From the University of the Western Cape, Department of Biotechnology, Bellville, Private Bag X17, 7535, South Africa
Received for publication, October 28, 2002, and in revised form, December 1, 2002
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ABSTRACT |
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Guanylyl cyclases (GCs) catalyze the
formation of the second messenger guanosine 3',5'-cyclic monophosphate
(cGMP) from guanosine 5'-triphosphate (GTP). While many cGMP-mediated
processes in plants have been reported, no plant molecule with GC
activity has been identified. When the Arabidopsis thaliana
genome is queried with GC sequences from cyanobacteria, lower and
higher eukaryotes no unassigned proteins with significant similarity
are found. However, a motif search of the A. thaliana
genome based on conserved and functionally assigned amino acids in the
catalytic center of annotated GCs returns one candidate that also
contains the adjacent glycine-rich domain typical for GCs. In this
molecule, termed AtGC1, the catalytic domain is in the N-terminal part.
AtGC1 contains the arginine or lysine that participates in hydrogen
bonding with guanine and the cysteine that confers substrate
specificity for GTP. When AtGC1 is expressed in Escherichia
coli, cell extracts yield >2.5 times more cGMP than control
extracts and this increase is not nitric oxide dependent. Furthermore,
purified recombinant AtGC1 has Mg2+-dependent
GC activity in vitro and >3 times less adenylyl cyclase activity when assayed with ATP as substrate in the absence of GTP.
Catalytic activity in vitro proves that AtGC1 can function either as a monomer or homo-oligomer. AtGC1 is thus not only the first
functional plant GC but also, due to its unusual domain organization, a
member of a new class of GCs.
Guanylyl cyclases
(GCs)1 (EC 4.6.1.2) catalyze
the formation of guanosine 3',5'-cyclic monophosphate (cGMP) from
guanosine 5'-triphosphate (GTP). cGMP acts as second messenger in
many prokaryotes and all eukaryotes (1) including plants. In higher
plants cGMP-mediated processes control
phytochrome-dependent gene expression required for
chloroplast development and anthocyanin biosynthesis (2-4). The light
down-regulated gene asparagine synthetase has been reported to be
controlled by a Ca2+/cGMP-dependent pathway
that activates other light responses and complementary loss- and
gain-of-function experiments have identified a 17 base pair
cis-element within the asparagine synthetase promoter that
is both necessary and sufficient for this regulation (5). This
cis-element may well be the target for a conserved
phytochrome-generated repressor whose activity is regulated by calcium
and cGMP (5).
In plants as well as in animals, nitric oxide (NO) is operating as a
redox-active signaling molecule, and NO donors have been shown to
induce expression of some defense-related genes, and tobacco mosaic
virus-dependent increases of NO synthase activity occur in
resistant plants only (6). NO does elevate cGMP levels in plants, and
NO-induced expression of some defense-related genes was found to be
mediated by the second messengers cGMP and cyclic ADP-ribose, both of
which also operate in animal responses to NO (6).
Cellular cGMP levels are also increased transiently after application
of the plant hormone gibberellic acid (GA) in barley aleurone layers
and GC inhibition prevents the GA-induced increase in cGMP and inhibits
GA-induced The regulation of ion transport is also in parts dependent on cyclic
nucleotides (8, 9). Such regulation can occur in plant voltage-gated
K+ channels where binding of cGMP modulates the
voltage/current relationship (8). Plants also contain cyclic
nucleotide-gated low affinity cation channels where binding of cAMP and
cGMP to the intracellular portion leads to direct gating (10).
Recently, voltage-independent channels without selectivity for
particular monovalent cations have been characterized in
Arabidopsis thaliana. Voltage-independent channels showed no
selectivity among monovalent cations, and their gating was found to be
voltage-independent, while micromolar concentrations of cAMP or cGMP at
the cytoplasmic side of the plasma membrane caused rapid decreases in
channel open probability (9). It was shown that short term
unidirectional Na+ influx is reduced in the presence of
cyclic nucleotides and that membrane-permeable cyclic nucleotide can
improve salinity tolerance presumably by reducing net Na+ uptake.
Cell-permeable cGMP and cAMP analogs elicit elevation of cytosolic
Ca2+ in tobacco protoplasts and cause a
physiological swelling response in plant protoplasts (11). Opening of
the stomatal pore, which results from a swelling of the two neighboring
guard cells, has been observed in response to cell-permeable cGMP
analogs and is suppressed by guanylyl cyclase inhibitors (12). Like
protoplast swelling, stomatal aperture regulation is likely to be tuned
by Ca2+ and cGMP cross-talk (13).
Since significant and transient increases in intracellular cGMP levels,
e.g. in response to the plant hormones GA (7) and cytokinins
(13) as well as vertebrate atrial natriuretic peptides and
immunoreactant plant natriuretic peptides (13-15), have been reported,
it is reasonable to presuppose GC activity in plants. However, no plant
molecule with GC activity has been identified to date, and this failure
could be explained by an unusually high level of divergence in plant
GCs that has put them outside the detection limit of "Blast"
searches or biochemical tools such as specific antibodies against GCs
from e.g. bacteria or animals. Alternatively, plant GCs may
not be homologous to currently annotated GCs and thus not easily
identified. In both cases, however, we hypothesized that plant GCs may
contain a significant degree of similarity to the catalytic center from
previously identified nucleotide cyclases and GCs in particular.
Consequently, we aligned designated catalytic domains (16-22) from
vertebrates, lower eukaryotes, and prokaryotes with a view to deduce a
GC catalytic domain search motif. Such a motif would then be used to do
pattern searches of the complete A. thaliana genomic
sequence to identify candidate proteins for functional testing.
Sequence Analyses--
GCs were retrieved from NCBI, and their
catalytic domains were used for Blast (23) queries of "The
Arabidopsis Information Resource" database and GenBankTM.
The catalytic domains were aligned using Clustal X (24), and the
alignment at the catalytic center of the catalytic domain was used to
derive the search motif. The derived search motif was tested for
accurate and specific detection of nucleotide cyclases by querying the
Protein Information Resource (www-nbrf.georgetown.edu) using the
Pattern Match option on the PIR-NREF link. The search motif was used to
query The Arabidopsis Information Resource database using the Patmatch
link in The Arabidopsis Information Resource.
Cloning and Protein Expression--
Total RNA was isolated from
3-week-old seedlings using the RNeasy plant mini kit (Qiagen GmbH,
Hilden, Germany) in combination with DNase treatment using RNase-free
DNase Set (Qiagen GmbH) according to the manufacturer's instructions.
First strand AtGC1 cDNA was synthesized from total RNA with 1 µM Primer GC1fwd (5'-CAC TGT GGA TCC ATG TGG
CCT CTT TGT TTT CTG-3') incorporating a 5' BamHI restriction
site (underlined), 1 µM Primer GC1rev (5'-CTG ACT
CTC GAG CTA ATA TCC GTT CTG GTT CC-3') incorporating a 5' XhoI restriction site (underlined) using reverse
transcriptase (Promega Corp.). Double-stranded AtGC1 cDNA
synthesis was done by PCR on first strand cDNA from above with 0.4 µM Primer GC1fwd and 0.4 µM Primer GC1rev
using the expand high fidelity PCR system kit (Roche Diagnostics South
Africa Pty. Ltd.) as instructed by the manufacturer, except that the
deoxynucleotide triphosphate concentration was changed to 100 µM for each deoxynucleotide triphosphate. The
AtGC1 cDNA was cloned as a
BamHI/XhoI fragment into pBluescript SK(+/ Cyclic Nucleotide Assays--
cGMP levels were measured by
radioimmunoassay using the cGMP (125I) assay system kit
(Amersham Biosciences UK Ltd.) as described in the supplier's manual
for the acetylation protocol. Purified GST:AtGC1 recombinant fusion
protein (140 µg) was used to determine AtGC1 guanylyl cyclase
activity in vitro (21). This was followed by measurements of
cGMP produced from the reaction using the cGMP (125I) assay
system kit. Cyclic AMP generated in vitro by GST:AtGC1 was
determined using the cAMP Biotrak enzyme immunoassay (Amersham Biosciences UK Ltd.).
Sequence alignment of representatives of annotated catalytic
domains of GCs from prokaryotes and eukaryotes are shown in Fig. 1. The residues implicated in catalysis
(18, 19) are indicated in red, and a 14-amino acid-long
search motif spanning the catalytic center was deduced (Fig. 1). An
isoleucine (Ile) and leucine (Leu) was added to positions 4 and
9, respectively, to include all three aliphatic amino acids with
non-polar side chains; this is in keeping with requirements of these
positions, since they are part of the hydrophobic pocket where the
purine moiety binds (18). The glutamic acid (Glu) implied in
Mg2+ binding (18) that is conserved in the present
alignment (Fig. 1) was not included in the search motif, since it is
not conserved in all GCs (19). The GC catalytic domain alignments also
revealed that GCs contain a glycine-rich domain N-terminal of the
catalytic center (Fig. 1), and this is the case in all currently
annotated GCs (not shown). This glycine-rich domain can be expressed as (G-{X}3,4-G-X{2,3}-G) and will
subsequently be referred to as glycine-rich motif and used as secondary
search parameter.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase synthesis and secretion (7). Both processes can
be restored by exogenous application of membrane-permeant analogs of
cGMP thus establishing cGMP as second messenger critical for
-amylase synthesis and/or secretion.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
(Stratagene) to construct pBS:AtGC1 and sequenced. The AtGC1 cDNA was subcloned from the pBS:AtGC1 construct
into the BamHI/XhoI sites of the glutathione
S-transferase (GST) fusion expression vector pGEX-6P-2
(Amersham Biosciences UK Ltd.) to make the GST:AtGC1 fusion expression
construct pGEX:AtGC1. E. coli BL21 (DE3) pLysS cells
(Invitrogen Ltd., Paisley, UK) were transformed with pGEX:AtGC1 for the
expression of the recombinant protein and with pGEX-6P-2 (Amersham
Biosciences UK Ltd.) for a positive control experiment. E. coli BL21 (DE3) pLysS cells were grown at 30 °C to an
OD600 of 0.8. The cells were then induced for expression of
GST:AtGC1 or GST by addition of
isopropyl-
-D-thiogalactopyranoside (IPTG) (Promega
Corp.) to a final concentration of 0.6 mM and growth at
30 °C for 60 min, with 3-isobutyl-1-methylxanthine (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) added to a final concentration of 1 mM to inhibit resident bacterial phosphodiesterases. To
determine NO dependence of AtGC1, the NO donor sodium nitroprusside was added to a final concentration of 1 mM to both uninduced
and induced cells 10 min before harvesting of the cells. Cells were
harvested by centrifugation at 4 °C at 10,000 rpm for 10 min, then
resuspended in ice-cold phosphate-buffered saline (140 mM
NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.3). Total E. coli
proteins were extracted by three freeze-thaw cycles in PBS, with
freezing done in liquid nitrogen and thawing done at 42 °C.
Purification of both recombinant GST:AtGC1 and GST was performed on a
glutathione-Sepharose 4B affinity column (Amersham Biosciences UK
Limited) and eluted in glutathione elution buffer (10 mM
reduced glutathione in 50 mM Tris-HCl, pH 8.0) according to
the protocol supplied by the manufacturer. The eluted proteins were
desalted and concentrated using Amicon® Ultra 10,000 MWCO 15-ml
centrifugal filter devices (Millipore Corp., MA).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of GC catalytic domains.
Edited Clustal X alignment of catalytic domains of guanylyl
cyclases is shown. The catalytic center is delineated by two
solid arrows, and the glycine-rich motif is delineated by an
open arrow. The deduced 14-amino acid-long search motif is
in bold, and substitutions are in square
brackets; X represents any amino acid, and curly
brackets define the number of amino acids. Red amino
acids are functionally assigned residues of the catalytic center, the
blue amino acid is the replacement of the conserved glycine
( ), and the underlined amino acids in positions 4 and 9 are the third branched aliphatic amino acid not appearing in the
alignment. The glutamic acid (E) implied in Mg2+
respectively Mn2+ binding (
) is not included in the
search motif. Asterisks mark conserved residues,
colons are conservative, and periods are
semiconservative substitutions, and the letter h stands for
hydrophobic residues forming the hydrophobic pocket. Accession numbers
of aligned sequences are as follows: I, BAB60905; II, NP_524603; III,
NP_494995; IV, NP_000171; V, BAA83786; VI (expressed sequence tag),
BI717053; VII (expressed sequence tag), AL132834; VIII, NP_440289; IX,
CAB42641; X, AY118140.
A pattern search of PIR with the catalytic center motif identifies 263 protein sequences of which 208 are annotated GCs and the rest are
either hypothetical proteins or annotated proteins with different
functions. A pattern search of the A. thaliana genome data
base with no substitution allowed returns no candidates. If a
replacement with any amino acid was permitted at the functionally unassigned but conserved glycine (Gly) residue in the catalytic center,
seven candidates molecules are returned. Of the seven A. thaliana proteins returned only one also contains the N-terminal glycine-rich motif. This A. thaliana molecule of unassigned
function was termed AtGC1. AtGC1 is 274 amino acids in length and
contains the search motif in its N-terminal region between residues 32 and 46 (Fig. 2). The functionally
unassigned but conserved glycine (Gly) in the catalytic center is
replaced by an aspartic acid (Asp) (Fig. 1). Homology searches with
AtGC1 return GCs, albeit with e-values 0.19, as well as
more closely related (<e
20) human, mouse,
mosquito, and Drosophila proteins without commonly known GC
domains and of currently unknown function.
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AtGC1 contains the conserved arginine (Arg) or lysine (Lys) (Fig. 1) that participates in hydrogen bonding with guanine (18, 19) and the cysteine (Cys) that confers substrate specificity for GTP (18, 19, 25). Finally, one of the amino acids that has been reported to stabilize the transition state in the conversion of GTP to cGMP, namely arginine (Arg), is conservatively replaced by lysine (Lys) (18, 19). Such a replacement is also seen in the Fugu (Takifugu rubripes) GC (Fig. 1). AtGC1 contains a glycine-rich domain N-terminal of the catalytic center (Fig. 1) and 17 amino acids N-terminal of the motif an arginine (Arg) (Fig. 2A) that is implied in PPi binding (18). This arginine is flanked by hydrophobic amino acids, and this together with the distance to the catalytic center is common in annotated class III nucleotide cyclases (18, 19).
The modeled secondary structure (26) (Fig. 2A) predicts a
-sheet spanning the purine-binding hydrophobic pocket, a feature that has been reported previously (18). Three-dimensional structure prediction (27) (not shown) indicates 18% identity of AtGC1 with the
P-loop in nucleotide triphosphate hydrolases. A domain comparison (Fig.
2B) between classical GCs and AtGC1 reveals the unusual
N-terminal position of the catalytic motif as well as the reduced size
of the domain. Pair-wise BLAST sequence comparison of AtGC1 against
sequences from the dimerization domain, kinase-homology domain,
trans-membrane domain, ligand-binding domain, and heme-binding domain
that interacts with NO indicated that AtGC1 does not have significant
sequence similarity with any of these domains.
To test biological activity of the candidate molecule, we have obtained
the cDNA by reverse transcriptase-PCR and expressed it in a
prokaryotic system (Fig. 3A).
Only in protein extracts from transformed induced bacteria do we
observe a fusion protein (GST:AtGC1) of the predicted molecular mass of
57 kDa. When equal amounts of cells were extracted and assayed for cGMP
with a radioimmunoassay, it was observed that the cGMP levels were
>2.5-fold elevated in extracts from transformed induced bacteria as
compared with both extracts from non-induced cells and the control
(Fig. 3B). The findings would suggest that the A. thaliana protein is either a bona fide GC or stimulates
resident E. coli GCs.
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Fig. 4 shows the purified recombinant
AtGC1 and results from in vitro testing of the recombinant
molecule. The GC activity of total extracted protein from
untransformed, but IPTG-induced, E. coli is less than 10 fmol/µg of protein and higher in the presence of Mn2+
than Mg2+. The purified fusion protein (Fig. 4A)
has an activity of >20 fmol/µg in the presence of Mg2+
and a drastically reduced activity (>50×) in the presence of Mn2+ (Fig. 4B). Both control extract from
un-transformed induced E. coli obtained after
glutathione-Sepharose 4B column purification and GST on its own show no
activity, thus indicating that the cGMP measured in the recombinant
protein preparation is solely due to the catalytic function of AtGC1. A
time course of GST:AtGC1-dependant cGMP formation shows that >80% of
the product was generated in less than 10 min and that no significant
amount of cGMP was produced from heat-inactivated recombinant protein
under the same experimental conditions (Fig. 4C). The
results obtained from in vitro testing are thus consistent
with monomeric or homo-oligomeric catalytic function that is
independent of ATP, activating proteins or co-factors other than
Mg2+. Finally, GST:AtGC1 was also tested for adenylyl
cyclase activity in vitro (Fig. 4D). The result
indicates that with ATP as substrate and in the absence of GTP, AtGC1
has adenylyl cyclase activity that is >3 times lower than the GC
activity and not significantly different in the presence of either
Mg2+ or Mn2+.
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DISCUSSION |
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Since there is compelling evidence that plants operate cGMP-dependent signaling pathways, we decided to use the resource of the complete A. thaliana genome sequence to search for GCs. The approach was based on the observation that known GC catalytic domains appear highly conserved between highly diverged organisms. We reasoned that plant GCs may contain at least some of the amino acid residues that are conserved in the catalytic center of known GCs, specifically the residues that are directly implicated in catalytic activity. Furthermore, the obligatory presence of the glycine-rich motif found adjacent to the N terminus of the catalytic center of all known GCs suggested inclusion of this feature as additional search criterium. In GCs the glycine-rich motif may play a similar role as in the P-loop of GTP-binding proteins where it gives flexiblity to the part of protein that interacts with the phosphate group in GTP, thus facilitating substrate binding (28). We anticipated that plant GCs would be significantly evolutionarily distinct and different from known GCs, since homology searches have failed to identify functional molecules in plants. Consequently, a search motif was constructed to allow conservative and semiconservative replacements of amino acid residues in the catalytic center. The glutamic acid (Glu) implied in Mg2+ binding (Fig. 1) (18) was not included in the search motif, since it is substituted by wide range of different amino acids (Asp, Ala, Gln, Cys, Thr, Arg, and Tyr) in prokaryotic guanylyl cyclases (19). In addition, the glutamic acid (Glu) is also replaced by aspartic acid (Asp) in some type I and type II adenylyl cyclases (18). This substitution pattern would thus suggest that polarity and charge of the amino acid side chain in this position is not restrictive for catalysis.
The fact that no A. thaliana protein contains the unsubstituted search motif prompted a decrease of the stringency of the motif allowing for a replacement of the functionally unassigned but highly conserved glycine in the catalytic center (Fig. 1). The modified search parameters yield seven A. thaliana proteins, and only one, AtGC1, fulfils the secondary criteria of the presence of the adjacent glycine-rich motif. It is concluded that the motif search with one substitution in the catalytic center is not highly selective for GCs, since 20 proteins out of 260 proteins identified in PIR that contain the motif are implicated in functions not related to known GC activity. However, the combination of the two search parameters, the catalytic center search motif and the glycine-rich motif, identify annotated GCs only. This would suggest that the use of both parameters combined is a reliable tool for the identification of candidate GCs.
AtGC1 contains the conserved residues that participate in hydrogen
bonding with guanine (18, 19) and the cysteine (Cys) that confers
substrate specificity for GTP rather than ATP (18, 19). This structural
feature is reflected in the in vitro activity of AtGC1 which
shows significantly more guanylyl than adenylyl cyclase activity. The
arginine (Arg) that stabilizes the transition state in the conversion
of GTP to cGMP is conservatively replaced by lysine (Lys) (18, 19), and
such a substitution is also observed in Fugu (T. rubripes)
(Fig. 1). AtGC1 also contains the hydrophobic pocket to which
nucleotide triphoshate purine moieties can bind (18), and the secondary
structure modeling predicts that this pocket is part of a -sheet
(Fig. 2A). A further N-terminal feature of GCs is the
presence of a pyrophosphate (PPi) binding motif that consists of an
arginine (Arg) flanked by aliphatic amino acids (18). AtGC1 contains
such a motif 22 amino acids from the catalytic center and hence in a
position that is conserved in PPi-binding sites of known GCs (Fig.
2A). The similarity between the predicted three-dimensional
structure of AtGC1 and that of the P-loop structure contained in
nucleotide triphosphate hydrolases (28, 29) is noteworthy, since
nucleotide triphosphate hydrolases can convert GTP to both GDP and GMP
(30, 31). In triphosphate hydrolases, the P-loop interacts with
Mg2+ for GTP binding and hydrolysis, and it may be argued
that the P-loop-like structure in AtGC1 has an analogous function, and this is supported by the observed dependence of the catalytic activity
of AtGC1 on Mg2+ (Fig. 4B).
AtGC1 contains a number of features that are unusual in currently annotated GCs. First, the catalytic center motif is found close to the N-terminal rather than the C-terminal or central part of the molecule where it is located in both particulate and soluble guanylyl cyclases (32-34). C-terminal location is the norm in particulate GCs where the N terminus functions as receptor and the C-terminal GC domain generates the cytosolic signal. In addition, in AtGC1 there is no evidence of a signal sequence for direction to the membrane and the designated PPi-binding arginine (Arg) is only 12 amino acids removed from the N-terminal end of the molecule. Other features typical of particulate GCs, the ligand-binding domain, transmembrane domain, kinase homology domain, and dimerization domain are also absent.
Contrary to the homodimeric particulate GCs, the soluble GCs form
/
heterodimers (33, 35). Soluble GCs are the principal effector
of the gaseous messenger NO. NO dependence of soluble GCs in turn
requires a heme-binding region, and this region is N-terminal of the
catalytic domain. NO binds covalently via two cystine-thioether bonds
that are provided by a conserved C-X-X-C-H motif
in soluble GCs (36). In AtGC1 there is no evidence of a heme-binding
motif that is required for NO binding. The absence of such a
heme-binding motif is thus consistent with the lack of NO inducibility
observed in AtGC1. From the sequence analysis, one would thus predict
that AtGC1 is not a membrane protein, not similar in domain structure
to known receptor or soluble GCs, and its lack of a heme-binding group
implies activity that is insensitive to NO, while catalytic activity
in vitro implies that it is active either as a monomer or
homo-oligomer.
The assay result obtained from transformed induced E. coli
demonstrates a significant increase in cGMP (Fig. 3). Since in this
assay cGMP was generated in intact bacteria and in the absence of added
substrate, it can be concluded that at least the N terminus of the
recombinant was present in the cytosol and thus supports the
predictions based on structural features. Since AtGC1 is likely to be
located in the cytosol, we tested whether the activity in vivo in E. coli cells was affected by the NO donor
sodium nitroprusside, and the observed NO insensitivity is consistent
with the structural features of AtGC1. NO independence in soluble GCs
does not appear to be the norm; however, an unusual soluble GC from
Manduca sexta (MsGC-3) that does not require dimerization
has been reported (37). In this molecule amino acids thought to
participate in heme binding are substituted with non-similar amino
acids (37). AtGC1 and MsGC-
3 are thus both soluble GCs that do not
depend on heterodimer formation for activity. Taken together, our
results imply that AtGC1 is active as a monomer, homodimer, or
homo-oligomer, with no obligatory dependence on membrane associations,
activation by ligands, ATP, or other co-factors with the exception of
Mg2+.
While homodimeric and oligomeric activity is common in particulate GCs (1, 17, 34), monomeric activity in a soluble GC has only recently been reported (38). Monomeric GC activity has also been demonstrated in a soluble GC (DdsGC) from Dictyostelium discoideum (39) that has in fact been identified as a homologue of an adenylyl cyclase. It is in keeping with this ancestry that DdsGC has two catalytic domains within a single molecule. The discovery of another GC from M. sexta (MsGC-I) has further complicated attempts to categorize GCs (40, 41). MsGC-I shows highest sequence identity with receptor GCs throughout its catalytic and dimerization domains but does not contain the ligand-binding, transmembrane, or kinase-like domains typical receptor GCs (40). Both AtGC1 and MsGC-I contain C-terminal extensions that are not present in other known guanylyl cyclases. MsGC-I shows no similarity to domains typical of soluble GCs but appears to exist as a soluble homodimer insensitive to NO stimulation (40); however, despite the absence of a transmembrane domain an as yet undefined membrane association in vivo has been suggested (40).
AtGC1 is adding to the steadily growing number of structurally diverse
molecules with GC activity and it appears that many more and diverse
GC-dependent physiological responses await discovery. We
are currently in the process of elucidating the biological role of
AtGC1 with particular emphasis on cGMP dependent promotion of stomatal
opening (12, 13) and responses to plant hormones such as kinetin and
immunoreactant plant natriuretic peptides (13-15).
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ACKNOWLEDGEMENTS |
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We thank Amanda Lochner (University of Stellenbosch) for help with the cGMP assay and Cathal Seoighe (South African National Bioinformatics Institute) for critical discussion.
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FOOTNOTES |
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* This work was supported by the National Research Foundation of South Africa.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/EBI Data Bank with accession number(s) AAM51559.
To whom correspondence should be addressed: University of the
Western Cape, Dept. of Biotechnology, Bellville, Private Bag X17, 7535, South Africa. Tel.: 27-21-959-2199; Fax: 27-21-959-1349; E-mail:
cgehring@uwc.ac.za.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210983200
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ABBREVIATIONS |
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The abbreviations used are:
GC, guanylyl
cyclase;
NO, nitric oxide;
GA, gibberellic acid;
GST, glutathione S-transferase;
IPTG, isopropyl--D-thiogalactopyranoside.
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