis of Rem expression in immune cells. However,
ribonuclease protection studies failed to detect Rem mRNA in immune
cell populations. The wide tissue distribution and relative expression
levels of Rem does however correlate with tissues that contain a high
proportion of vascular cells. Because LPS is known to regulate vascular
endothelial cells, we are currently examining whether Rem is expressed
in this cell type.
The biochemical function of Rem proteins remains to be defined and will be the focus of future work. The unique structure of Rem, its enrichment in tissues with a large number of vascular endothelial cells, its ability to specifically bind GTP, and its regulation by LPS suggest that it may control cellular pathways in endothelial cells. Although a large number of GTP-binding proteins have been identified, they have diverse cellular functions, and the presence of this biochemical activity in itself does not define a cellular role for Rem. However, the extensive information gained from the biochemical characterization of Ras and other GTP-binding proteins may facilitate studies of Rem. Indeed, recent studies with Kir expression in yeast suggest that members of the RGK Ras subfamily act as molecular switches controlling specific aspects of cell physiology through a mitogen-activated protein kinase cascade. Additional studies using dominant-negative and constitutively active forms of Rem may help to answer whether it regulates such a putative pathway.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U91601.
We thank M. Kindy for providing the
LPS-treated mouse tissues used in these studies; D. Noonan for the
TNF- and rpL32 riboprobe plasmids, macrophage cell lines, and advice
on ribonuclease protection assays; Dr. Kevin Sarge for mouse testis
cDNA; Dr. Debra J. Wolgemuth (Columbia University College of
Physicians and Surgeons) for the mouse testis cDNA library; Drs. C. Snow and D. Cohen for isolated T- and B-cells and mouse macrophages and
helpful advice; and Dr. S. Whiteheart and members of the laboratory for
helpful comments on the manuscript.