(Received for publication, April 14, 1995; and in revised form, June 8, 1995)
From the
We describe the characterization of a novel G protein
subunit, G
. cDNA encoding this subunit was cloned from
the central nervous system of the mollusc Lymnaea stagnalis.
The deduced protein contains all characteristic guanine
nucleotide-binding domains of G
subunits but shares only a limited
degree of overall sequence identity with known subtypes (
30%).
Moreover, two of the nucleotide-binding domains exhibit salient
deviations from corresponding sequences in other G protein
subunits. The A domain, determining kinetic features of the GTPase
cycle, contains a markedly unique amino acid sequence (ILIIGGPGAGK). In
addition, the C domain is also clearly distinct (DVAGQRSL). The
presence of a leucine in this motif, instead of glutamic acid, has
important implications for hypotheses concerning the GTPase mechanism.
In contrast to other G
subtypes, G
has no
appropriate N-terminal residues that could be acylated. It does contain
the strictly conserved arginine residue that serves as a cholera toxin
substrate in G
and G
but lacks a site
for ADP-ribosylation by pertussis toxin. In situ hybridization
experiments indicate that G
-encoding mRNA is expressed
in a limited subpopulation of neurons within the Lymnaea brain. These data suggest that G
defines a
separate class of G proteins with cell type-specific functions.
Heterotrimeric G proteins (G) form a family of
molecular go-betweens that couple stimulus-triggered cell surface
receptors to response-generating effectors within the
cell(1, 2, 3) . In reconstitution systems,
specific G protein subtypes can interact with more than one receptor or
effector subtype(1) . Such a promiscuity might, in fact, allow
the integration or distribution of extracellular signals in
vivo(4) . To address this issue, one needs to study intact
cells since their complex plasma membrane organization furnishes a
specificity of signaling that is significantly higher than that of
reconstituted systems(5) . Moreover, since signaling pathways
may differ considerably in different cells, it is crucial to work with
unambiguously identified cell types.
One system offering such
opportunities is the simple central nervous system of the pond snail, Lymnaea stagnalis, which we use to study the function of G
protein-mediated signaling networks in neuronal information processing.
The Lymnaea brain contains large neurons that can be
reproducibly identified from animal to animal, manipulated in
situ, and cultured and studied in
vitro(6, 7) . Previously, we have cloned a
diverse set of snail G subunits, i.e. G
,
G
, G
, and G
, as well
as a G
subunit (8, 9, 10) . All of these Lymnaea subunits appear to be remarkably similar to their
mammalian counterparts (76-82% amino acid sequence identity).
Taking into consideration such a striking resemblance and the fact that
at least 16 G
subtypes exist in mammals (forming four classes), it
seemed reasonable to assume that Lymnaea expresses more than
merely four G protein
subunits (belonging to three classes).
Here, we describe the cloning of a novel G subtype,
G
, which has hitherto not been reported in any other
species. Although clearly a G protein
subunit, it is unlike
subtypes described to date. Two of its nucleotide-binding domains
deviate markedly from analogous sequences in other G
proteins.
These differences will probably have significant functional
consequences. In situ hybridization shows that the
G
gene is specifically expressed in a minority of
neurons within the Lymnaea central nervous system, suggesting
that this atypical G protein subunit has a cell-specific function. Our
findings indicate that the G protein family is yet more elaborate,
implying that additional members remain to be discovered.
PCR was carried out with one-tenth of an
animal equivalent of cDNA and 10 µg/ml each of GAL5 and either GAL6
or GAL7 (in 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5
mM MgCl, 0.01% (w/v) gelatin, 0.1% Triton X-100,
200 µM of each dNTP, 0.2 units of Super Taq polymerase (HT Biotechnology); 40 cycles of 1 min at 94 °C, 1
min at 40 °C, and 1 min at 72 °C). Under these conditions, the
GAL5-GAL6 combination yielded, among others, a product of an
appropriate size (
240 base pairs), whereas the GAL5-GAL7
combination did not. The GAL5-GAL6 product was subcloned using standard
procedures(13) . Inserts were sequenced with vector-specific
primers using Sequenase 2.0 according to the manufacturer's
instructions (U. S. Biochemical Corp.).
In a collection of 34 clones analyzed, we encountered
multiple known Lymnaea cDNA sequences, coding for G (8 clones), G
(5 clones), or G
(3 clones). The ratio of these clones is roughly similar to their
relative abundance in the Lymnaea brain (data not shown). The
other 18 clones, however, contained an identical and novel sequence.
Upon conceptual translation it appeared to encode (part of) a G protein
subunit, as deduced from the presence of hallmark amino acid
motifs directly flanking the residues encoded by the PCR primers. The
predicted amino acid sequence was clearly different from that of all Lymnaea G
subunits characterized by us
before(8, 10) . Moreover, it did not resemble any
other G
sequence described to date.
Figure 1: Schematic representation and restriction sites of the GAAL1 and GAAL2 cDNAs. Openboxes represent protein-coding sequences; straightlines represent untranslated sequences, and the wavyline indicates retained intron sequences within the GAAL 1 cDNA. B, BamHI; C, ClaI; E, EcoRI; N, NcoI; P, PstI; S, SmaI.
Partial nucleotide sequence
analysis revealed that the GAAL1 insert (3.5 kilobases) corresponds to
an immature RNA transcript, as it starts with (part of) a retained
intron within the domain A-encoding part (Fig.2A). The
3`-intron-exon boundary follows a sequence of highly repetitive nature
and exhibits a sequence (cag/GT) that complies to the consensus for
splice acceptor sequences (17) . The position of the intron is
identical to that of intron 1 in G and G
class genes as well as G
genes of Caenorhabditis
elegans(18, 19, 20) . In general, this
intron tends to be large. Indeed, as pGAAL1 misses the 5`-part of the
intron, the length of the latter should exceed 0.5 kilobases (Fig.2A).
Figure 2:
Characterization of
G-encoding cDNAs. A, partial nucleotide
sequence of the GAAL1 cDNA. The 5`-part of GAAL1, harboring the
downstream part of a putative retained intron and the 3`-intron-exon
boundary, was sequenced. Intron sequences are given in lowercase letters, and exon sequences and the encoded amino
acids are given in upper case letters. B, nucleotide
sequence of the GAAL2 cDNA and conceptual translation. The regions that
were used to devise degenerate PCR primers GAL5 (sense) and GAL6 or
GAL7 (antisense) are overlined. Triangles indicate
the position of the intron in the GAAL1 cDNA, and an asterisk denotes the stop codon. The arginine residue that is conserved in
all G protein
subunits (Arg-212 in G
) and serves
as a cholera toxin substrate in G
and G
has been circled. Numbering of the nucleotide sequence
is relative to the start codon.
The GAAL2 cDNA does contain a complete
open reading frame. Fig.2B shows the nucleotide
sequence and conceptual translation of the 1541-base pair insert. No bona fide polyadenylation signal can be found at the 3`-end of
GAAL2, which probably arose from internal priming by the oligo(dT)
primer used for reverse transcription. Since the GAAL1 cDNA contains
approximately 1.9 kilobases of trailer sequences and open reading frame
analysis of GAAL2 revealed a putative coding region of 1158 base pairs,
the corresponding mRNA could be as long as 3 kilobases provided that
alternative splicing does not occur. The GAAL2 cDNA specifies a
386-amino acid protein with a theoretical molecular weight of 44,666.
The reading frame starts with a methionine codon in an appropriate
sequence context for translation initiation(21) . Within the
deduced protein sequence, domains A, C, G, and I of the putative
guanine nucleotide-pocket can be easily discerned ((16) ; see
also Fig.3). The novel G protein subunit was designated
G
.
Figure 3:
Comparison of G with
other Lymnaea G protein
subunits. Alignment of the amino
acid sequence of the G
protein with that of Lymnaea G
, G
, and
G
(8) as well as Lymnaea G
(10) is shown. Numbers are relative to the G
sequence. Guanine nucleotide-binding motifs A, C, G, and I (16) are indicated by thickoverlining. The
helical domain (24) has been indicated by singleoverlining, whereas linkers L1 and L2, which connect the
helical domain to the rest of the protein, are indicated by dottedoverlining. Asterisks indicate identical amino
acid residues, dots indicate conserved substitutions, and dashes indicate gaps to allow for optimal alignment. Amino
acid residues that appear to be conserved in G protein
subunits
are indicated by trianglesunderneath the compared
sequences.
G is most distinct in the so-called helical
domain (24) , encompassing amino acids 54-218. This
domain is the most heterogenous part of G protein
subunits and
includes a region of structural disparity between G
and G
1, which might be related to their
interaction with different proteins (25) . The G
helical domain appears to bear an insert when compared with other
G
proteins (Fig.3).
Different as G may
be, it does contain 41 out of 63 residues that are conserved between
members of the G
, G
,
G
, and G
classes (not shown). When
taking into account the G
subunits of the slime mold Dictyostelium(26) and the G
protein
of the trematode Schistosoma mansoni(27) , 27 residues
obey a new consensus of 31 invariant amino acids (indicated by triangles in Fig.3). Thus, G
harbors
virtually all residues which, as deduced from their strict
conservation, appear to play pivotal roles in G
function.
The
arginine residue that is conserved in all G protein subunits and
serves as a substrate for ADP-ribosylation by cholera toxin in
G
and G
(1) is also present
in G
. Solely on the basis of the primary structure of
G
, it cannot be concluded, however, whether the
protein can actually be modified by the toxin. Another bacterial toxin,
pertussis toxin, modifies some G
class members through
ADP-ribosylation of a cysteine residue four residues away from the C
terminus(1) . Since G
lacks a cysteine at this
position, the G
protein will subserve pertussis
toxin-insensitive functions.
Figure 4:
In situ hybridization of the Lymnaea central nervous system with a GAAL2-specific probe.
Sections of the Lymnaea brain were incubated with a cRNA probe
prepared from the complete GAAL2 cDNA. A, overview. C, cerebral ganglia; Pl, pleural ganglia; Pa, parietal ganglia; V, visceral ganglion; R, right; L, left. The pedal ganglia are not present
in the section shown. B, detail of a visceral ganglion. Arrows indicate yellow cells within a sectioned nerve (N). C, detail of visceral (V) and right
parietal (RPa) ganglia. Arrows indicate neurons that
appear to highly express G-specific mRNA; arrow
heads indicate cells that do not hybridize with the GAAL2 cRNA
above background.
In this paper, we describe a novel G protein subunit,
G
, which is expressed differentially in the central
nervous system of the pond snail, L. stagnalis. We feel
confident that we have indeed cloned the
subunit of a
heterotrimeric G protein rather than a member of the Ras family of
small guanine nucleotide-binding proteins. First, its putative
nucleotide-binding domains resemble those of G
proteins much more
than those of the Ras family. This also holds true for the sequence
directly following domain C, which constitutes a major conformational
switch domain of G protein
subunits(34) . Second, its
molecular mass (44.7 kDa) is within the range of G
proteins
(39-45 kDa) but differs significantly from that of the Ras family
(21 kDa). Third, G
contains many amino acids that
appear to be invariant in G protein
subunits. Among these is the
strictly conserved arginine residue (35) (Arg-212 in
G
) that serves as a cholera toxin substrate in
G
and G
(1) and appears to
play a key role in stabilizing the transition state during GTP
hydrolysis(25, 36) . G
also contains
the conserved threonine and glycine residues (Thr-217 and Gly-237),
which initiate the conformational changes that occur upon guanine
nucleotide exchange(34) , and the crucial glutamic acid
(Glu-270) that propagates these changes to other parts in the molecule.
The glutamine residue (Gln-238) that is supposed to fulfill a key role
during GTP hydrolysis (25, 36) has also been
preserved. Amino acid sequence comparison indicates that G
resembles other G
subtypes described to date (27-33%
identity) but clearly defines a distinct subclass.
Of the
nucleotide-binding motifs, the A domain is involved in binding
phosphate groups(24, 25) . Although the core sequence
of this domain in G (GGPGAGK) differs considerably
from known G
A domains, it fits the G(G/P/A)PGXGK
consensus for another group of nucleotide-binding proteins, the
adenylate kinases(37) . Nonetheless, G
subunits with A
domain sequences differing from the formerly canonical GAGESGK motif
exhibit deviating nucleotide affinities and kinetics of GTP
hydrolysis(38, 39) . G
may therefore
exhibit a GTPase cycle with greatly distinct features.
The C domain
of G protein subunits coincides by and large with an important
conformational switch element (switch II) (34) and is highly
conserved. Surprisingly, G
violates the consensus
sequence (DVAGQR instead of DVGGQR). Such a deviation is also present
in the G
6 and G
7 subunits of Dictyostelium(26) . The DVGGQR motif appears to
propagate GTP-induced conformational changes to the switch II
region(24, 34) . It has been suggested that the two
glycines of the motif provide the necessary freedom for this
action(24) . Indeed, the latter is hampered by mutation of the
second glycine to alanine(40) . It is, however, highly unlikely
that substitution of alanine for the first glycine has similar effects,
since it has now been found to occur naturally in three independent
G
subunits. Moreover, the strict conservation of alanine in an
analogous position in Ras(-like) proteins, and the fact that mutations
in this position are implicated in cellular
transformation(41) , point to a functional significance of the
presence of such a residue in Lymnaea G
.
Another salient deviation of G is found immediately
downstream of the DVAGQR motif. Hitherto, the glutamic acid in the
sequence SERKKWIH appeared to be strictly conserved among all G
subunits. On the basis of crystal structure studies, it was predicted
that this residue acts as a general base during GTP hydrolysis,
activating a water molecule for a nucleophilic attack on the
-phosphate group(24) . However, G
has
leucine at this position, and it is very difficult to imagine that such
a residue could perform a similar task. Indeed, a mutational study of
the importance of the pertinent glutamic acid refutes the
hypothesis(43) . Moreover, reports on the structure of
GDP
AlF4
-bound G
subunits (mimicking the
transition state) (25, 36) support the notion that it
is the conserved DVXGQR glutamine that plays an important
role(35) .
Overall, the helical domain is the most
heterogenous part of G proteins. It is thought that this domain
constitutes an independent, built-in GTPase-activating
domain(35, 42) . In addition, the helical domain acts
as an intrinsic guanine nucleotide dissociation inhibitor by burying
the guanine nucleotide-binding pocket(24) . Its unique amino
sequence in G
suggests that there are few constraints
on its (primary) structure. Such a sequence diversity might be
exploited for subtype-specific interactions with other proteins.
The
functions of G are beyond speculation as yet but are
likely to involve transmembrane signaling. It will be very interesting
to elucidate which receptors and effectors are coupled by this G
subtype. Biochemical and mutational studies of G
and
G
have implicated the extreme C terminus, the
equivalents of G
residues 347-364, and possibly
the N terminus in receptor coupling(44, 45) . The
corresponding regions of G
do not resemble their
counterparts in any other G
protein. A similar conclusion may be
drawn with respect to the effector-interaction interface. Effectors of
G
(adenylyl cyclase) and G
(cGMP-specific phosphodiesterase) interact with discrete regions
on one face of the
subunits(46, 47, 48) . These regions
encompass their
2-
4,
3-
5, and
4-
6
parts(24, 34) , corresponding to G
residues 245-251, 270-297, and 330-350,
respectively. G
is significantly dissimilar in the
latter two regions. Thus, although our understanding of the G
domains that interact with receptors and effectors is far from
complete, it appears that G
would functionally link
other receptors and effectors than hitherto described G
subtypes.
Although transmembrane signaling is an obvious possibility in terms of
G
function, it cannot be excluded that G
is involved in other processes, like intracellular
trafficking(49, 50, 51) .
Irrespective of
the precise nature of its tasks, G appears to subserve
cell-specific functions. G
-specific mRNA is
specifically expressed in a restricted set of cell types within the Lymnaea brain. Among these are the yellow cells, which are
considered to be involved in osmoregulation(52) . Yet, at this
stage it is hard to relate G
expression in these cells
to specific function(s). In view of the apparent cell type specificity,
it will be instructing to know whether or not G
expression is specifically restricted to the central nervous
system. We also need to assess whether G
is Lymnaea- or mollusc-specific or whether similar proteins exist
in higher organisms like vertebrates. It will be very interesting to
express the G
protein and determine its biochemical
properties and to perturb its function with antisense DNA or antibodies
and assess its cellular role(s).
G is the fifth Lymnaea G protein
subunit identified by our group. Even
the unicellular slime mold, Dictyostelium discoideum, has a
G
family consisting of at least 8 members(26) . In Drosophila, atypical G protein
subunits like concertina
and G
have been discovered (22, 23) .
Since the PCR might very well miss certain G
subtypes, especially
when these subunits harbor a different sequence in the parts that were
used to develop degenerate primers, we surmise that a range of Lymnaea G
proteins might have escaped detection. The
identification of G
, a G
subunit with clearly
distinct properties, indicates that the ever increasing array of G
subtypes has not yet met its limit.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) Z47551[GenBank].
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