(Received for publication, March 10, 1995; and in revised form, August 24, 1995)
From the
cDNA clones of two novel Ras-related GTP-binding proteins (RagA
and RagB) were isolated from rat and human cDNA libraries. Their
deduced amino acid sequences comprise four of the six known conserved
GTP-binding motifs (PM1, -2, -3, G1), the remaining two (G2, G3) being
strikingly different from those of the Ras family, and an unusually
large C-terminal domain (100 amino acids) presumably unrelated to GTP
binding. RagA and RagB differ by seven conservative amino acid
substitutions (98% identity), and by 33 additional residues at the N
terminus of RagB. In addition, two isoforms of RagB (RagB and RagB
) were found that differed only by an
insertion of 28 codons between the GTP-binding motifs PM2 and PM3,
apparently generated by alternative mRNA splicing. Polymerase chain
reaction amplification with specific primers indicated that both long
and short form of RagB transcripts were present in adrenal gland,
thymus, spleen, and kidney, whereas in brain, only the long form
RagB
was detected. A long splicing variant of RagA was not
detected. Recombinant glutathione S-transferase (GST) fusion
proteins of RagA and RagB
bound large amounts of
radiolabeled GTP
S in a specific and saturable manner. In contrast,
GTP
S binding of GST-RagB
hardly exceeded that of
recombinant GST. GTP
S bound to recombinant RagA, and RagB
was rapidly exchangeable for GTP, whereas no intrinsic GTPase
activity was detected. A multiple sequence alignment indicated that
RagA and RagB cannot be assigned to any of the known subfamilies of
Ras-related GTPases but exhibit a 52% identity with a yeast protein
(Gtr1) presumably involved in phosphate transport and/or cell growth.
It is suggested that RagA and RagB are the mammalian homologues of Gtr1
and that they represent a novel subfamily of Ras-homologous GTP binding
proteins.
Ras-homologous GTPases constitute a large family of signal
transducers that alternate between an activated, GTP-binding, and an
inactivated, GDP-binding state (Hall, 1990; Bourne et al.,
1990; Boguski and McCormick, 1993). These proteins represent cellular
switches that are operated by GTP-exchange factors and factors
stimulating their intrinsic GTPase activity. To date, five subfamilies
of the Ras superfamily are known: Ras, Rho, Rab, Ran, and ARF ()proteins. These subfamilies are not only characterized by
common structural features but also by a similar function, e.g. regulation of growth (Ras) (Egan and Weinberg, 1993), cytoskeleton
organization (Rho) (Aktories et al., 1992), or vesicle
transport (Rab and ARF) (Novick and Brennwald, 1993; Kahn et
al., 1993). All GTPases of the Ras superfamily have in common the
presence of six conserved motifs involved in GTP/GDP binding, three of
them as phosphate/magnesium binding sites (PM1-PM3), and the other
three as guanine nucleotide binding sites (G1-G3) (Valencia et
al., 1991). Therefore, the sequences of the least related GTPases
comprise approximately 20-30% identical amino acids, whereas the
sequence similarity is considerably higher within subfamilies (e.g. >40% identity in the Rab family, >50% in the ARF family)
(Kahn et al., 1992).
The application of the PCR method has
greatly facilitated the identification of novel cDNAs belonging to
larger families of homologous genes, e.g. Ras-homologous
GTPases (Chavrier et al., 1992; Clark et al., 1993).
Constructing degenerate oligonucleotide primers from the conserved PM1
and PM3 domains, we have previously employed this approach to cloning
of novel members of the ARF family (Schürmann et al., 1994; Cavenagh et al., 1994). In addition to
the known ARF and several unknown ARF-like isoforms, we isolated a
single clone of a cDNA fragment that appeared to encode a GTP-binding
protein, but it did not resemble any of the known subfamilies. Here we
report the isolation and characterization of full-length cDNAs encoding
two novel GTP-binding proteins (RagA and RagB) that, on the basis of
structural criteria, constitute a novel subfamily of mammalian
Ras-homologous GTPases. RagB is unique among small GTP-binding proteins
in that its mRNA is expressed as two splicing variants differing by an
insertion of 28 codons within the coding region. Interestingly, this
insertion is located between the PM2 motif and the strand, which are presumably involved in nucleotide or effector
binding.
Figure 1:
Nucleotide and deduced amino acid
sequence of rat RagA. The deduced amino acid sequence is shown in
single letter code above the respective codons. The translation start
was assigned to the first AUG codon of the clone. The sequence of the
PCR product that was isolated from 3T3-L1 cells (mouse) and used for
screening of the cDNA libraries is underlined; a single
nucleotide differing from the rat sequence (321C T) is shown
below. The domains presumably involved in GTP binding are boxed.
A human cDNA of RagA was isolated from a fetal brain cDNA library, and its missing 5`-end (132 bp) was completed with the rapid amplification of cDNA ends procedure. The nucleotide sequence of the open reading frame was 92.7% identical (not shown), and the deduced amino acid sequence was 100% identical with that of the rat sequence. There was no significant homology in the 5`-untranslated region. Thus, it appears unlikely that the open reading frame extends beyond the presumed translation start.
Figure 2: Nucleotide and deduced amino acid sequence of rat RagB. The deduced amino acid sequence is shown in single-letter code above the respective codons. The translation was started at the first AUG codon of the clone following a stop codon. The translation start matching that of RagA is boxed; the first 33 amino acids not present in RagB are highlighted by italics. The insertion of 84 nucleotides (28 amino acids), which is alternatively spliced, is underlined.
Figure 3:
a. Sequence alignment of human and rat
ragA and ragB. The alignment was performed with the CLUSTAL
program. Hyphens represent gaps introduced for optimal
alignment. Amino acids identical in all Rag isoforms are denoted by asterisks; amino acids identical in the human and rat RagB are
designated by periods. Note that the amino acid sequences of
human and rat RagA are 100% identical and that only the long isoforms
of RagB (RagBs) are given. b, sequence alignment of
RagA/B
with the yeast protein Gtr1. Gtr1 (Bun-ya et
al., 1992) was identified as the closest relative of Rag by a data
bank search (Swiss-prot, EMBL), and the deduced amino acid sequences of
Rag and Gtr1 were aligned with the aid of the PALIGN program. Hyphens
represent gaps introduced for optimal alignment (open gap cost, 10;
unit gap cost, 2). Amino acid residues differing in RagB are given on top of the RagA sequence. The putative consensus domains for
phosphate/magnesium binding (PM1-3) and guanine nucleotide
binding (G1-3) are depicted on the top of the alignment.
Identical amino acids are denoted by vertical lines. c, alignment of the GTP-binding domain (amino acids
34-261) of RagB with Ras. The putative GTP-binding motif and the
position and sequence of the 28-amino acid insertion in RagB
is given on top of the alignment. Identical amino acids
are highlighted by vertical lines; conservative substitutions
are depicted by colons.
Figure 5:
PCR amplification of the alternatively
spliced domain in RagA and RagB. The domain including the 84-bp
insertion of RagB was amplified with oligonucleotide primers
specifically matching the sequences of either RagA (A) or RagB (B) as described under ``Materials and Methods.''
Total RNA from rat thymus (Th), adrenal gland (Ad),
spleen (S), brain (B), and kidney (K) was
reverse transcribed and used as template. The products were separated
on agarose gels, transferred on to nylon membranes, and probed with
cDNA fragments of RagA (upper panel) and RagB (lower
panel). Co, plasmid DNA of either RagA or RagB was used as template.
Based on the comparison
with RagA, the translation start of RagB might be assigned to
nucleotide 572. However, there is an alternative start codon in RagB at
nucleotide 473 that extends the open reading frame by 33 codons.
Therefore, a human cDNA clone was isolated and sequenced in order to
compare the presumed translation starts. This clone represented the
short isoform RagB (nucleotide sequence not shown); the
sequence of the 84-bp insertion was obtained by PCR cloning. As
illustrated in Fig. 3a, the open reading frame of the
human sequence is homologous to that of the extended rat sequence
starting at nucleotide 473, with 27 identical amino acids and five
conservative substitutions in the N terminus. Furthermore, the human
sequence lacks the second start codon of the rat sequence (methionine
34; Fig. 3a), which corresponds with the translation
start of RagA. Thus, it appears reasonable to conclude that the N
terminus of RagB is 33 amino acids longer than that of RagA.
Except
for the extended N terminus and the insertion in RagB, the
deduced amino acid sequences of RagA and RagB were nearly identical (7
conservative substitution, 97.8% identity). Because of this high
similarity, the two isoforms were designated A and B (instead of 1 and
2) according to previously established guidelines (Kahn et
al., 1992). The identity of the nucleotide sequences was 83.8%
within the coding region, but only 51% or less in the 3`-untranslated
regions and before the translation start.
The alignment of the first 200 amino
acids of RagA/B with prototypes of the five subfamilies of
Ras-homologous GTPases revealed that the similarity was low (17.5%
identity with Ras, 17.6% with Rho1, 18% with Rab1, 17.5% with Ran, 21%
with ARF1, 17% with ARL1; PALIGN program). However, as is illustrated
by an alignment of RagB with Ras (Fig. 3c), the
GTP-binding domain of Rag is homologous to Ras in order, distance, and
structure of the conserved motifs of GTP binding. Based on this
similarity, Rag can be assigned to the superfamily of Ras-related
GTPases. The alignment also illustrates the position of the insertion
in RagB, which disrupts the PM2 motif and the
strand.
The low structural similarity of Rag with other members of the Ras superfamily is illustrated by a multiple alignment and the resulting tree (Fig. 4). On the basis of this comparison, it is evident that the Rag isoforms cannot be assigned to any of the known subfamilies. Thus, the proteins appear to represent a novel subfamily of the GTP-binding proteins; we designated them Rag in order to emphasize both their unique structure and the remote similarity with the Ras, Rab, and Ran families.
Figure 4: Dendrogram of an alignment of rag with prototypes of the other subfamilies of Ras-homologous GTPases. The dendrogram was constructed with the CLUSTAL program (gap penalty, 5; open gap cost, 10; unit gap cost, 10). Similarities were calculated from the matrix of the pairwise similarity scores. References for the known sequences: Ras, (Ruta et al., 1986); Rho, (Chavrier et al., 1990); Rab1, -2, and -4 (Touchot et al., 1987); Rab6, (Zahraoui et al., 1989); Ran, (Drivas et al., 1990); ARF1, (Bobak et al., 1989); ARL1, 4, (Schürmann et al., 1994); Gtr1, (Bun-ya et al., 1992)
Fig. 6, upper panel, illustrates a Northern
blot of several rat tissues probed with a cDNA fragment of RagA. A
single transcript with an approximate size of 1.8 kb was identified in
most tissues with highest levels in adrenal gland; lower levels of mRNA
were also detected in brain, skeletal muscle, fat cells, liver, spleen,
testis, ovary, thymus, and lung. When a full-length cDNA was used as
the probe (data not shown), additional, presumably nonspecific bands
appeared; the pattern of tissue distribution of the 1.8-kb band was
identical to that shown in Fig. 6. Three different probes
derived from the RagB cDNA were employed in the Northern
blot analysis. Two of these probes (full-length cDNA and
3`-untranslated region) failed to detect any specific signal. With a
cDNA fragment comprising a portion of the coding region, two very
weakly hybridizing transcripts (2.5 and 3.8 kb) were detected against
the background in brain, testis, adrenal gland, and thymus (Fig. 6, lower panel). Thus, the mRNA of RagB appeared
much less abundant than that of RagA, and exhibited a somewhat
different tissue distribution.
Figure 6: Northern blot analysis of the expression of RagA and RagB in different rat tissues. Total RNA from the indicated tissues was hybridized with probes generated from a cDNA fragment of RagA (upper panel; underlined sequence in Fig. 1) or RagB (lower panel; 1.1-kb EcoRI fragment). B, total brain; H, heart; M, skeletal muscle; A, fat cells; Li, liver; K, kidney; S, spleen; T, testes; Ad, adrenal gland; O, ovary; Th, thymus; I, intestine; Lu, lung. As judged from ethidium bromide staining (not shown) the amounts of RNA in each lane were essentially identical with the exception of kidney (partial degradation).
Figure 7:
Binding and dissociation of GTPS
from recombinant ragA and ragB. Panels A and B, GST
and recombinant GST-Rag fusion proteins (GST, hollow circles;
RagA, filled circles; RagB
, filled
squares; RagB
, hollow squares) were prepared
as described, and samples (2 µg of protein) were incubated with
tracer GTP
S. The bound tracer was assayed at the indicated times
after separation by filtration on nitrocellulose membranes. Panels
C and D, samples of the fusion proteins of RagA and
RagB
were loaded with tracer GTP
S for 60 min.
Nucleotide exchange was initiated by the addition of buffer with (filled triangles) or without (hollow triangles) GTP
(final concentration, 1 mM), and bound tracer was assayed at
the indicated time points.
The lower panel of Fig. 7illustrates experiments designed to study the nucleotide
exchange on RagA and RagB. Fusion proteins were loaded with GTPS
for 60 min, and nucleotide exchange was started by the addition of
buffer with or without added GTP. On both fusion proteins, bound tracer
GTP
S decreased rapidly after the addition of unlabeled GTP. Thus,
RagA and RagB
appear to exchange bound GTP
S in the
absence of any catalyzing factor.
GTPase activity of Rag was tested
by loading of the protein with [-
P]GTP.
After an additional incubation period of 0-60 min, the bound
tracer nucleotides were separated by thin-layer chromatography. During
the 60-min incubation period, there was no detectable decrease in the
amount of bound tracer GTP, nor any formation of GDP. Thus, both RagA
and RagB
appear to lack an intrinsic GTPase activity in the
absence of an activating factor.
RagA and RagB are novel GTP-binding proteins with the striking feature that one isotype, RagB, was found as two mRNA species differing by an insertion of 28 codons within the reading frame. Surprisingly, the nucleotide sequence of ragB does not appear to contain the consensus sequences for splicing in the region of the insertion. However, we have repeatedly found both isoforms of RagB by PCR with cDNA from rat as well as from human tissues. The identity of the PCR products (109/193 bp and 996/1080 bp) was confirmed by sequencing; their sequences including portions of the 3`-untranslated region were identical except for the 84-bp insertion. Thus, it appears reasonable to assume that the two isoforms were derived from the same gene by alternative mRNA splicing. The splicing site near the PM2 domain of Rag (threonine 42) is not unexpected, because it corresponds exactly with well conserved exon borders of ras (McGrath et al., 1983) and ARF-2 (Serventi et al., 1993), and is only 12 codons downstream of the border between exon 2 and 3 in Rab isotypes (Wichmann et al., 1989).
Specific and saturable
binding was observed with recombinant GST-fusion proteins of RagA. In
contrast, preparations of GST-RagB bound only minute
amounts of GTP, which hardly exceeded those bound by GST preparations,
but deletion of the 84-bp insert from the inactive RagB
generated a fully active, GTP-binding fusion protein of
RagB
. Thus, it is concluded that the large difference in
GTP binding between RagB
and RagB
is due to the
insertion of 28 amino acids between PM2 and the
strand. Because the insertion might affect the orientation of PM2
(threonine 42), it appears plausible that it affects nucleotide
binding, exchange, and/or hydrolysis. Furthermore, it was recently
shown that the
strand in the Ras homologue Rap is
involved in binding of the effector molecule Raf (Nassar et
al., 1995). However, the possibility cannot be excluded that the
RagB
protein is not properly folded in the bacterial
expression system or that it requires the extended N terminus for
proper folding and GTP binding. Thus, although it is tempting to
speculate that the 28-amino acid insertion may produce an important
functional alteration, the exact nature of this alteration remains to
be determined.
RagA and B differ slightly in the tissue distribution of their mRNA. According to the Northern blot analysis, ragA was expressed in most tissues, but it was most abundant in adrenal gland, ovary, and testis. Rag B appeared most abundant in brain and testis, but mRNA levels were much lower than those of RagA. Like RagA, RagB isoforms could be detected by PCR amplification in all tissues investigated. However, the alternative splicing of ragB was tissue-specific in that brain expressed only its long form. This tissue specificity is an indication that the alternatively spliced insertion in RagB may confer a functional modification of the GTP-binding protein.
Several structural criteria have previously been
established for entry of a novel protein into the Ras superfamily: a
size of 20-29 kDa, the presence of the consensus motifs for GTP
binding, the presence of other sequence motifs characteristic for one
of the subfamilies, and an at least 30% identity with other members of
the family (Valencia et al., 1991; Kahn et al.,
1992). The novel gene product Rag comprises four of the known
GTP-binding motifs (PM1-PM3 and G1). In addition, a G2 domain (HKMD)
appears to be present but differs from that found in all other Ras
homologues (NKXD) by a substitution of asparagine for
histidine. A potential G3 domain, tentatively assigned to the TSI motif
is unrelated to those found in other isotypes. It should be noted that
G3 is the domain least conserved among other subfamilies and that it
appears to be only indirectly involved in GTP binding (Valencia et
al., 1991). The distances between the GTP-binding domains in Rag
were essentially identical to those in other members of the Ras
superfamily. Because of the presence of the GTP-binding domains in
conserved order and distances, it appears reasonable to assume that the
tertiary structure of the main part of Rag providing the interaction
with GTP/GDP (amino acids 1-200) is similar to that of Ras (Pai et al., 1990) and ARF (Amor et al., 1994) and that
its basic function is that of a GTP/GDP dependent switch. However,
there are several striking differences to other members of the
Ras-family. First, Rag is considerably bigger than most other known
mammalian Ras homologues (calculated molecular mass 36.6 kDa (RagA),
40.2 kDa (RagB), or 43.2 kDa (RagB
)). This
extraordinary size is due to an additional domain of approximately 100
amino acids at the C terminus; similar additional domains have been
found in the yeast counterparts of Ras (Powers et al., 1984;
Dhar et al., 1984) and in the ARF-related GTPase ARD (Mishima et al., 1993). Second, other characteristic motifs, e.g. the lipid modification motifs commonly encountered in Ras
homologues except in Ran are absent. Finally, the overall amino acid
identity of Rag to other members of the Ras family is low (highest to
ARF1, 21%) and is restricted to the GTP binding motifs. In spite of
these differences, we suggest that Rag is assigned to the superfamily
of Ras-homologous GTPases. It is obvious, however, that the protein
does not belong to any of the known subfamilies and may thus represent
a sixth subfamily conferring a unique specialized function.
The deduced amino acid sequence of Rag is similar (51% identity) to that of a gene from yeast (GTR1), which has been identified as an open reading frame near the TUB3 locus on chromosome XIII (Bun-ya et al., 1992). The gene is located in close proximity (1.1 kb) of a phosphate transporter (PHO84), and its disruption produced phenotypes resembling those of PHO84 mutants. Thus, it was concluded that the function of the Gtr1 protein is related to that of the phosphate transporter PHO84. Furthermore, disruption of GTR1 resulted in moderately reduced growth at lower temperature. It should be noted, however, that our present findings raise the possibility that an isoform of GTR1 similar to ragB might have in part compensated for the disruption of GTR1. Because of the similarities between rag and GTR1, the possibility has to be considered that rag is the mammalian counterpart of GTR1. Further studies might therefore focus on an involvement of Rag in transport of phosphate or other ions. However, there are some distinct structural differences between Rag and Gtr1. Whereas the identity in the overall GTP-binding domain is 60%, the C-terminal 100 amino acids, which are probably not involved in GTP-binding show only a 35% identity. In contrast, the similarity between other yeast and mammalian GTPases with closely identical related functions is higher, e.g. 75% identity in the case of Ypt/Rab1 (Zahraoui et al., 1989). Thus, Gtr1 and Rag could have identical GTP-binding characteristics, but their functions might have diverged during evolution.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X85183[GenBank], X85184[GenBank], X90529[GenBank], and X90530[GenBank].