Preliminary characterization of two atypical soluble guanylyl cyclases in the central and peripheral nervous system of Drosophila melanogaster
Departments of Integrative Biosciences and Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR 97239, USA
* Author for correspondence (e-mail: mortonda{at}ohsu.edu)
Accepted 13 April 2004
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Summary |
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Key words: cGMP, guanylyl cyclase, nitric oxide, Drosophila melanogaster, peripheral nervous system, central nervous system, NO-insensitive, sensilla, chemosensory
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Introduction |
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Guanylyl cyclases, the enzymes that catalyze the synthesis of cGMP,
generally fall into one of two classes: the integral membrane receptor
guanylyl cyclases and the cytoplasmic soluble guanylyl cyclases
(Lucas et al., 2000). Soluble
guanylyl cyclases are classically obligate heterodimers composed of an
subunit and a ß subunit. The
/ß heterodimers are potently
activated by the gaseous messenger nitric oxide (NO) via a prosthetic
heme group that binds to the heterodimer in the N-terminal regulatory domain
(Lucas et al., 2000
). Receptor
guanylyl cyclases, by contrast, are homodimeric proteins that are activated by
extracellular ligands or intracellular calcium binding proteins
(Lucas et al., 2000
).
Recent reports have described a number of soluble guanylyl cyclases that
exhibit significantly different properties compared with the conventional
/ß heterodimers (Morton,
2004
). One of these, MsGC-ß3, was identified in the insect
Manduca sexta (Nighorn et al.,
1999
). Expression of MsGC-ß3 in COS-7 cells yielded moderate
levels of guanylyl cyclase activity in the absence of additional subunits and
this activity was not stimulated by NO donors
(Nighorn et al., 1999
). Gel
filtration data demonstrated that MsGC-ß3 formed homodimers
(Morton and Anderson, 2003
).
Another atypical soluble guanylyl cyclase is the rat ß2 subunit, which is
also active in the absence of other subunits, although this activity is
slightly sensitive to NO stimulation
(Koglin et al., 2001
).
Orthologues of MsGC-ß3 have been identified in searches of the genomes
of Caenorhabditis elegans and Drosophila melanogaster
(Morton, 2004). The C.
elegans MsGC-ß3 orthologue, GCY-31, and the other six soluble
guanylyl cyclases from C. elegans have all been predicted to be NO
insensitive (Morton et al.,
1999
). In addition to the previously studied conventional
and ß subunits, the Drosophila genome contains three additional
soluble cyclase subunits that have all been predicted to be insensitive to NO
(Morton and Hudson, 2002
;
Morton, 2004
). One of these,
CG4154, is over 80% identical to MsGC-ß3 and is predicted to form active
homodimers (Morton and Hudson,
2002
). The other two, CG14885 and CG14886, have been predicted to
require an additional subunit for activity, potentially forming active
heterodimers with either CG4154 or the conventional
subunit,
Gyc
-99B (Morton and Hudson,
2002
). Rather than continue to use the CG numbers to designate
these guanylyl cyclases, we propose the following designations based on their
chromosomal locations: Gyc-88E for CG4154, Gyc-89Da for
CG14885 and Gyc-89Db for CG14886. This nomenclature
is also consistent with the names of the Drosophila NO-sensitive
subunits, Gyc
-99B and
Gycß-100B. Preliminary results
(Morton, 2004
) confirmed that
Gyc-88E yielded basal activity when expressed alone and Gyc-89Db was inactive
when expressed alone. Furthermore, these studies showed that Gyc-89Db could
form an active enzyme when co-expressed with Gyc-88E, although it was not
tested with Gyc
-99B (Morton,
2004
). These studies also highlighted an unusual property of
Gyc-88E; when expressed either alone or co-expressed with Gyc-89Db, it was
slightly activated by the NO donor sodium nitroprusside (SNP) but not by
DEA-NONOate or the NO-independent soluble guanylyl cyclase activator YC-1.
These findings suggested that it was not NO itself that activated Gyc-88E but
rather an additional breakdown product of SNP. The present study expands on
these preliminary findings with further biochemical studies that strongly
suggest that NO does indeed activate Gyc-88E and Gyc-89Db and shows that both
are expressed in the central nervous system (CNS) and co-expressed in a subset
of peripheral putative chemosensory neurons.
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Materials and methods |
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RNA collection
Animals were staged according to the method described by Campos-Ortega and
Hartenstein (1997). Animals of
selected stages were frozen and pulverized in liquid nitrogen in a pestle and
mortar. Total RNA was isolated from the resulting powder with Trizol®
reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's
instructions. Poly(A)+ RNA was isolated from total RNA using
oligo(dT) cellulose (Ambion, Austin, TX, USA) according to the supplied
protocol.
RT-PCR cloning of Gyc-88E and splice variant analysis
Superscript II RNase H reverse transcriptase (Invitrogen) was used in a
reverse transcription (RT) reaction using an oligo(dT)1218
primer (Invitrogen) to synthesize cDNA that was used in subsequent PCR
reactions. The composition of the reaction mixture was: 50 mmol l-1
Tris-HCl (pH 8.3), 75 mmol l-1 KCl, 5 mmol l-1
MgCl2, 10 mmol l-1 dithiothreitol (DTT), 0.5 µg total
RNA (from a mix of larval and pupal animals), 1 µl
oligo(dT)1218 primer (500 µg ml-1), 1 µl
dNTP mix (10 mmol l-1 each) in a total volume of 20 µl. The RT
reaction was carried out at 50°C for 50 min, followed by a 15-min
inactivation step at 70°C. Three primers were designed using the
Drosophila genomic sequence located at FlyBase (accession numbers:
AE003707, AE002708 and AE014297) and were used in two semi-nested PCR
reactions to clone the entire open reading frame (outer primer set
5'-CAATGTCAGCCAAGTGAAG-3',
5'-TACATATACCCTCTCATTAGC-3'; inner primer set
5'-GAGGAAGTGGATCCATG-3', 5'-TACATATACCCTCTCATTAGC-3')
in two rounds of PCR using the Expand High Fidelity PCR System (Boehringer
Mannheim, Indianapolis, IN, USA), consisting of 30 cycles with an annealing
temperature of 51°C for 25 s. A 0.5 µl aliquot of the PCR reaction was
used as template for subsequent amplification with the inner primers. The
resulting 3 kb product was cloned into the TOPO II vector (Invitrogen).
Sequencing of multiple clones revealed the existence of two splice variants
(Gyc-88E-L and Gyc-88E-S) that differed by 21 bp, depending
on how the 10th and 11th exons are spliced together. To determine if the
splice variants were expressed in other stages, RT reactions were performed as
described above on total RNA prepared from mixed larval stages or adults. Two
nested sets of primers that were designed to amplify across the junction
between the 10th and 11th exons were used in two rounds of PCR to produce a 70
bp or 91 bp band corresponding to Gyc-88E-S or Gyc-88E-L,
respectively. The outer set of primers were
5'-GCACCAGCCAGAGAAACG-3' and
5'-TACATATACCCTCTCATTAGC-3'; the inner set of primers were
5'-GCAGTGCATCATTGGATC-3' and 5'-GCAGTTGGAGTGGTTGCA-3'.
The composition of the reaction mixture was: 20 mmol l-1 Tris-HCl
(pH 8.4), 50 mmol l-1 KCl, 1.5 mmol l-1
MgCl2, 200 µmol l-1 each dNTP, 500 nmoles of each
primer, 0.5 µl reverse transcription reaction and 2.5 units of Taq DNA
polymerase (Invitrogen) in a 50 µl reaction for 30 cycles with an annealing
temperature of 51°C for 25 s. A 0.5 µl aliquot of the PCR reaction was
used as the template for subsequent amplification, with the inner primers
using the same reaction conditions. A 3% NewSieve GQA agarose gel (ISC
BioExpress, Kaysville, UT, USA) was used to distinguish the two PCR
products.
Northern blots
Poly(A)+-selected RNA (1.5 µg) from selected stages was
separated on a 1% denaturing formaldehyde agarose gel as previously described
(Sambrook et al., 1989) and
transferred to a Nytran SuperCharge nylon membrane using a Turboblotter
(Schleicher and Schuell BioScience, Keene, NH, USA). A digoxygenin
(DIG)-labeled RNA probe was generated using full-length Gyc-88E or a
portion of the ribosomal protein RP49 (used for a loading control) with the
Megascript Kit (Ambion) using DIG-UTP (Roche, Indianapolis, IN, USA). The
resulting probe was hybridized to the membrane-bound transcript at 68°C
using UltraHyb (Ambion), with a final probe concentration of 0.1 µmol
l-1. Hybridized membranes were washed for 2x10 min with
low-stringency wash (2xSSC buffer, 0.1% SDS) and for 2x15 min with
high-stringency wash (0.1xSSC, 0.1% SDS) at 68°C. Membranes were
then incubated with Fab fragments of sheep anti-DIG-AP (alkaline phosphatase)
antibody (Roche) at 1:1000 dilution in maleic acid buffer (Roche) for 1 h,
followed by two 15-min washes with wash buffer (Roche). AP was detected by
applying CDP-Star chemiluminescent substrate (Roche) to the membrane.
Transient expression of Gyc-88E and Gyc-89Db and guanylyl cyclase assay
To examine the enzyme activities of Gyc-88E, the full open reading frame
(ORF) of Gyc-88E-L and Gyc-88E-S were subcloned into the
mammalian expression vector pcDNA3.1 (Invitrogen) utilizing ApaI and
KpnI restriction enzyme sites. A cDNA of Gyc-89Db that
contained the full ORF was obtained as an expressed sequence tag (EST) cDNA
(clone ID: GH09958) from the Berkeley Drosophila Genome Project and
subcloned into pcDNA3.1 utilizing EcoRI restriction enzyme sites.
COS-7 cells were transiently transfected with constructs and the homogenates
assayed for guanylyl cyclase activity as described previously
(Morton and Anderson, 2003).
1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) was dissolved in dimethyl
sulfoxide (DMSO; Sigma, St Louis. MO, USA) prior to use in guanylyl cyclase
assays and was used at a final concentration of 100 µmol l-1. NO
donors were dissolved in distilled water or DMSO just prior to use in guanylyl
cyclase assays. Sodium nitroprusside (SNP; Sigma), 3-morpholinosydnonimine
(SIN-1; Calbiochem, San Diego, CA, USA),
5-[1-(phenylmethyl)-1H-indazol-3-yl]-2-furanmethanol (YC-1; Calbiochem),
S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem),
S-nitrosoglutathione (SNOG; Calbiochem),
2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NONOate; Calbiochem),
1-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC-12;
Calbiochem) and
(Z)-1-{N-(3-ammoniopropyl)-N-[4-(3-aminopropylammonio)
butyl]-amino}diazen-1-ium-1,2-diolate (spermine NONOate; Calbiochem) were used
at a final concentration of 100 µmol l-1.
Whole-mount in situ hybridization of embryos and larval CNS
Whole mount in situ hybridization was used to identify the spatial
expression pattern of Gyc-88E and Gyc-89Db during
embryogenesis and in the larval CNS. DIG-labeled RNA probes were generated as
described above for the northern blots and were fragmented with a carbonate
buffer (60 mmol l-1 Na2CO3, 40 mmol
l-1 NaHCO3, pH 10.2) for 20 min at 65°C. Mixed
stages of embryos were collected and fixed as described
(Sullivan et al., 2000) and
stored in 100% ethanol until use. Before use, the embryos were rehydrated with
PBT (phosphate-buffered saline with 0.1% Tween 20) and post-fixed for 30 min
with 4% formaldehyde. The larval CNS was removed and fixed in 4%
paraformaldehyde for 45 min, washed for 4x15 min with PBT and used the
same day. Samples were prehybridized with hybridization buffer [5xSSC,
50% formamide, 0.1 mg ml-1 heparin sulfate, 0.1 µg
ml-1 sonicated salmon sperm DNA (Invitrogen), 0.1% Tween 20, pH
5.2] for 1 h prior to adding probe at a final concentration of 1.5 ng
µl-1 followed by overnight incubation at 60°C. Samples were
washed for 4x1 h at 60°C in 5xSSC, 50% formamide followed by
4x15 min in PBT and blocked for 1 h with PBT plus 10% bovine serum
albumin. Fab fragments of sheep anti-DIG-AP antibody (Roche) were incubated
with samples at 1:1000 with PBT overnight at 4°C and washed for 4x15
min with PBT. DIG-labeled RNA probes were detected with NBT/BCIP one-step
alkaline phosphatase substrate (Pierce, Rockford, IL, USA) and the reaction
stopped with five rinses of 100% ethanol. For in
situ/immunocytochemical double-label experiments, the neuronal marker
antibody 22C10 (Developmental Studies Hybridoma Bank) was added (1:200) at the
same time as the anti-DIG-AP antibody and was detected with horseradish
peroxidase anti-mouse antiserum (Jackson ImmunoResearch, West Grove, PA, USA)
at 1:1000 in PBT for 1 h. After 4x15 min washes with PBT, 22C10 was
visualized with 0.5 mg ml-1 diaminobenzidene (DAB; Sigma) plus
0.003% hydrogen peroxide. DAB reactions were stopped with five consecutive
washes with PBT. Anti-DIG antibody was then visualized as above. The samples
were then dehydrated in ethanol, cleared in methyl salicylate and mounted in
Permount (Fisher Scientific, Fairlawn, NJ, USA).
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Results |
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An unrooted phylogenetic tree generated from a ClustalX analysis illustrates the relationships between the ß and ß-like subunits of the soluble guanylyl cyclases (Fig. 2). MsGC-ß3 clusters with its orthologues, Gyc-88E, CP12881 and the C. elegans gene GCY-31. Gyc-89Db clusters with Gyc-89Da, P3998 (the Anopheles gambiae orthologue to Gyc-89Da and Gyc-89Db) and the C. elegans gene GCY-33. The conventional NO-sensitive ß1 subunits from vertebrates and invertebrates form a distinct cluster, while the remaining five C. elegans soluble guanylyl cyclase subunits and the mammalian ß2 subunits form two additional separate groups.
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Both Gyc-88E splice variants are expressed in larvae and adults
A northern blot, using Gyc-88E as a probe, revealed a single
Gyc-88E transcript of approximately 6 kb
(Fig. 3A). This is about twice
the size of the coding region of the transcript, indicating extensive 5'
and/or 3' untranslated regions. This transcript was present in both
larval and adult stages, with apparently higher levels of transcript present
in the adult (Fig. 3A).
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To determine whether there was any developmental regulation of the different Gyc-88E splice variants, we used RT-PCR to examine their expression in larvae and adults (Fig. 3B). Two pairs of nested primers were designed to amplify a region across the splice junction to yield a 70 bp or 91 bp product, corresponding to Gyc-88E-S and Gyc-88E-L, respectively. A high-resolution 3% agarose gel was then used to resolve the two products. We detected PCR products corresponding to both Gyc-88E-L and Gyc-88E-S in samples from both larvae and adults (Fig. 3B). The 140 bp fragment present in all lanes corresponds to genomic DNA contamination in the samples.
Guanylyl cyclase activity of Gyc-88E and Gyc-89Db
To test the predictions described above, we subcloned the Gyc-88E
and Gyc-89Db open reading frames into the mammalian expression vector
pcDNA3.1. These constructs were then transiently transfected in COS-7 cells
and the resulting extracts assayed for guanylyl cyclase activity. As expected,
and confirming previously reported preliminary data
(Morton, 2004), Gyc-88E
displayed basal activity in the absence of other subunits. Both splice
variants of Gyc-88E were also active in the absence of additional subunits and
both yielded similar levels of activity
(Fig. 4A). All previously
described guanylyl cyclases require a metal ion co-factor (Mg or Mn) for
activity, with Mn yielding higher levels of activity compared with Mg
(Lucas et al., 2000
). Gyc-88E
exhibited similar properties, with both slice variants yielding significantly
higher levels of activity in the presence of Mn compared with Mg
(Fig. 4A). As a comparison, we
also transfected COS-7 cells with a plasmid coding for the Manduca
guanylyl cyclase, MsGC-ß3 (Nighorn et
al., 1999
). The activity of MsGC-ß3 in the presence of either
Mg or Mn was at least 10-fold higher than that of Gyc-88E
(Fig. 4A), but whether this was
due to an intrinsically higher level of specific activity or whether it
reflected higher levels of protein expression is not known. To further
investigate possible enzymatic differences between the two Gyc-88E splice
variants, we assayed cell extracts for guanylyl cyclase activity in the
presence of differing concentrations of GTP and either Mg or Mn
(Fig. 4B). MichaelisMenten kinetics analysis was applied to the results to examine
differences in the Km or Vmax between
the splice variants. Estimates for the value of Km for
both splice variants were similar to each other in the presence of Mg
(Gyc-88E-S, 2.8±0.8 mmol l-1; Gyc-88E-L, 2.5±0.6 mmol
l-1) and Mn (Gyc-88E-S, 0.03±0.02 mmol l-1;
Gyc-88E-L, 0.02±0.02 mmol l-1). The values for
Vmax of the splice variants were also the same as each
other in the presence of Mg (Gyc-88E-S, 6.5±0.7 pmol cGMP
min-1 mg-1 protein; Gyc-88E-L, 6.0±0.6 pmol cGMP
min-1 mg-1 protein) and Mn (Gyc-88E-S, 5.1±0.2
pmol cGMP min-1 mg-1 protein; Gyc-88E-L, 5.2±0.1
pmol cGMP min-1 mg-1 protein).
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Previous studies showed that the NO donor SNP slightly activated Gyc-88E
(Morton, 2004).
Fig. 4C shows that this is also
true for both of the splice variants of Gyc-88E, and again no differences are
seen between the two variants. As shown previously, this increase was much
smaller (23-fold) than seen with conventional
/ß subunits
such as the Manduca MsGC-
1/MsGC-ß1 heterodimers shown
here. The preliminary report on the properties of Gyc-88E showed that,
although SNP was an effective activator, another NO donor (DEA-NO) was
ineffective, as was YC-1 (Morton,
2004
), an NO-independent activator of conventional soluble
guanylyl cyclases (Friebe and Koesling,
1998
). These data suggested that NO was not the active component
of SNP breakdown. To further investigate this possibility, we tested whether
the SNP-stimulated activity was sensitive to the conventional soluble guanylyl
cyclase inhibitor ODQ. Fig. 4C
shows that ODQ was ineffective at inhibiting the SNP-stimulated activity of
Gyc-88E, although it was a potent inhibitor of the activation of
MsGC-
1/MsGC-ß1.
To examine the guanylyl cyclase activity of Gyc-89Db, we subcloned its
coding sequence, obtained as an EST clone from the Berkeley
Drosophila Genome Project (BDGP), into pcDNA3.1 and transiently
expressed it into COS-7 cells. As previously demonstrated
(Morton, 2004), Gyc-89Db
displayed no activity when expressed in the absence of additional subunits
(Fig. 4D) whether Mg or Mn was
included as the metal cofactor. One possible heterodimer partner is the
Drosophila
subunit, Gyc
-99B
(Morton and Hudson, 2002
).
However, when we co-expressed Gyc
-99B (also obtained as an EST clone
from BDGP) with Gyc-89Db, we again failed to detect any enzyme activity
(Fig. 4D). To test that our
Gyc
-99B cDNA was expressed properly, we also cloned the
Drosophila conventional ß subunit, Gycß-100B, using RT-PCR,
subcloned it into pcDNA3.1 and co-expressed it with Gyc
-99B. As
expected, this gave significant basal activity that was enhanced in the
presence of Mn (Fig. 4D). The
only other subunit that was predicted to contain the necessary residues to
form an active enzyme with Gyc-89Db was Gyc-88E
(Morton and Hudson, 2002
).
When these two subunits were co-expressed, the level of basal activity, in the
presence of either Mg or Mn, was higher than when Gyc-88E was expressed alone
(Fig. 4D), suggesting that
Gyc-88E and Gyc-89Db are capable of forming active heterodimers and might be
partners in vivo. No difference was seen in the levels of activity
when either splice variant of Gyc-88E was co-expressed with Gyc-89Db (data not
shown). As with all previously described guanylyl cyclases, the level of
activity of the heterodimer was enhanced in the presence of Mn compared with
Mg.
As described above, earlier studies showed that some, but not all,
activators of conventional soluble guanylyl cyclases were capable of
activating Gyc-88E and Gyc-88E/Gyc-89Db
(Morton, 2004). To gain
further insight into these differences, we tested several different classes of
NO donors in guanylyl cyclase assays. We found that only SNP and the two
S-nitroso compounds SNAP and SNOG were able to significantly stimulate Gyc-88E
(Fig. 4E). As previously
reported, DEA-NONOate was ineffective at stimulating Gyc-88E
(Morton, 2004
). We repeated
this experiment and also tried two additional NONOates, NOC-12 and spermine
NONOate, but no members of this class of NO donors were effective at
stimulating Gyc-88E. In addition, again as previously reported, the
NO-independent soluble guanylyl cyclase activator YC-1 was also ineffective at
stimulating Gyc-88E. When Gyc-88E was co-expressed with Gyc-89Db, activity was
significantly stimulated by all of the NO donors except DEA-NONOate
(Fig. 4F). Not only was the
basal activity of Gyc-88E enhanced when it was co-expressed with Gyc-89Db but
the SNP-, SNAP- and SNOG-stimulated activity was also significantly increased.
Interestingly, whereas two of the NONOates, NOC-12 and spermine NONOate, and
the unrelated NO donor SIN-1 were ineffective at stimulating Gyc-88E, all
three stimulated the Gyc-88E/Gyc-89Db co-expression samples. This was in
contrast to DEA-NONOate, which was ineffective at stimulating Gyc-88E when
expressed alone or when co-expressed with Gyc-89Db. Similarly, YC-1 was
ineffective at stimulating either the Gyc-88E or the Gyc-88E/Gyc-89Db samples.
By contrast, all the NO donors and YC-1 were potent activators of the
Drosophila conventional soluble guanylyl cyclase,
Gyc
-99B/Gycß-100B, and at the concentration used here (100 µmol
l-1) they were all similarly effective
(Fig. 4G).
Gyc-88E and Gyc-89Db are expressed in the peripheral and central nervous system
To determine the cellular localization of Gyc-88E and
Gyc-89Db, we performed in situ hybridization using
fragmented DIG-labeled RNA probes on whole Drosophila embryos and 3rd
instar larval central nervous systems. Gyc-88E expression was
detected in a segmental pattern in the ventral nerve cord (VNC) and in the
brain in embryos, beginning at stage 15 or 16 and continuing through stage 17
(Fig. 5A horizontal
view; Fig. 5C lateral
view). Gyc-89Db showed a similar expression pattern in the VNC and
brain but could be detected as early as stage 13 (data not shown) and also
continued through stage 17 (Fig.
5B horizontal view;
Fig. 5D lateral view).
In stage 17 embryos, the total number of cells that expressed Gyc-88E
was noticeably higher than the number of cells that expressed
Gyc-89Db, especially in the brain (compare
Fig. 5C and
Fig. 5D). Stained single cells
visible in the anterior and posterior of the embryo in
Fig. 5C,D are not part of the
CNS and are discussed below. Application of a sense riboprobe generated from
Gyc-88E or Gyc-89Db yielded a low level of background
staining throughout the embryos with no cells stained
(Fig. 5E
Gyc-88E; Fig. 5F
Gyc-89Db).
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We also examined Gyc-88E and Gyc-89Db expression in the CNS of wandering 3rd instar larvae (Fig. 6). Expression of both guanylyl cyclases was observed in single cells scattered throughout the brain lobes and VNC. In the brain, expression of Gyc-88E and Gyc-89Db was most prominent in a small cluster of cells located in the anterior medial region of each lobe (Fig. 6A Gyc-88E; Fig. 6B Gyc-89Db). In the VNC, Gyc-88E and Gyc-89Db expression was found in both lateral and midline cells. In the ventral region of the VNC, Gyc-88E (Fig. 6C) and Gyc-89Db (Fig. 6D) expression was found in a similar number of individual cells, with more prominent cell clusters located in the more anterior part of the VNC. In the dorsal region of the VNC, Gyc-88E (Fig. 6E) expression was found in a large number of cells in the lateral regions while Gyc-89Db (Fig. 6F) was found in noticeably fewer cells. The uniform background staining, which was most prominent in the brain lobes, was also observed in samples hybridized with sense probe (data not shown), indicating non-specific staining similar to that observed in embryo preparations (Fig. 5E,F).
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In stage 17 embryos, Gyc-88E and Gyc-89Db were also expressed in a number of cells that appeared to be associated with the peripheral nervous system (Fig. 7). The overall pattern of this peripheral staining was very similar for probes to both Gyc-88E and Gyc-89Db. Both Gyc-88E (Fig. 7A horizontal view; Fig. 7C lateral view) and Gyc-89Db (Fig. 7B horizontal view; Fig. 7D lateral view) were expressed in two cells on each side of segments T1, T2 and T3 and in one cell on each side of A1 and A2. The cells in segment A2 often stained very weakly and were not always detected. The positions of the stained cells in the thoracic segments are similar to the locations of the embryonic/larval basiconical sensilla. As a preliminary test to determine whether both guanylyl cyclase subunits were co-expressed in the same cells, we hybridized embryos to both probes simultaneously. In these experiments, the number of lateral cells that stained was the same as when each probe was used individually: two cells on each side of segments T1, T2 and T3 and one cell on each side of A1 and A2 (Fig. 7E; Table 2).
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In addition to the lateral cells, both probes hybridized to cells that appeared to be associated with the ganglia that innervate the head sensory organs (Fig. 7F Gyc-88E; Fig. 7G Gyc-89Db). Gyc-88E hybridized to a single pair of cells whereas Gyc-89Db was expressed in 25 closely grouped cells on each side of the embryo. In addition, the Gyc-89Db probe stained 45 closely grouped cells per side located in a more posterior position (not visible in the focal plane shown in Fig. 7G). These cells will be described in more detail later. Finally, Gyc-88E and Gyc-89Db were also found in 12 cells in segments A8 and A9 (telson; Fig. 7H Gyc-88E; Fig. 7I Gyc-89Db; three focal planes shown for each probe) in an apparently overlapping pattern. These cells appear to be associated with the clusters of neurons that innervate the 10 external sensory cones found in segments A8 and A9. To determine if both probes hybridized to the same cells, we examined the preparations where both Gyc-88E and Gyc-89Db probes were used simultaneously, counted the number of cells that stained and compared these results with the number of cells stained using a single probe. We counted the same number of cells (12) in segments A8 and A9 (Fig. 7J; three focal planes shown) in the double-labeled embryos as in embryos labeled with Gyc-88E or Gyc-89Db probes individually. This suggests that both guanylyl cyclases were co-expressed in the same cells in these segments. In the anterior region of the embryo, where Gyc-89Db labels more cells than Gyc-88E, we never observed more labeled cells using both probes together than the maximum number of cells observed in the Gyc-89Db single probe preparations. This suggests that the cells that expressed Gyc-88E also expressed Gyc-89Db, but there were some cells that expressed only Gyc-89Db. These data are summarized in Table 2.
To determine if the peripheral cells that expressed Gyc-88E and
Gyc-89Db in stage 17 embryos were neurons of the peripheral nervous
system, we combined in situ hybridization with immunocytochemistry
using the neuron-specific antibody 22C10
(Fig. 8). These experiments
demonstrated that the Gyc-88E- and Gyc-89Db-expressing cells
were always stained with 22C10 and by their positions were identified as
peripheral neurons that innervate various external sensory organs and the
trachea. Comparison of our data with detailed diagrams of the peripheral
nervous system (Stocker, 1994;
Bodmer and Jan, 1987
;
Brewster and Bodmer, 1995
)
allowed us to more specifically identify the cell or cell cluster.
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In segments T2 and T3, Gyc-88E and Gyc-89Db were
co-expressed in one of the three neurons in the lateral and ventral clusters
(Fig. 8A
Gyc-88E, lateral; Fig.
8B Gyc-89Db, lateral;
Fig. 8C
Gyc-88E, ventral; Fig.
8D Gyc-89Db, ventral) that innervate the
basiconical sensilla, which are external sensory organs with a putative
chemosensory role (Stocker,
1994). In the lateral T2 and T3 clusters (upper row in
Fig. 7C,D), Gyc-88E
and Gyc-89Db were expressed in one of the three les neurons (lateral
external sensilla-innervating) (Fig.
8A Gyc-88E;
Fig. 8B
Gyc-89Db). The collected dendrites from the three les neurons were
observed to project upwards to the location of the basiconical sensillum
(Fig. 8A,B). In the ventral T2
and T3 clusters (lower row in Fig.
7C,D), Gyc-88E and Gyc-89Db were expressed in
one of the three (ves) neurons (ventral external sensilla-innervating)
(Fig. 8C
Gyc-88E; Fig. 8D
Gyc-89Db). In the thoracic segments, the position of the
single guanylyl cyclase-expressing cell in the cluster of three external
sensilla neurons was variable. In segment A1 and A2, Gyc-88E and
Gyc-89Db were expressed in one of the two v'td neurons (ventral
tracheal dendrite) (Fig. 8E
Gyc-88E; Fig.
8F Gyc-89Db), which wrap their projections around
specific tracheal branches (Bodmer and Jan,
1987
). The stained neuron in A1 and A2 was always in the
anterior-most position of the two v'td neurons.
In the anterior region of the embryo, Gyc-89Db was expressed in
more cells than Gyc-88E. Gyc-89Db was expressed in four neurons in
the dorsal ganglion (Fig. 8G).
These cells correspond to the cells that were out of the plane of focus in the
preparation shown in Fig. 7F.
The dorsal ganglion innervates a sensory structure, known as the dorsal organ,
that is thought to be the main site of olfaction in larvae
(Stocker, 1994;
Heimbeck et al., 1999
).
Gyc-89Db was also expressed in three large neurons and up to two more
weakly stained cells in the terminal ganglion of the maxillary organ
(Fig. 8H), a structure that
includes several types of sensilla
(Stocker, 1994
).
Gyc-88E was also expressed in the terminal ganglion but was only
expressed in a single neuron (Fig.
8I). The cells of the terminal ganglion that express
Gyc-88E and Gyc-89Db are the same cells that are in the
focal plane in preparations shown in Fig.
7F,G.
In segments A8 and A9, Gyc-88E and Gyc-89Db were
expressed in a subset of neurons that innervate the five sensory cones, which
also have putative chemosensory roles
(Stocker, 1994). The five
sensory cones are named according to their positions one caudal, two
dorsal caudal, one dorsal lateral and one dorsal medial and contain a
combination of trichoid and basiconical sensilla
(Stocker, 1994
). A single
neuron in each of these sensory cones was observed that expressed
Gyc-88E and Gyc-89Db
(Fig. 8J
Gyc-88E; Fig. 8K
Gyc-89Db). These neurons correspond to the cells numbered 2,
3 and 512 in Fig.
7GI. In some cases, it was possible to trace the dendrite
from the neuron that stained for Gyc-88E or Gyc-89Db to the
tip of the sensory cone (Fig.
8K), acharacteristic of chemosensory neurons
(Dambly-Chaudiere et al.,
1992
). The remaining two neurons, numbered 1 and 4 in
Fig. 7GI, had dendrites
that projected in a posterior direction, but we could not determine their
target of innervation because it was not possible to follow them to their
terminus.
![]() |
Discussion |
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---|
A structural feature that Gyc-88E shares with MsGC-ß3 and CP12881 is a
long C-terminal extension (Fig.
1) that is not found in ß1 subunits. The C-terminal
extensions of MsGC-ß3, Gyc-88E and the Anopheles orthologue
CP12881 are highly divergent, except for two conserved stretches of 21 and 10
amino acids, suggesting that these regions play an important role in enzymatic
regulation. Removing the entire C-terminal extension from MsGC-ß3
decreased the Km in the presence of Mg, while no change
was measured in the presence of Mn (Morton
and Anderson, 2003). These results suggested that the C-terminal
domain formed an auto-inhibitory domain in MsGC-ß3
(Morton and Anderson, 2003
).
The estimated values for Km for Gyc-88E were similar to
those for MsGC-ß3, in particular the almost 20-fold reduction in the
presence of Mn compared with Mg, suggesting that the C-terminal domain of
Gyc-88E might also be inhibitory. However, removal of this domain in Gyc-88E
did not produce any change in the kinetic parameters (K.K.L. and D.B.M.,
unpublished data). An interesting feature with a potential role in regulation
that is not found in the C-terminal extensions of MsGC-ß3 or CP12881 are
the phosphorylation motifs located in the seven additional residues found in
the Gyc-88E-L splice variant (Fig.
1A).While no differences in the activity or kinetics were found
between the splice variants in this study, it is possible that phosphorylation
of this site alters Gyc-88E-L activity.
Biochemical properties of Gyc-88E and Gyc-89Db
Gyc-88E shares a number of unusual sequence and structural features with
MsGC-ß3. Firstly, like the receptor guanylyl cyclases and unlike all
known ß1 subunits, both Gyc-88E and MsGC-ß3 possess all of the
residues thought to interact with the Mg-GTP substrate
(Fig. 1B; for a detailed
discussion of homodimer/heterodimer predictions and a model of the catalytic
site, see Morton and Hudson,
2002). Previous studies showed that MsGC-ß3 does yield basal
activity in the absence of other subunits
(Nighorn et al., 1999
) and
forms homodimers (Morton and Anderson,
2003
). These studies formed the basis for the prediction that
Gyc-88E would also yield basal activity in the absence of other subunits, a
prediction demonstrated to be correct in this and a previous study
(Fig. 4;
Morton, 2004
). While
MsGC-ß3 yielded higher levels of basal activity than Gyc-88E, the basal
activity of Gyc-88E was nevertheless similar to that of the Manduca
1/ß1 heterodimer. Unlike all known ß1 subunits and the
mammalian ß2 subunits, both Gyc-88E and MsGC-ß3 have substitutions
at two cysteine residues known to be crucial for heme binding and NO
activation in the rat ß1 subunit
(Friebe et al., 1997
;
Fig. 1B). Extracts made from
COS-7 cells transiently transfected with MsGC-ß3 yielded no increase in
activity over basal levels when NO donors were applied
(Nighorn et al., 1999
). This
observation, together with its sequence features, led to the prediction that
Gyc-88E would also be NO-insensitive
(Morton and Hudson, 2002
).
Preliminary data suggested that this was in fact the case. Although SNP
slightly activated Gyc-88E, another structurally unrelated NO donor,
DEA-NONOate, was ineffective (Morton,
2004). Furthermore, an NO-independent activator of conventional
soluble guanylyl cyclases, YC-1, was also ineffective
(Morton, 2004
). This suggested
that the mechanism of activation of Gyc-88E was quite distinct from that of
conventional
/ß heterodimers and that it was likely to be another
breakdown product of SNP and not NO that activated Gyc-88E. Results from the
present study, however, suggest that NO might be capable of activating
Gyc-88E. Although DEA-NONOate was again found to be ineffective, several other
structurally unrelated NO donors yielded a small but significant increase in
activity. However, compared with the 1020-fold stimulation of
conventional
/ß heterodimers, stimulation of Gyc-88E was only
23-fold. Four different structural classes of NO donors were tested for
their ability to activate Gyc-88E. SNP and two S-nitroso compounds
(SNAP and SNOG) stimulated Gyc-88E whereas three different NONOates and an
unrelated compound, SIN-1, were ineffective.
As predicted and previously reported
(Morton, 2004), Gyc-89Db
showed no activity when expressed alone but formed an active guanylyl cyclase
when co-expressed with Gyc-88E. Here, we show that Gyc-88E and Gyc-89Db are
likely to form heterodimers in vivo. Not only are they co-expressed
in many of the same cells but no activity was detected when Gyc-89Db was
co-expressed with its other predicted partner, the conventional
subunit, Gyc
-99B. It is possible that Gyc-88E and Gyc
-99B
dimerize, as do MsGC-ß3 and MsGC-
1
(Morton and Anderson, 2003
),
but, possibly due to misfolding, these heterodimers are inactive. In addition
to increased basal activity, Gyc-88E and Gyc-89Db yielded higher levels of
activity in the presence of NO donors compared with when Gyc-88E was expressed
alone. As with Gyc-88E, not all NO donors were effective at stimulating the
activity of the Gyc-88E/Gyc-89Db heterodimers, but some compounds (SIN-1 and
two of the NONOates) that were ineffective at stimulating Gyc-88E did
stimulate the heterodimer. Although DEA-NONOate failed to significantly
increase the activity of the heterodimer, it did appear to have a small
positive effect. YC-1 was also ineffective at stimulating the heterodimer.
These results contrast with the effects on the conventional
/ß
heterodimer, where all the compounds tested were effective at stimulating
guanylyl cyclase activity. The fact that several different structural classes
of NO donors were capable of activating both Gyc-88E and the Gyc-88E/Gyc-89Db
heterodimers suggests that NO, rather than another breakdown product of these
compounds, was the active component in these experiments. This conclusion was
strengthened by our finding that 1 mmol l-1 sodium cyanide (SNP
breakdown also produces cyanide ions) failed to stimulate Gyc-88E (data not
shown).
It is unclear why SNP and the S-nitroso compounds were able to
significantly stimulate Gyc-88E while other compounds failed to do so. Also
unknown are why some of the NONOates were capable of stimulating the
Gyc-88E/Gyc-89Db heterodimer whereas DEA-NONOate was ineffective and why none
of the NONOates were capable of activating Gyc-88E. It is notable that SNAP
and SNOG have a considerably longer half-life of NO release than the other NO
donors used (hours rather than minutes), and DEA-NONOate has the shortest
half-life of the NONOates tested. All these compounds were approximately
equally effective at stimulating the conventional
Gyc-99B/Gycß-100B heterodimers at the concentrations used. If this
concentration is supramaximal for the conventional subunits, but submaximal
for Gyc-88E and Gyc-89Db, then the lower concentration of free NO with some of
the donors could explain the data. In addition, although there was no
statistically significant increase in activity of Gyc-88E in the presence of
the NONOates, they all showed a slight increase. Further doseresponse
studies of the different donors might resolve this issue.
Our experiments with ODQ further illustrate differences between the heme
group/regulatory domains of the two soluble guanylyl cyclases studied in this
report and conventional /ß heterodimers. ODQ, which is known to
block NO stimulation of the
/ß heterodimers by oxidation of the
heme group, had no inhibitory effect on the SNP stimulation of Gyc-88E. The
regions of the regulatory domain in Gyc-88E and/or Gyc-89Db that are
responsible for these biochemical differences remain to be discovered.
As the NO donors at most only weakly stimulate Gyc-88E and Gyc-89Db, and
MsGC-ß3 is insensitive to NO donors, it seems unlikely that NO is the
activator of members of the ß3 family in vivo. Although the
nature of the in vivo activator is unknown, there are some
suggestions based on studies of MsGC-ß3. There is circumstantial evidence
that MsGC-ß3 is activated by the neuropeptide eclosion hormone
via a pathway that might be mediated by protein kinases (see
Morton and Simpson, 2002). The
cells that express Gyc-88E, however, do not appear to include likely eclosion
hormone target cells in Drosophila
(Baker et al., 1999
).
Nevertheless, the location of potential phosphorylation sites in conserved
regions of the C-terminal domain of Gyc-88E suggests that phosphorylation
might be a mechanism of activation of this family of guanylyl cyclases.
Localization of Gyc-88E and Gyc-89D expression
Evidence that supports the formation of Gyc-88E and Gyc-89Db heterodimers
in vivo was provided by in situ hybridization using probes
to both subunits simultaneously. In these experiments, the use of both probes
did not label an increased number of cells in the peripheral nervous system
compared with using a single probe. The number of cells stained in thoracic
and abdominal segments was identical when either probe was used individually
or when both probes were used together, suggesting that all these peripheral
cells expressed both Gyc-88E and Gyc-89Db (see
Table 2). In the head segment,
however, the total number of cells that we detected was more variable.
Nevertheless, we never observed more stained cells in the double-probe in
situ experiments than the maximum number of cells observed when probing
for Gyc-89Db alone (see Table
2). These experiments suggest that some of the anterior cells
express both Gyc-88E and Gyc-89Db while others express only
Gyc-89Db. Co-immunoprecipitation experiments are needed to
definitively demonstrate heterodimer formation.
Although there are many cells that appear to co-express Gyc-88E
and Gyc-89Db, there are several places where only one of the cyclases
is expressed. In addition to the peripheral cells in the head segments,
Gyc-89Db was expressed in the CNS at earlier stages than
Gyc-88E. Because Gyc-89Db was only active in the guanylyl cyclase
assays co-expressed with Gyc-88E, it is not clear what the function of
Gyc-89Db is when expressed alone. It is possible that the Gyc-88E
transcript was present in these cells but was present at levels too low to be
detected with in situ experiments or that the Gyc-89Db
transcript was present but was not translated. Alternatively, Gyc-89Db may be
playing a dominant negative role, by heterodimerizing with other soluble
guanylyl cyclase subunits and thus preventing them from dimerizing with
subunits that would yield an active guanylyl cyclase dimer. A similar
situation has been found with MsGC-ß3, which will form inactive
heterodimers in vitro with MsGC-1 and MsGC-ß1
(Morton and Anderson,
2003
).
In the present study, we have primarily focused on the expression of
Gyc-88E and Gyc-89Db in the embryonic peripheral nervous
system, where they are co-expressed in a subset of peripheral neurons. We were
able to identify several of the cells staining for both guanylyl cyclases in
segments T2 and T3 as one of three neurons (les in the lateral cluster and ves
in the ventral cluster) that innervate the basiconical sensilla. Basiconical
sensilla are external club-like structures with pore-like openings to the
outside environment and have a putative hygroreceptor or chemosensory role
(Stocker, 1994;
Younossi-Hartenstein and Hartenstein,
1997
). We also found neurons staining for both guanylyl cyclases
in segment A8 and A9 that innervate all of the posterior cone-shaped external
sensilla (Campos-Ortega and Hartenstein,
1997
). We could not, however, specifically name these neurons, as
they are part of large clusters that have not been characterized in great
detail. These sensory cones possess both trichoid and basiconical sensilla
(Campos-Ortega and Hartenstein,
1997
). Consistent with a role in chemoreception, we observed a
neuronal cell body stained for guanylyl cyclase that extended a single
dendrite to the very tip of a sensillum
(Fig. 8K), a distinguishing
feature of chemosensory neurons
(Dambly-Chaudiere et al.,
1992
). In segments A1 and A2, Gyc-88E and
Gyc-89Db were expressed in the anterior-most of the two v'td neurons,
each of which innervates specific non-overlapping tracheal branches. The
function of these neurons is not known. The double-label experiments also
allowed us to determine the location of the guanylyl cyclase-expressing cells
we observed in the head segment. Gyc-88E stained one neuron in each
of the two terminal ganglia while Gyc-89Db stained 25 neurons
in each of the terminal ganglia and 45 neurons in each of the two
dorsal ganglia. The terminal ganglion innervates the maxillary organ, which is
known to serve a gustatory function and has at least six different types of
sensilla (Stocker, 1994
;
Heimbeck et al., 1999
;
Oppliger et al., 2000
). The
dorsal ganglion innervates the dorsal or antennal organ, which consists of
seven different sensilla and is the main olfactory organ in larval
Drosophila (Stocker,
1994
; Heinbeck et al., 1999;
Oppliger et al., 2000
). It was
not possible to determine which sensilla were innervated by the neurons that
expressed Gyc-88E and Gyc-89Db.
Expression of Gyc-88E and Gyc-89Db in peripheral neurons
that innervate various external sensilla and the trachea was also detected in
newly hatched first-instar larvae (data not shown), suggesting that these
guanylyl cyclases play a role in sensory transduction during larval life.
Guanylyl cyclases and cGMP signaling have been demonstrated to play an
important role in several types of sensory transduction in both vertebrates
and invertebrates (reviewed in Kramer and
Molokanova, 2001; Morton and
Hudson, 2002
). For example, cGMP produced by a receptor guanylyl
cyclase is the primary signal molecule in vertebrate phototransduction
(Kramer and Molokanova, 2001
).
In Drosophila, cGMP appears to play a modulatory role in
phototransduction and olfaction, rather than being involved in the primary
transduction pathway (Bacigalupo et al.,
1995
; Morton and Hudson,
2002
). In the silkmoth (Bombyx mori), soluble and
particulate guanylyl cyclase activity was measured in the antennae, and in
Manduca the receptor-like guanylyl cyclase MsGC-I was detected in
olfactory receptor neurons (Nighorn et
al., 2001
). In C. elegans, several different receptor
guanylyl cyclases are expressed in olfactory neurons
(Yu et al., 1997
). Two of
these, ODR-1 and DAF-11, are co-expressed in the chemosensory neuron AWC, and
mutations to either gene resulted in the abolishment of chemotaxis to all
AWC-sensed odorants (Birnby et al., 2001;
L'Etoile and Bargmann,
2000
).
Another possible role for the cGMP formed in neurons by Gyc-88E and
Gyc-89Db is axonal path-finding (Schmidt
et al., 2002). While Gyc-88E expression was detectable
only in later embryonic stages when most axonal path-finding events have
already occurred, Gyc-89Db expression was detectable in the
peripheral nervous system at stages that coincide with axonal path-finding
events (stage 1316; Campos-Ortega
and Hartenstein, 1997
). Thus, it is possible that Gyc-89Db plays a
role in both peripheral nervous system development and sensory
transduction.
Both Gyc-88E and Gyc-89Db were also expressed in the
embryonic and larval central nervous systems. Gyc-89Db expression in
the CNS began as early as stage 12 (data not shown) and continued at a
constant level through embryogenesis. Gyc-88E expression in the CNS
was first detectable at stage 15 or 16. Gyc-88E expression continued
throughout embryogenesis but expanded to an increased number of cells
throughout the CNS by the end of stage 17. Both Gyc-88E and
Gyc-89Db were expressed in a number of cells in the brain and VNC of
third-instar larvae. At this point it is difficult to speculate on the
function of these guanylyl cyclases in the nervous system during embryonic
development and in larvae, but roles for cGMP signaling have been demonstrated
in axon guidance (Nishiyama et al.,
2003), synapse formation
(Leamey et al., 2001
;
Gibbs et al., 2001
) and cell
migration (Haase and Bicker,
2003
).
The discovery of atypical soluble guanylyl cyclases that are insensitive or relatively insensitive to NO in Drosophila, Manduca and C. elegans suggests the existence of novel pathways upstream of soluble guanylyl cyclase that do not involve NO. The presence of atypical guanylyl cyclases in neurons of the peripheral nervous system of Drosophila that are amenable to physiological and genetic experimentation should provide new avenues to examine the function and regulation of these unusual enzymes.
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Acknowledgments |
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