From the Arizona Research Labs, Division of
Neurobiology, The University of Arizona, Tucson, Arizona 85721 and
the § Department of Biological Structure and Function,
Oregon Health Sciences University, School of Dentistry,
Portland, Oregon 97201-3097
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
We have identified a novel guanylyl cyclase,
named MsGC-I, that is expressed in the nervous system of Manduca
sexta. MsGC-I shows highest sequence identity with receptor
guanylyl cyclases throughout its catalytic and dimerization domains but
does not contain the ligand-binding, transmembrane, or kinase-like
domains characteristic of receptor guanylyl cyclases. In addition,
MsGC-I contains a C-terminal extension of 149 amino acids that is not present in other receptor guanylyl cyclases. The sequence of MsGC-I contains no regions that show similarity to the regulatory domain of
soluble guanylyl cyclases. Thus, MsGC-I appears to represent a member
of a new class of guanylyl cyclases. We show that both a transcript and
a protein of the sizes predicted from the MsGC-I cDNA are present
in the nervous system of Manduca and that MsGC-I is
expressed in a small population of neurons within the abdominal ganglia. When expressed in COS-7 cells, MsGC-I appears to exist as a
soluble homodimer with high levels of basal guanylyl cyclase activity
that is insensitive to stimulation by nitric oxide. Western blot
analysis, however, shows that MsGC-I is localized to the particulate
fraction of nervous system homogenates, suggesting that it may be
membrane-associated in vivo.
The intracellular messenger 3',5'-cyclic guanosine monophosphate
(cGMP) plays an important role in numerous physiological functions,
including visual and chemosensory signal transduction, control of fluid
and ion transport, smooth muscle relaxation, and the modulation of
synaptic efficacy (1-4). The enzyme responsible for cGMP synthesis is
guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2).
Currently, guanylyl cyclases are classified as one of two distinct
enzymatic forms, soluble or receptor, based upon their cellular
distribution and structure. Soluble guanylyl cyclases are localized
within the cell cytoplasm, where they function as heterodimers composed
of Receptor guanylyl cyclases are transmembrane proteins thought to act
primarily as homodimers although they have been shown to form trimers,
tetramers, and other higher oligomer units (8). All known receptor
guanylyl cyclases are glycoproteins containing five functional domains:
an extracellular ligand-binding domain, a single transmembrane domain,
a kinase-like regulatory domain, a dimerization domain, and a catalytic
domain (9, 10). At present there are seven known mammalian receptor
guanylyl cyclase isoforms, named GC-A through GC-G (3, 13), and
recently a family of at least 26 different putative receptor guanylyl
cyclases has been identified in Caenorhabditis elegans (3).
Receptor guanylyl cyclases are generally activated through the binding of an extracellular peptide ligand, although the retinal guanylyl cyclases can be activated through a decline in intracellular calcium levels (11). In addition, ATP, which binds to a distinct site within
the kinase-like domain (12), can also modulate their activity.
Recent reports have suggested the existence of additional forms of
guanylyl cyclase that are cytoplasmically localized yet insensitive to
NO. An unusual guanylyl cyclase, designated ksGC (kinase-like domain containing soluble
guanylyl cyclase), has been cloned from rat
kidney cells (14). From DNA sequence analysis, this clone appears to
contain the kinase-like, dimerization and catalytic domains
characteristic of receptor guanylyl cyclases but contains no
ligand-binding or transmembrane domains. This indicates that ksGC is a
cytoplasmically localized guanylyl cyclase that is insensitive to NO.
These sequence-based predictions have not yet been tested, as the
putative protein encoded by this cDNA is enzymatically inactive
when expressed in heterologous cells. Another cytoplasmically localized
NO-insensitive guanylyl cyclase activity has also recently been
reported in the nervous system of lobsters, which can be separated from
a less prevalent NO sensitive form by anion exchange high performance
liquid chromatography (15).
Here we describe the cloning and characterization of a novel form of
guanylyl cyclase, from the nervous system of the insect, Manduca
sexta. This cyclase, named MsGC-I, shows highest sequence identity
with the receptor guanylyl cyclase, GC-B, throughout its catalytic and
dimerization domains, but does not contain the other domains associated
with receptor guanylyl cyclases. The discovery and characterization of
this novel guanylyl cyclase has the potential to expand the known
mechanisms of cGMP regulation.
Animals--
The rearing and staging of M. sexta have
been described previously (16).
Isolation of the MsGC-I cDNA Clone--
MsGC-I was isolated
using a degenerate oligonucleotide reverse transcriptase-polymerase
chain reaction approach to identify guanylyl cyclases in the abdominal
nervous system of M. sexta. Degenerate oligonucleotide
primers were designed against the amino acid sequences DVYKVETI
(CCRAAIARRCARTAICKNGGCAT) and MPRYCLFG (GAYGTITAYAARGTIGWIACNAT) from
the catalytic domain common to both soluble and receptor guanylyl
cyclases. RNA isolation, reverse transcriptase-polymerase chain
reaction, cDNA library construction and screening, sequencing, and
Northern blot analysis were carried out using conventional procedures
described previously (16).
COS-7 Cell Expression, Guanylyl Cyclase Activity Assay, and cGMP
Measurement--
The full-length open reading frame of MsGC-I was
subcloned into the mammalian expression vector pcDNA3.1
(Invitrogen) and transiently transfected into COS-7 cells (1 µg of
DNA/35-mm dish) using LipofectAMINE (Life Technologies, Inc.). Cells
were harvested in homogenization buffer (50 mM Tris-HCl, pH
7.9) 60 h after transfection and assayed for guanylyl cyclase
activity as described previously (16). To separate soluble and
particulate fractions, 0.25 M sucrose was included in the
homogenization solution, and the homogenate was centrifuged at
100,000 × g for 1 h at 4 °C. To assay cGMP levels in intact COS-7 cells, cells were plated onto 24-well plates, and each well was transfected with 0.2 µg of plasmid. After 3 days,
the cells were incubated in saline (120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 25 mM Tris-HCl, 15 mM glucose, pH 7.4) for 30 min
at 37 °C followed by a further 5 min in either the presence or
absence of 1 mM sodium nitroprusside (SNP). The saline was then removed and the cells lysed in acidified ethanol (ethanol:1 M HCl, 100:1). Following centrifugation, the supernatant
was lyophilized, redissolved in 50 mM sodium acetate, pH
6.2, and assayed for cGMP content using a commercial radioimmunoassay
kit (NEN Life Science Products).
Size Exclusion HPLC--
Transfected COS-7 cells were harvested
in separation buffer (100 mM NaCl, 25 mM HEPES,
pH 7.4, and 10 mM dithiothreitol) containing a protease
inhibitor mixture (4-(2-aminoethyl) benzenesulfonyl fluoride,
trans-epoxysuccinyl-L-leucylamido (4-guanidine) butane, bestatin, leupeptin, aprotinin, and sodium EDTA; 0.1 mg/ml, Sigma) at a
concentration of 2.3 × 107 cells/ml. After
homogenization, cells were centrifuged for 5 min at 1,000 × g to remove large particulate matter followed by further
centrifugation of the supernatant at 100,000 × g for
15 min at 4 °C. The resulting high speed supernatant was further separated using a Bio-Sil SEC-125 size-exclusion column in separation buffer using a flow rate of 0.5 ml/min. Fractions were collected every
0.2 min (100 µl) and tested for guanylyl cyclase activity.
Antiserum Production--
A glutathione S-transferase
(GST) fusion protein of MsGC-I was made by ligating a 3' fragment of
the MsGC-I cDNA clone (bases 1172-2560, see Fig. 1) into the
GST-fusion vector pGEX 4T-2 (Amersham Pharmacia Biotech). The resulting
protein was analyzed by Western blot using anti-GST antisera (Amersham
Pharmacia Biotech) and shown to be of the predicted 42-kDa size. The
protein preparation was lyophilized and sent to HTI Bio-Products Inc.
(Ramona, Ca) for the production of antisera.
Western Blot Analysis--
For Manduca nervous system
Western blots, 20 abdominal central nervous systems were homogenized in
1 ml of buffer (50 mM Tris-HCl, pH 7.9, 0.25 M
sucrose) plus protease inhibitor mixture (described above). The samples
were separated by SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride membrane. Primary antiserum
was used at 1:1000 and secondary antibody (goat anti-rabbit-HRP, Jackson Laboratories) at 1:10,000. The location of the immunoreactive bands was detected through chemiluminescence and the subsequent exposure of the blot to film. Western blot analysis of transfected COS-7 cells was performed in the same manner except that six 35-mm plates were harvested and homogenized in 1 ml of buffer, and primary antiserum was diluted 1:20,000.
Riboprobe Generation and in Situ
Hybridization--
Digoxigenin-labeled riboprobes were generated for
use in in situ hybridization as described by Komminoth (17).
The entire linearized cDNA clone for MsGCI was used as a template.
The resulting riboprobes were hydrolyzed to an average size of 150 nucleotides using alkaline hydrolysis. Fragment size was evaluated by
formaldehyde-agarose gel electrophoresis. Probe concentration was
estimated by dot blot assay (Boehringer Mannheim) and used at a
concentration of 40-400 pg/ml.
For in situ hybridization, we used a method modified from
that of Bhatt et al. (18). Abdominal central nervous tissue
from prepupal insects was fixed overnight at 4 °C in 4%
paraformaldehyde. Modifications to the permeabilization protocol
included: addition of a collagenase treatment (2 mg/ml, Sigma type IV)
for 30 min at room temperature prior to exposing the tissue to HCl and
increasing the proteinase K concentration to 100 mg/ml. The composition
of the hybridization solution was also modified to: 5× saline/sodium phosphate/EDTA, 50% formamide, 5% dextran sulfate, 1× Denhardt's solution, 500 mg/ml sonicated salmon sperm DNA, 250 mg/ml yeast tRNA.
Tissue was pre-hybridized in this solution for 1-2 h at 50 °C,
denatured probe was then added, and samples were incubated overnight at
50 °C. Wash steps following RNase treatment were modified by the
addition of a wash in 1× SSC at room temperature for 30 min, followed
by three washes in 0.1× SSC for 20 min each at 50 °C prior to
blocking. Two-percent cold-water fish gelatin was added to the blocking
solution. Alkaline phosphatase-conjugated anti-digoxigenin antibody was
used at a 1:2,000 dilution and incubated overnight at 4 °C.
Following the wash steps, the alkaline phosphatase-conjugated antibody
was detected using a BCIP/nitro blue tetrazolium substrate solution
(Amresco), incubated in the dark for between 1-4 h.
Immunocytochemistry--
Immunocytochemical staining of
Manduca ventral nerve cords was performed using a
whole-mount protocol adapted from Davis et al. (19). Tissue
was fixed overnight at 4 °C in 4% paraformaldehyde followed by
extensive washing in phosphate-buffered saline containing 0.5% Triton
X-100 (PBST). Tissue was blocked in PBST containing 10% normal goat
serum followed by incubation in primary antiserum at 1:2,500 at room
temperature overnight. The secondary antibody (rhodamine-labeled goat
anti-rabbit) was used at 1:250 at room temperature overnight. Digitized
images were prepared using a Bio-Rad MCR 600 laser-scanning confocal microscope.
Cloning and Sequence Analysis of MsGC-I--
We have used RT-PCR
with degenerate oligonucleotides designed to a conserved region of the
catalytic domain to identify guanylyl cyclases from the nervous system
of M. sexta and have isolated fragments of eight different
cyclases. Cloning and characterization of three of these, which belong
to the soluble guanylyl cyclase class, have already been reported (16,
20). Here we report the cloning of a novel receptor-like guanylyl
cyclase, named MsGC-I. Based on sequence analysis, MsGC-I appears to
create a new class of guanylyl cyclase, which does not fit into either
the soluble or receptor guanylyl cyclase class.
We have screened two independent cDNA libraries made from
Manduca abdominal central nervous tissue and obtained five
identical full-length copies of the MsGC-I clone. The full-length
MsGC-I cDNA is 2,560-base pairs long and contains a 1,500-base pair
open reading frame. The open reading frame begins with an initiator methionine at position 108 and ends with a stop codon at position 1,608 followed by a 3'-untranslated region and a poly(A)+ tail.
One in-frame and several out-of-frame stop codons precede the start of
the open reading frame. The full-length sequence of MsGC-I has been
placed into GenBankTM under accession number AF073342.
The MsGC-I open reading frame translates into a predicted 500-amino
acid protein that displays highest similarity to atrial natriuretic
peptide receptor-B (GC-B) by BLAST analysis. Fig. 1 shows the protein sequence alignment of
MsGC-I to GC-B. As can be seen in this figure, MsGC-I shows high
similarity (76-77% identity) to GC-B within the catalytic domain
(GC-B, 840-1047; MsGC-I, 144-350), and putative dimerization domain
(GC-B, 798-839; MsGC-I, 102-143). There are, however, only 101 amino
acids preceding the start of the dimerization domain of MsGC-I, and
these do not appear to contain a signal sequence or the ligand-binding,
transmembrane, or kinase-like domains characteristic of receptor
guanylyl cyclases. The MsGC-I protein appears to begin within the
C-terminal region of the kinase-like domain of GC-B. Throughout this
region MsGC-I shows only 12% identity with the GC-B kinase-like
domain, and visual examination of the sequence reveals no consensus ATP
binding site (G-X-X-X-G; Ref 12). A
novel sequence is also found at the C terminus of MsGC-I, which extends
149 amino acids beyond the end of the catalytic domain. This domain
does not show significant homology to any proteins found within the
NCBI data base.
A phylogenetic tree analysis of receptor and soluble guanylyl cyclase
catalytic domains groups the catalytic domain of MsGC-I with the
catalytic domains of receptor guanylyl cyclases. When compared in a
pairwise fashion, MsGC-I shows only 33% identity with the catalytic
domain of soluble cyclases compared with 77% identity with GC-B. The
N-terminal end of MsGC-I also shows no significant similarity with the
sequences containing the heme-binding and heterodimerization regions of
the soluble guanylyl cyclases (21, 22). Thus, sequence analysis
suggests that MsGC-I is a novel guanylyl cyclase that is most similar
to receptor cyclases within the catalytic and dimerization domains but
does not contain sequences that would allow either membrane
localization or stimulation by ligand binding.
Northern Blot Analysis of MsGC-I Transcripts--
To confirm that
the MsGC-I cDNA represented a full-length clone rather than a
truncated version of a receptor guanylyl cyclase, we performed Northern
blot analysis using a series of probes designed to hybridize to all
portions of the MsGC-I cDNA. These results are shown in Fig.
2. All of the probes hybridized to a
2.5-kb transcript, the size predicted from the MsGC-I cDNA. An
additional, longer transcript of 4 kb was also labeled by probes made
to the highly conserved catalytic domain. We do not know what this
larger transcript represents. These results demonstrate that a
transcript of the predicted size for MsGC-I is made within the
Manduca nervous system and suggest that MsGC-I is not a
truncated version of a larger receptor guanylyl cyclase.
Expression of MsGC-I in COS-7 Cells--
To examine the enzymatic
properties of MsGC-I, we subcloned its open reading frame into the
mammalian expression vector pcDNA3.1 and transiently transfected
COS-7 cells with this construct. Cell extracts were examined for
guanylyl cyclase activity in the presence of both Mg2+ and
Mn2+ as guanylyl cyclases show different levels of activity
in the presence of these two cations (23). No guanylyl cyclase activity could be detected in untransfected COS-7 cells, cells treated with
LipofectAMINE alone, or cells transfected with the control pcDNA3.1
vector (data not shown). By contrast, Fig.
3A shows that COS-7 cells
transfected with the MsGC-I construct have significant guanylyl cyclase
activity. The guanylyl cyclase activity of MsGC-I is much higher in the
presence of Mn2+ compared with Mg2+, and in
neither case is this activity stimulated by the NO donor, SNP. As a
positive control for SNP stimulation, we also tested the activity of
COS-7 cells co-transfected with vectors containing the
Manduca homologues of mammalian
Loss of heme, and thus loss of NO sensitivity, is a common phenomenon
that occurs during purification of soluble guanylyl cyclase (22). To
determine whether a similar phenomenon was responsible for the lack of
NO stimulation of MsGC-I, we measured the NO sensitivity of MsGC-I in
intact COS-7 cells. We exposed intact transfected COS-7 cells to SNP
and then determined the level of cGMP within the cells. Again
MsGC-I-transfected COS-7 cells showed no response to SNP (Table
I), whereas COS-7 cells co-transfected
with the M. sexta
To determine the cellular localization of MsGC-I in these transfected
COS-7 cells, we used ultracentrifugation to separate the homogenates
before assaying the guanylyl cyclase activity. Fig. 3B shows
that the majority of the activity is in the supernatant, suggesting
that MsGC-I is cytoplasmically localized in this heterologous cell system.
MsGC-I contains a sequence with high similarity to a region in GC-A
known to function as a dimerization domain (9). To determine whether
MsGC-I functioned as a homodimer, we measured its apparent molecular
weight using high performance liquid chromatography gel filtration. The
results of this experiment are shown in Fig. 3C. The
predicted size of the MsGC-I monomer protein, based on sequence
analysis, is 55 kDa. The majority of the enzyme activity in transfected
COS-7 cell homogenates eluted in fractions corresponding to the
predicted sizes of both a dimer (Mr = 110,000)
and a trimer (Mr = 165,000), with no activity
detectable in fractions eluting at the predicted elution time for a
monomer protein.
Western Blot Analysis of the MsGC-I Protein--
To determine
whether a protein of the predicted size is present in the nervous
system of Manduca, we generated antisera to the C-terminal
portion of the protein and carried out Western blot analysis. Fig.
4A shows the results of
Western blot analysis on COS-7 cells that had been transfected with
either MsGC-I, or one of the Manduca soluble guanylyl
cyclase subunits (MsGC-
Using the same antiserum to examine MsGC-I protein products in
Manduca abdominal nervous system extracts (Fig.
4B), we again detected two bands. One of these was of the
predicted 55-kDa size, whereas the other was much smaller, less than 36 kDa. In the absence of protease inhibitors, the abundance of the
smaller band was greatly enhanced while the abundance of the 55-kDa
band was decreased, indicating that the smaller band was the product of
protease activity (data not shown). No bands were detected by
incubation of the blot with pre-immune serum. Furthermore,
pre-incubation of the antisera with the GST-MsGC-I fusion protein
almost completely abolished both the 55- and the 33-kDa bands. The
presence of a band with the predicted size for the MsGC-I protein
product in both transfected COS-7 cells and Manduca nervous
tissue extracts strongly supports the idea that the MsGC-I mRNA is
translated into a protein of the predicted size within the
Manduca central nervous system. We also examined the
localization of the MsGC-I protein by separating extracts of
Manduca nervous tissue into soluble and particulate
fractions. Although MsGC-I was localized in the 100,000 × g supernatant in COS-7 cells, as shown by both activity
measurements (Fig. 3B) and Western blots (data not shown), in Manduca nervous system extracts it appears to be present
in the particulate fraction (Fig. 4B).
Localization of MsGC-I Transcript and Protein within the Abdominal
CNS--
We used in situ hybridization and
immunocytochemical staining to localize the expression of MsGC-I within
abdominal ganglia of Manduca. In situ
hybridization was performed using riboprobes made in both the sense and
antisense directions to the full-length MsGC-I cDNA. Incubation of
abdominal ganglia with riboprobe made in the antisense direction
revealed a distinct set of neuronal somata staining in each abdominal
ganglion. The most consistent staining of MsGC-I transcript was seen in
the second through the fifth abdominal ganglia, where two to five
bilateral pairs of cells, localized in the lateral/posterior portion of
each ganglion, were stained (Fig.
5A). In some preparations,
other larger, potential lateral neurosecretory cells were also
detected, as were some bilateral midline cells. No staining of somata
was ever detected when using a control riboprobe made in the sense
direction (Fig. 5B).
We have also examined localization of the MsGC-I protein within the
nervous system through immunocytochemistry. This method showed that
localization of the MsGC-I protein was generally consistent with the
localization of the MsGC-I transcript. Strong staining was consistently
observed within somata localized to the lateral/posterior portions of
each abdominal ganglion (Fig. 5C). In addition, in many
preparations, cells localized in the anterior parts of the ganglia were
also detected. Projections from stained neurons leading into the
neuropil, and the neuropil itself, were also frequently stained,
suggesting that the MsGC-I protein may be localized within neuronal
projections as well as in cell bodies. No staining in somata or
neuropil was ever detected in response to incubation of tissue with
pre-immune serum (Fig. 5D).
This paper describes the cloning and characterization of a novel
guanylyl cyclase isoform, which we have named MsGC-I. Previously described guanylyl cyclases have been classified as either receptor or
soluble, based on their intracellular localization and general structure. Receptor guanylyl cyclases are membrane bound and primarily activated by ligand binding. Soluble guanylyl cyclases are
cytoplasmically localized and primarily activated by NO. MsGC-I does
not fit into either of these classifications and thus may define a new
class of guanylyl cyclase. The catalytic domain of MsGC-I appears most similar to those of receptor cyclases, specifically GC-B, but it does
not contain a signal sequence or the ligand-binding, transmembrane and
kinase-like domains of previously identified receptor guanylyl cyclases. In addition, MsGC-I shows no similarity to the regulatory domain of soluble guanylyl cyclases. It also contains a 149-amino acid
extension beyond the catalytic domain that has no similarity to any
protein in the data bases and has no known function.
The guanylyl cyclase most similar in domain structure to MsGC-I is
ksGC, cloned from rat kidney cells (14). Sequence analysis of ksGC
cDNA shows that it is most closely related to receptor guanylyl
cyclases and contains catalytic, dimerization and kinase-like domains
yet contains no ligand-binding or transmembrane domains. It has been
suggested, however, that ksGC is a partial-length cDNA of longer
guanylyl cyclase, specifically the recently described GC-G (13). This
is based on both sequence similarity between the two cDNAs and the
fact that ksGC has not been shown to produce a functionally active
protein. MsGC-I, on the other hand, is clearly not a cloning artifact.
The cDNA was independently isolated five times from two different
cDNA libraries, and both a transcript and a protein of the
predicted sizes for MsGC-I have been shown to be present in the
Manduca nervous system using northern and Western blot analysis.
In situ hybridization and immunocytochemistry show that
MsGC-I is expressed in a small population of neurons within the
abdominal ganglia. In situ hybridization identifies two to
five pairs of cells in the lateral posterior portion of the ganglia
that express MsGC-I. Immunocytochemical localization also reveals these
cells and some additional cells in the anterior portion of the ganglia. These cells are predicted to be interneurons based upon their size and
the fact that no labeled process could be seen leaving the ganglia via
the nerve roots. The localization of the posterior lateral cells
suggests that they might be lateral neurosecretory cells although
unambiguous identification of these cells is not possible at this time.
We have also shown that the cDNA for MsGC-I encodes a functionally
active guanylyl cyclase by expressing it in COS-7 cells. In COS-7
cells, MsGC-I shows high basal guanylyl cyclase activity, which is much
higher when Mn-GTP is provided as a substrate rather than Mg-GTP. This
is a similar property to the enzyme activity seen for receptor guanylyl
cyclases (23). Although it is common for guanylyl cyclases to show
higher basal activity when Mn-GTP is provided as a substrate, it is
likely that Mg-GTP is the substrate used in vivo. To examine
the activity of MsGC-I within COS-7 cells, we also measured the
accumulation of cGMP in intact COS-7 cells that had been transfected
with MsGC-I. These experiments showed that MsGC-I had unexpectedly high
levels of basal activity Based on the sequence of MsGC-I, we predicted that it cannot be
activated by either extracellular ligand-binding or stimulation by NO,
the most well known mechanisms known to increase guanylyl cyclase
activity. Experiments using transfected COS-7 cells support these
predictions, that MsGC-I activity is in the soluble fraction of cell
homogenates and MsGC-I cannot be stimulated by exposure to SNP. In
addition, the accumulation of cGMP in intact COS-7 cells is insensitive
to SNP stimulation. Another sequence-based prediction is that MsGC-I
should form homodimers. Wilson and Chinkers (9) have demonstrated that
a 42-amino acid region within the receptor guanylyl cyclase, GC-A, is
capable of mediating homodimer formation and appears to form an
amphipathic helix. Of these 42 amino acids, 40 are conserved in GC-B,
suggesting that this region in GC-B is also capable of mediating
homodimer formation. Thirty-two of these amino acids are conserved in
MsGC-I, and molecular modeling predicts that they form an Ultracentrifugation of COS-7 cell homogenates shows that MsGC-I is
located in the cytoplasm of these cells. Western blot analysis, however, shows that, in the nervous system of Manduca,
MsGC-I is present in the particulate fraction suggesting association with membranes. Although there are two potential sites for fatty acylation within the MsGC-I sequence, they are not localized at either
end of the protein, suggesting that neither represent true fatty
acylation sites. In addition, fatty acyl-mediated protein localization
has been demonstrated in heterologously expressed proteins in COS-7
cells (e.g. Ref. 24), yet in COS-7 cells MsGC-I is
cytoplasmically located. Thus, it seems more likely that, in vivo, MsGC-I is localized to the membrane through an interaction with another protein. One possible candidate for this protein is a
receptor guanylyl cyclase. In addition to forming homodimers, receptor
guanylyl cyclases can also form heterodimers (8), indicating that
MsGC-I could be localized to membranes by forming a heterodimer with an
endogenous receptor guanylyl cyclase. Another possibility is that the
unique C-terminal domain of MsGC-I interacts with an unknown membrane protein.
The photoreceptor-specific receptor guanylyl cyclases, GC-E and GC-F
(RetGC-1 and RetGC-2 in humans) are regulated by their interactions
with a heterologous class of proteins In summary, these findings identify MsGC-I as a member of a new class
of guanylyl cyclase. Based on its structure and enzyme activity in
heterologous cells, we have shown that it cannot be directly activated
by either extracellular peptide ligands or NO. This suggests a novel
mechanism for the regulation of intracellular cGMP.
INTRODUCTION
Top
Abstract
Introduction
References
and
subunits. Each subunit consists of a regulatory domain,
which contains sequences responsible for heme binding and heterodimer
formation, and a catalytic domain (5). The soluble guanylyl
cyclases contain an attached protoporphyrin-IX-type heme prosthetic
group that is required for activation. The best characterized activator
of soluble guanylyl cyclase is nitric oxide
(NO)1 which binds to the iron
within the attached heme moiety, resulting in a conformational change
in the protein and increased enzymatic activity (6). Other factors such
as carbon monoxide have also been shown to activate soluble guanylyl
cyclase (7) by a similar mechanism.
MATERIALS AND METHODS
RESULTS
View larger version (60K):
[in a new window]
Fig. 1.
A, schematic diagram of the predicted
protein structure of MsGC-I compared with the receptor guanylyl
cyclase, GC-B. Domains are identified and percent identity between the
two sequences is shown below (SS, signal sequence;
TM, transmembrane domain; Dimer., dimerization
domain). Also shown is the region of the protein used in constructing
the GST fusion protein. B, predicted amino acid sequence of
MsGC-I and alignment with GC-B. Shaded residues are
conserved between the two proteins.
View larger version (75K):
[in a new window]
Fig. 2.
A, schematic diagram of the relationship
between MsGC-I and three of the probes used for Northern blot analysis.
The upper figure is a schematic of the entire MsGC-I
cDNA showing the open reading frame. The probes are positioned
below the regions that they were designed to complement.
Probe a was designed to complement base pairs 1-623;
b, base pairs 720-844; and c, base pairs
1491-2560. Also shown is the position of the polymerase chain reaction
fragment amplified from the highly conserved catalytic region.
B, Northern blot analysis of the MsGC-I transcript in the
M. sexta abdominal central nervous system shows the presence
of a 2.5-kb transcript with all three probes. Five µg of
poly(A)+ RNA was separated on a 1% formaldehyde-agarose
gel and blotted onto a Zetaprobe membrane and probed with
32P-labeled DNA probes shown in panel A. All
probes hybridized to a band of the predicted 2.5-kb size, whereas those
designed against the conserved catalytic domain also hybridized to a
larger 4-kb band.
1 and
1 soluble
guanylyl cyclase subunits (16). In this case, clear SNP-stimulated
guanylyl cyclase activity could be measured in the presence of
Mg2+.
View larger version (26K):
[in a new window]
Fig. 3.
A, guanylyl cyclase activity in
transfected COS-7 cells. COS-7 cells were transiently transfected with
either MsGC-I or co-transfected with MsGC- 1 and MsGC-
1 in the
expression vector pcDNA3.1. Enzyme activity was measured in the
presence of either 4 mM Mg2+ or 4 mM Mn2+ as shown and in the presence
(filled bars) or absence (open bars) of 100 µM SNP. Values reported are the mean ± S.E. of four
to nine determinations. B, subcellular localization of
MsGC-I in COS-7 cells. COS-7 cells were transiently transfected with
MsGC-I, homogenized, and separated by centrifugation at 100,000 × g for 1 h at 4 °C. Guanylyl cyclase assays were
performed on the whole homogenate (Homog.), the supernatant
(Sup.), and the pellet in the presence of 4 mM
Mn2+. Values are the mean ± S.E. of four
determinations. C, gel filtration of MsGC-I. COS-7 cells
were transfected and homogenized, and the particulate and soluble
fractions were separated. The supernatant was further separated by gel
filtration, and fractions were assayed for guanylyl cyclase activity in
the presence of 4 mM Mn2+. Molecular weights of
the active fractions were calculated based on the elution times of
known standards run under the same conditions (see
inset).
1 and
1 subunits clearly showed SNP-stimulated activity under these same conditions. It is interesting to note the high basal guanylyl cyclase activity of MsGC-I under these
conditions: cells transfected with MsGC-I accumulated a similar level
of cGMP compared with cells cotransfected with MsGC-
1 and MsGC-
1
and then stimulated with SNP.
Activity of MsGC-I in intact COS-7 cells
1, MsGC-
1, and MsGC-
3). Two bands were
detected in COS-7 cells that had been transfected with MsGC-I. One of
these bands was of the predicted size for the MsGC-I protein product,
approximately 55 kDa, whereas the other band was smaller, appearing to
be slightly less than 44 kDa. No immunoreactive bands were detected in
any of the samples from COS-7 cells transfected with other cloned guanylyl cyclases nor when the blot was incubated with pre-immune serum, suggesting that the antiserum was specific for MsGC-I.
View larger version (73K):
[in a new window]
Fig. 4.
A, Western blot analysis of transfected
COS-7 cells. Cells were transfected with MsGC-I or the
Manduca soluble guanylyl cyclase subunits, MsGC- 1,
MsGC-
1, and MsGC-
3 as shown. The first lane was
incubated in pre-immune serum, whereas the last four
lanes were incubated with anti-GST-MsGC-I antiserum. Molecular
mass markers (in kDa) are also shown. COS-7 cells transfected with
MsGC-I show an immunoreactive band at the predicted size of 55 kDa.
B, Western blot analysis of Manduca abdominal
nervous system homogenates. Tissue extracts, whole homogenates
(H), supernatant (S), or pellet (P),
were separated by SDS-polyacrylamide gel electrophoresis, transferred
to membrane, and incubated with either pre-immune serum or with
anti-GST-MsGC-I antiserum. Samples of whole homogenates and pellets
show the presence of an immunoreactive band at the predicted size of 55 kDa.
View larger version (95K):
[in a new window]
Fig. 5.
Localization of cells expressing MsGC-I.
A and B, localization of the MsGC-I transcript by
in situ hybridization. MsGC-I riboprobes were made in both
the sense (A) and antisense (B) directions and
in situ hybridization performed on prepupal
Manduca abdominal ganglia. Two groups of cells in the
lateral/posterior portion of the ganglion are seen upon hybridization
with the antisense riboprobe. No cell body staining is seen with the
control sense probe. C and D, localization of the
MsGC-I protein with immunocytochemistry. Manduca abdominal
ganglia were incubated with antiserum raised against the GST-MsGC-I
fusion protein (C) or pre-immune serum (D). Cell
bodies in the lateral/posterior portion of the ganglion are stained
most intensely upon exposure to antisera. Staining is also seen in more
anterior cell bodies and neuropil. No staining is seen in response to
incubation with pre-immune serum. Scale bar = 200 µm.
DISCUSSION
cells transfected with MsGC-I synthesized
similar amounts of cGMP to cells transfected with the
Manduca homologues of
1 and
1 subunits that had been
stimulated by SNP. It would seem unlikely that this high level of basal
activity reflects the basal activity of MsGC-I in the nervous system of
Manduca. It is possible that in vivo MsGC-I is
inhibited and activation is achieved by release of this inhibition.
helix
(data not shown). Many of the conserved residues are hydrophobic,
suggesting that this region of MsGC-I may form an amphipathic helix and
could mediate dimerization. As one test of this possibility, we
determined the apparent molecular weight of MsGC-I, when expressed in
COS-7 cells, using gel filtration. These results showed that no enzyme
activity eluted at the expected position of MsGC-I monomers, whereas
most of the activity eluted at a position consistent with either
homodimer or homotrimer formation. Although homodimerization of MsGC-I
appears the most likely explanation of these results, it is also
possible that it forms complexes with endogenous proteins in the COS-7 cells.
the guanylyl cyclase activating
proteins (GCAPs, Ref. 25), which interact with the cyclases at some
part of their intracellular domain (11). At high calcium
concentrations, the GCAPs inhibit the retinal guanylyl cyclases, and
when calcium concentrations drop within the photoreceptors, this
inhibition is relieved and the cyclases are activated (11). Recently,
GCAPs have also been shown to regulate olfactory cell-specific guanylyl
cyclases in a similar manner (26). MsGC-I could also be regulated by a
GCAP, inhibiting its activity while also localizing it to the membrane.
When the cells containing MsGC-I are stimulated, this inhibition could be relieved. This could also cause dissociation of MsGC-I from the
membrane, allowing it to form highly active, cytoplasmically localized homodimers.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Norm T. Davis for technical assistance with the immunocytochemical methods and for sharing expertise in the anatomy of the M. sexta nervous system, Sharon Hesterlee for technical assistance in confocal microscopy, and Meredith Calvert for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This project was supported by grant number NS29740 from NINDS, National Institutes of Health and by grant 9604536 from National Science Foundation.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/EMBL Data Bank with accession number(s) AF073342.
¶ To whom correspondence should be addressed: Dept. of Biological Structure and Function, Oregon Health Sciences University, School of Dentistry, 611 SW Campus Drive, SD, Portland, OR 97201-3097. Tel.: 503-494-8596; Fax: 503-494-8554; E-mail: mortonda{at}ohsu.edu.
The abbreviations used are: NO, nitric oxide; cGMP, 3',5'-cyclic guanosine monophosphate; GST, glutathione S-transferase; SNP, sodium nitroprusside; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; kb, kilobases(s); GCAP, guanylyl cyclase activating protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|