Drosophila CG8422 encodes a functional diuretic hormone receptor
1 Department of Anatomy and Neurobiology, Washington University School of
Medicine, Saint Louis, MO 63110, USA
2 Department of Cell Biology, Duke University, Durham, NC 27710,
USA
Author for correspondence (e-mail:
taghertp{at}pcg.wustl.edu)
Accepted 3 December 2003
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Summary |
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Key words: neuropeptide, GPCR, receptor, Drosophila, diuretic hormone, ß-arrestin-2, GFP, cAMP
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Introduction |
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Where examined, the different neuropeptides produce additive effects but,
in some cases, they may act synergistically (e.g.
Coast et al., 1999). In several
species, these factors are expressed throughout the central nervous system
(CNS) and gut, often within identified neuroendocrine neurons
(Cantera and Nässel, 1992
;
Chen et al., 1994
;
Patel et al., 1994
;
Iaboni et al., 1998
;
Te Brugge et al., 1999
;
Veenstra and Hagedorn, 1991
;
Tamarelle et al., 2000
;
Wiehart et al., 2002a
). In
some instances, they are co-expressed in the same cells
(Thompson et al., 1995
).
In pioneering work, Reagan
(1994) used expression cloning
to identify a receptor for the CRF-like DH of Manduca and later of
the cricket Acheta (Reagan,
1996
). These DH-Rs are related to the secretin (Type II) family of
G-protein-coupled receptors (GPCRs): for example, in its transmembrane
domains, the Acheta receptor is 53% identical to the Manduca
DH-R and 38% identical to the human CRF receptor. Activation of both
Manduca and Acheta DH-R by DH led to stimulation of
adenylate cyclase, which is consistent with the activity of this peptide
in vivo in Malpighian tubules
(Coast, 1996
). In both animals,
DH-R is expressed in the Malpighian tubules, but its complete
expression pattern has not yet been reported in any insect. A related receptor
is present in the silkmoth Bombyx
(Ha et al., 2000
), although
its functional properties have not yet been described.
In spite of its diminutive size, Drosophila presents a useful
model for the study of endocrine physiology because of its advanced genetics
and fully sequenced genome. By phylogenetic analysis, Drosophila
contains 44 genes encoding putative peptide GPCRs
(Hewes and Taghert, 2001), of
which 39 belong to the rhodopsin family (Type I) and five belong to the
secretin (Type II) family. Among Type II receptors, two paralogous genes,
CG8422 and CG12370, appear orthologous to DH-R. In
the present study, we describe further studies of CG8422 and test the
hypothesis that it is a receptor for Drosophila DH. Based on its
properties when functionally expressed in mammalian tissue culture cells, we
have developed two independent lines of evidence to support the identification
of CG8422 GPCR as a Drosophila DH-R. We also include data to
indicate that CG8422 is reliably expressed in vivo.
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Materials and methods |
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Transfections and cell culture
HEK-293 cells were transfected with lipofectamine using 10 mg DNA per
4x106 cells. Cells were transfected with a 5:1 ratio of
CG8422 DNA to ß-arrestin-2-GFP
(ßarr2-GFP) DNA. Stable lines expressing CG8422 were
generated through selection of resistance to hygromyocin B. Cells were
maintained in a humidified incubator under 5% CO2 atmosphere at
37°C and split 1:5 every three days. The growth medium was Dulbecco's
modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
and antibiotics.
ßarr2-GFP translocation assay
We used methods previously described by Johnson et al.
(2003b). Briefly, HEK-293
cells were transfected as described above and plated onto 35 mm dishes with a
centered glass cover slip to facilitate imaging. Growth media was removed and
replaced with serum-free media [minimum essential media (MEM), without phenol
red] thirty minutes prior to assays. Peptides were dissolved in the same
medium and added at room temperature. Images were collected using 488
nmexcitation and a 505 nm long-pass filter on a Zeiss laser scanning
microscope or on an Olympus laser scanning microscope. Images were imported
into Adobe Photoshop and adjusted for contrast.
cAMP assays
To monitor changes in intracellular cAMP levels, HEK-293 cells were
transiently co-transfected with receptor cDNA and a multimerized
CRE-luciferase reporter gene. They were assayed 24 h
post-transfection for luciferase activity with a LucLite Kit using the
manufacturer's recommendations (Perkin Elmer, Waltham, MA, USA). Luminescence
was measured on a Victor Wallac 2 plate reader (Perkin Elmer). EC50
values were calculated from concentration response curves using computerized
nonlinear curve fitting (PRISM 3.0; GraphPad, San Diego, CA, USA).
Ca2+ assays
We used methods previously described by Johnson et al.
(2003a). In brief, following
selection with antibiotic, HEK-293 cells stably expressing CG8422
were assayed for receptor activation dependent upon ligand application. Cells
were then loaded with 5 mmol l-1 of the calcium-sensitive
fluorescent dye FLUO3-AM (Molecular Probes, Eugene, OR, USA). The dye was
dissolved in DMSO/pluronic acid mixture in a Hank's balanced salt solution
(HBSS) containing 20% Hepes buffer and 2.5 mmol l-1 probenecid
(Sigma, St Louis, MO, USA). A secondary incubation for 30 min at37°C
followed. Cells were washed three times with HBSS/Hepes/probenecid solution
and then placed in a microplate reader (Victor Wallac 2; Perkin-Elmer) to
measure fluorescent signals.
Peptides
Dromyosuppressin (DMS), Drosophila adipokinetic hormone (AKH),
crustacean cardioactive peptide (CCAP), Drosophila ecdysis triggering
hormone (ETH) and Drosophila pigment dispersing factor (PDF) were
purchased from Multiple Peptide Systems, San Diego, CA, USA.
Drosophila allatostatin A (AstA-1), allatostatin C (Ast-C), and
Drosophila FMRFamide (DPKQDFMRFamide) were purchased from BACHEM
(King of Prussia, PA, USA). Proctolin and corazonin were purchased from Sigma.
Drosophila diuretic hormone 31 (DH-II) and diuretic hormone 44 (DH)
were obtained from Julian Dow, Drosophila tachykinin (DTK1) from Dick
Nässel, Drosophila allatostatin B (AstB-1) and IFamide from Jan
Veenstra, Drosophila Neuropeptide F (NPF) from Joe Crim, and
Drosophila sex peptide (SP) from Erik Kubli.
Statistics
Statistical analyses were performed on the effects of DH on HEK
CRE-luciferase levels and on CG8422 expression levels using
the computer program Instat (Graphpad) using P<0.05 as
significant.
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Results |
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HEK-293 cells transiently expressing the receptor encoded by
CG8422 displayed clear translocation of ßarr2-GFP to the
membrane within a few minutes of exposure to 1 µmol l-1 DH
(Fig. 1). Such a saturating
dose triggers desensitization, a process underlying the translocation of GFP:
even at such high doses, the response is highly specific to potent agonists
(Barak et al., 1997,
1999
;
Kim et al., 2001
;
Oakley et al., 2001
). Lower
doses can be effective in this assay (e.g.
Johnson et al., 2003b
), but we
used the assay here as a primary screen and so relied only on the 1 µmol
l-1 dose. Notably, translocation did not occur in cells expressing
CG8422 in response to the application of any of 16 other
neuropeptides. Likewise, translocation did not occur in HEK cells tested with
DH that were not expressing CG8422 (data not shown). Additionally,
after 20 min exposure to DH, the ßarr2-GFP lost its association with cell
membranes and became internalized within large, vesicular compartments
(Fig. 2).
|
|
To evaluate this indication of DH binding to CG8422, and to assess the
possible nature of CG8422 signaling, we monitored changes in cAMP and
calcium levels due to CG8422 receptor activation. In cells
transiently co-expressing CG8422 and CRE-luciferase,
DH-stimulated adenylate cyclase, as indicated by a >5-fold increase in
luciferase levels (Fig. 3).
This effect displayed an EC50 value of 1.47 nmol l-1.
Cells that expressed only the CRE reporter did not produce this
response to DH. Using FLUO3-AM as an indicator, we found a small effect of
CG8422 activation on calcium levels. 10-6 mol l-1 DH
caused a 37.5±2.9% increase in calcium levels of
CG8422-expressing HEK cells, but 10-7 mol l-1
was ineffective (data not shown). 10-6 mol l-1 DH caused
a 2.4±0.6% increase in calcium levels of naive HEK cells. By contrast,
10-8 mol l-1 proctolin caused a 165.7±1.2%
increase in calcium levels in proctolin receptor-expressing HEK cells
(Johnson et al., 2003a).
|
The in vivo expression of CG8422 was established by
measuring transcript levels using microarray analysis of adult head RNA
populations. We mined data from 60 array experiments reported by Lin et
al. (2002
; raw data available
at
http://circadian.wustl.edu),
in which adult head RNA from control and period mutant stocks were
studied under cycling (light:dark) and constant (dark:dark) conditions.
CG8422 receptor levels were detected in each of the four conditions:
CG8422 was scored `Present' by Affymetrix (Santa Clara, CA, USA)
software in
40% of experiments. Their mean levels were not significantly
different between conditions (Fig.
4).
|
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Discussion |
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The DH-R gene appears conserved across several insect orders:
additional representatives have been identified by sequence analysis in the
moth Bombyx (Ha et al.,
2000) and in Drosophila
(Hewes and Taghert, 2001
). In
the transmembrane domains, the DH-Rs of Manduca and Acheta
are, respectively, 50% and 52% identical with the deduced ORF encoded by
Drosophila CG8422. Consistent with the predictions based on
phylogenetic analysis (Hewes and Taghert,
2001
), we have presented three lines of pharmacological evidence
to indicate that DH is an endogenous ligand for the Drosophila CG8422
GPCR. First, we demonstrated ßarr2-GFP translocation in specific response
to DH application. Second, CG8422 co-expressed in HEK-293 cells with
a CRE-luciferase reporter caused a marked increase in luciferase
levels in response to that peptide. Third, HEK cells stably expressing
CG8422 elevated intracellular calcium in response to DH. Hence, we
conclude that CG8422 is a functional DH-R in Drosophila. Whether
CG8422 serves to regulate diuresis within tubules must await more detailed
physiological analysis. In Drosophila, the CG12370 paralog
displays 59% identity with CG8422 in its transmembrane domains.
Whether the CG12370 receptor is also responsive to DH remains to be
determined.
We found that the ßarr2-GFP initially translocated to the membrane
following DH exposure and subsequently internalized to large vesicular
compartments. This particular pattern of ßarr2-GFP internalization
(vesicle forming) corresponds to that seen for many other GPCRs. For both
mammalian and Drosophila receptors, internalization patterns fall
into two categories: Class A receptors maintain ßarr2 at the membrane,
while Class B receptors internalize the arrestins with the receptor into
vesicular compartments (Oakley et al.,
2001). These differing patterns of receptor-ßarr2
associations correlate with differential re-sensitization and MAP-kinase
signaling properties (Oakley et al.,
2001
; Tohgo et al.,
2003
). The patterns observed for CG8422 are typical for
Class B receptors. The significance of this observation for CG8422
signaling in vivo will have to be re-evaluated following its
activation in Drosophila tissues.
To verify results from the ßarr2-GFP translocation assay, we extended
our observations to consider possible CG8422 signaling via
cAMP. That property is predicted based on previous functional expression of
DH-R orthologs (Reagan,
1994,
1996
) and on the fact that, in
Drosophila, as in all other insects examined to date, CRF-diuretic
related peptides stimulate fluid secretion via cAMP
(Cabrero et al., 2002
). In line
with such predictions, we found strong stimulation of adenylate cyclase
following CG8422 activation. However, we note that our
EC50 value (
1 nmol l-1) is two orders of magnitude
more sensitive than values derived from in vitro studies of
Malpighian tubules in Drosophila. That discrepancy may be reconciled
by any of several explanations. For example, expression levels in a cell line
may exceed native expression levels or there may be differing sensitivities in
the assays employed; alternatively, such a discrepancy may reflect the fact
that another DH-R, and not CG8422, is normally expressed in
Drosophila tubules. Furthermore, the estimated
1 nmol
l-1 EC50 value agrees with previous estimations from
studies of receptor orthologs expressed in heterologous systems (Reagan,
1994
,
1996
) and with the
EC50 estimation for DH-stimulated fluid secretion in vitro
by Malpighian tubules in Tenebrio (Weihart et al., 2002b).
Our demonstration of calcium signaling through CG8422 suggests
that this receptor may activate multiple second messengers. We note that the
release of intracellular calcium caused by DH exposure only occurred at
relatively high doses and hence may not be physiologically significant. In
Drosophila tubules, DH did not cause substantial increases in
intracellular calcium as measured by UAS-aequorin reporter gene
(Cabrero et al., 2002).
However, DH-IIs affect both cAMP levels and calcium levels, dependent upon
species (Coast et al., 2001
).
Interestingly, in the mosquito Aedes, CRF affects tubule fluid
secretion via cAMP at lower concentrations and via calcium
at high concentrations (Clark et al.,
1998a
,b
).
DH directly stimulated a doubling of cAMP phosphodiesterase levels in
Drosophila tubules (Cabrero et
al., 2002
): we did not test whether this regulative process is
also downstream of CG8422 activation.
By microarray analysis, CG8422 transcripts were low but reliably
detected in RNA derived from adult heads. In addition, transcript levels did
not vary as a function of the environmental conditions or genotypes tested.
Beyond this confirmation of in vivo gene expression, precise
definition of neuronal and non-neuronal expression of this receptor will need
to be evaluated using techniques that offer greater cellular resolution. In
Drosophila, the DH peptide is restricted to a small set of
neuroendocrine cells (Cabrero et al.,
2002) and, unlike the situation seen in other insects, is
conspicuously absent in abdominal neuroendocrine cells.
Drosophila DH (Cabrero et al.,
2002) and DH-II (Coast et al.,
2001
) peptides have the functional attributes predicted for
CRF-related and calcitonin-related insect diuretic hormones. The
identification of a functional Drosophila DH-R presented here adds to
this base of information regarding Drosophila diuretic hormone
signaling. It will facilitate the introduction of genetic analyses to examine
diuretic hormone physiology in vivo.
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Acknowledgments |
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Footnotes |
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