Zoologisches Institut, Universitaet Zurich, Winterthurerstrasse 190, CH 8057, Zurich, Switzerland
* Author for correspondence (e-mail: ahajnal{at}zool.unizh.ch)
Accepted 18 March 2003
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SUMMARY |
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Key words: RAS, MAPK, G protein, Caenorhabditis elegans
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INTRODUCTION |
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Several GPCRs and heterotrimeric G proteins were found to control cellular
proliferation and differentiation
(Dhanasekaran et al., 1995;
Lorenz et al., 2000
;
Moolenaar, 1991
;
Post and Brown, 1996
;
Pouyssegur et al., 1988
;
van Corven et al., 1989
).
Moreover, activated G
proteins can cause malignant transformation of
cultured cells and have been implicated in the formation of human cancer
(Dhanasekaran et al., 1995
).
These observations have suggested a possible link between GPCR signalling
pathways and the RAS/mitogen-activated protein kinase (MAPK) pathway that is a
key regulator of cellular proliferation and differentiation
(Peyssonnaux and Eychene,
2001
; Schramek,
2002
). Different pathways linking GPCR signalling to the RAS/MAPK
cascade have been reported (Belcheva and
Coscia, 2002
; Gutkind,
2000
; Hur and Kim,
2002
; Lowes et al.,
2002
; Luttrell et al.,
1997
; Marinissen and Gutkind,
2001
; Sugden and Clerk,
1997
). For example, in Saccharomyces cerevisiae MAPK is
regulated by a heterotrimeric G protein when the mating pheromone binds to a
GPCR (Herskowitz, 1995
;
Metodiev et al., 2002
). In
mammalian cells, the autophosphorylation of the PDGFR or the EGFR can be
induced by stimulation of the angiotensin II receptor or by GPCR agonists like
endothelin 1, lysophosphatic acid and thrombin
(Daub et al., 1996
;
Linseman et al., 1995
).
Furthermore, heterotrimeric G proteins can control RAS signalling by
activating a RAS guanine-nucleotide releasing factor through a protein kinase
C pathway (Marais et al.,
1998
; Mattingly and Macara,
1996
), via PI3K
(Lopez-Ilasaca et al., 1997
)
or by regulating the RAP-1 small GTPase that acts as a RAS antagonist
(Kitayama et al., 1989
).
However, the exact biochemical routes linking the various GPCR pathways to the
RAS/MAPK cascade and the in vivo significance of many of the postulated
interactions remain to be investigated.
In C. elegans, the RAS/MAPK pathway is used multiple times during
development to control diverse processes
(Kayne and Sternberg, 1995;
Riddle, 1997
;
Sternberg and Han, 1998
). In
particular, RAS-mediated signalling controls the cell fate specification
during the development of the hermaphrodite vulva, and it is required for the
olfaction of volatile attractants (Hirotsu
et al., 2000
). During vulval development, a signal from the
gonadal anchor cell induces three out of six equipotent vulval precursor cells
(VPCs) to adopt the vulval cell fates
(Kornfeld, 1997
;
Sternberg and Han, 1998
;
Wang and Sternberg, 2001
). The
anchor cell signal activates in the three proximal VPCs (P5.p, P6.p and P7.p)
the conserved RTK/RAS/MAPK signalling pathway to specify the primary (1°)
and secondary (2°) vulval fates (Hill
and Sternberg, 1992
). The remaining VPCs that do not receive the
inductive signal (P3.p, P4.p and P8.p) adopt the non-vulval tertiary (3°)
cell fate and fuse with the hypodermis. After the vulval fates have been
specified, P6.p, P5.p and P7.p divide in an invariant pattern and
differentiate yielding 22 descendants that form the vulva. Mutations that
reduce the activity of the RAS pathway cause a vulvaless (Vul) phenotype
because P5.p, P6.p or P7.p adopt the 3° uninduced instead of the 1° or
2° vulval cell fates. Conversely, mutations that hyperactivate the RAS
pathway result in a multivulva (Muv) phenotype where more than three VPCs can
adopt 1° or 2° vulval cell fates.
C. elegans can detect various water-soluble and volatile
substances in its environment (Bargmann et
al., 1993; Dusenbery,
1974
; Ward, 1973
).
Two pairs of bilaterally symmetric neurones in the head, the AWA and AWC
neurones, mediate the response to volatile attractants
(Bargmann et al., 1993
). When
an animal is exposed to volatile attractants such as isoamylalcohol and
diacetyl, which are sensed by the AWC and AWA chemosensory neurones,
respectively, MPK-1 MAPK is rapidly activated in these neurones
(Hirotsu et al., 2000
).
Mutations that reduce the activity of the RAS/MAPK pathway at the level or
downstream of RAS decrease the response of the animals to volatile attractants
(Hirotsu et al., 2000
). Thus,
RAS-mediated signalling in AWA and AWC is required to elicit a prolonged
response that results in the chemotaxis towards the source of the attractant.
A large number of GPCRs that are expressed in sensory neurones and are
candidate chemosensory receptors have been identified in C. elegans
(Bargmann and Kaplan, 1998
;
Troemel et al., 1995
). They
have been divided into six gene families (sra, srb, srd, sre, srg and
sro). It was estimated that a single chemosensory neurone expresses
up to 20 GPCRs (Mombaerts,
1999
). In addition, the C. elegans genome encodes 20
G
, two Gß and two G
subunit proteins
(Jansen et al., 1999
). One
homologue exists for each of the four classes of G
proteins found in
mammals, plus 16 G
genes that do not belong to a specific class.
Although little is known about the function of the putative GPCRs in
chemosensation, except for the diacetyl receptor ODR-10
(Sengupta et al., 1996
),
several of the G
subunits have been shown to regulate olfaction
(Jansen et al., 1999
).
However, if and how G proteins regulate the RAS/MAPK signalling pathway in the
olfactory neurones is not known.
Here, we report the identification of the GPCR SRA-13 as a negative
regulator of the RAS/MAPK pathway during vulval induction and olfaction.
SRA-13 acts predominantly through the GPA-5 G protein to inhibit MAPK
activation in the AWA and AWC chemosensory neurones. Surprisingly, SRA-13 and
GPA-5 play a similar role in negatively regulating the RAS/MAPK pathway during
vulval induction, suggesting that a common mechanism of crosstalk functions in
both tissues. During vulval development, the SRA-13/GPA-5 signal may serve to
adapt the activity of the RAS/MAPK pathway to environmental conditions. Our
data demonstrate the in vivo significance of the connection between GPCR
signalling pathways and the RAS/MAPK cascade.
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MATERIALS AND METHODS |
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LGI: mek-2(n2678), che-3(e1124)
LGII: ga145 (G.B. and A.H., unpublished), sra-13(zh13) (this study), let-23(sa62), let-23(sy1)
LGIII: unc-119(e2498), daf-2(e1370)
LGIV: let-60(n2021), let-60(n1046), let-60(n2031dn)
(Beitel et al., 1990),
let-60(n1876) (Beitel et al.,
1990
), let-60(ga89)
(Eisenmann and Kim, 1997
),
lin-45(sy96), gpa-7(pk610)
(Jansen et al., 1999
)
LGV: gpa-2(pk16), gpa-3(pk35)
(Jansen et al., 1999),
gaIs36[hs::mpk-1] (Lackner and
Kim, 1998
)
LGX: sem-5(n2019), gap-1(ga133)
(Hajnal et al., 1997),
gpa-5(pk376) (Jansen et al.,
1999
), osm-5(p813), syIs1[lin-3(xs)]
Unless noted in the table legends, all experiments were conducted at
20°C. Transgenic lines were generated by injecting the DNA at a
concentration of 100 ng/µl into both arms of the syncytial gonad as
described (Mello et al.,
1991). pUNC119 (20 ng/µl) and pTG96 (100 ng/µl) were used as
a transformation markers (Maduro and
Pilgrim, 1995
; Yochem et al.,
1998
).
Identification of sra-13
During the positional cloning of a gene defined by the ga145 and
zh17 mutations that cause a Muv phenotype in a gap-1(0)
background and map on chromosome II between dpy-10 and
unc-4, we observed that multicopy extrachromosomal arrays consisting
of the YAC Y24H1 and the cosmid F49E12 partially rescued the ga145;
gap-1(0) Muv phenotype, reducing the penetrance from 77% to 25%. However,
the subsequent genetic mapping and the molecular cloning indicated that the
ga145 and zh17 mutations are alleles of the puf-8
gene located on the YAC Y7B4 (Fig.
1A; G. Battu and A. Hajnal, unpublished).
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Plasmid constructs
The sra-13::gfp translational reporter was generated by PCR
amplification of a 2.4 kb genomic sra-13 fragment containing 900 bp
of 5' promoter sequences and the entire open reading frame, using the
primers OGB61(AAT CTA GAA TTC GTG AAA AAG TCC ACC) and OGB62 (CGT CTA GAT ATT
TCC ACG CTG AAT TG) followed by XbaI restriction and ligation into
the XbaI site of the vector pPD95.75 (a gift from Andrew Fire). The
transcriptional sra-13::nls::gfp reporter was generated by ligating a
XbaI-BamHI restricted 900 bp genomic fragment containing the
5' promoter elements of F49E12.5 into the XbaI-BamHI
site of pPD96.04 (a gift from Andrew Fire). The 900 bp fragment was PCR
amplified using the primers OGB61 and OAH150 (TTT GGA TCC GTT GAG GAA GGA GTT
GCC AT). The sra-13(xs) fragment was generated by PCR amplification
using the primers OGB 7 (GTC CAC CGA ATT GGC AAA GAA ATG G) and OGB 29 (ATG
CGT GTC AGT CAT CCT TTT GG) of a 3.7 kb genomic fragment encompassing the
entire sra-13 ORF, 0.9 kb of upstream and 1.3 kb of downstream
sequences. To generate the sra-13(mut) construct, two complementary
primers, OGB 138 (GTT AAG TCA ACC GGA TCC GTG CTT) and OGB139 (AGC ACG GAT CCG
GTT GAC TTA AC), bearing a stop mutation after residue 21 followed by a
BamHI site were used in two separate PCR reactions with primers OGB7
and OGB29 (see above), respectively, to generate two DNA fragments, one
containing the upstream sequences, the first 21 amino acids and a stop codon,
and the other starting from the stop codon and containing the remaining
sra-13 open reading frame and downstream sequences. These two
fragments were ligated at the BamHI sites and cloned into the pGEMT
vector (Promega), yielding an insert identical to the sra-13(xs)
fragment, except for the stop mutation and a BamHI site. The
hs::sra-13 construct was generated by ligating a 1.9 kb
BamHI-KpnI fragment amplified with the primers OGB113 (AAT
ATC GGA TCC ATG GCA ACT CCT TCC TCA ACT) and OGB140 (AAA AAA GGT ACC CGG TGT
GAT TGA TGA GTT GGA) covering the sra-13-coding sequence and 350 bp
of the 3' UTR into the BamHI-KpnI site of vector
pJK465, which contains a hsp16 heat shock promoter upstream of the
BamHI site.
Chemotaxis assays
Chemotaxis assays were performed as described
(Bargmann et al., 1993). Around
100 animals that had been washed three times with M9 buffer were placed along
a line in the centre of a 90 mm phosphate-buffered agar plate onto which the
attractant and a neutral chemical, each diluted in ethanol, had been spotted
together with 1 M sodium azide on either side of the plate and equidistant
from the worms. After allowing the animals to crawl for 1 hour, the numbers of
animals at the attractant and at the neutral chemical were counted. The
chemotaxis index was calculated as I=(the number of animals at the
attractant)-(the number of animals at the neutral chemical)/(total number of
animals on the plate). Chemotaxis towards NaCl was assayed as described
(Bargmann and Horvitz, 1991
).
All experiments were performed at least in triplicate and the mean index and
standard deviation were calculated. Except for the assays shown in Fig.
3A,3B,
isoamylalcohol and diacetyl were used at dilutions of 10-2 and
10-3, respectively.
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|
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For the starvation assays, the worms were hypochlorite treated to release
the eggs, which were allowed to hatch overnight in M9 buffer as described
(Riddle et al., 1997). The
synchronized L1 larvae were fed with E. coli OP50 for 3.5 hours until
they reached the end of the L1 stage. The late L1 larvae were washed 3 times
with M9 buffer and put on NGM plates containing 50 µg/ml ampicillin (to
prevent the growth of attached E. coli) without food for 36 hours.
The larvae were then re-fed until they reached the L4 stage and their vulval
induction index could be scored as described above. At the late L1 to early L2
stage, vulval induction is sensitive to food starvation, although in larvae
that had been grown in the presence of food until they reached the mid-to
late-L2 stage (9 hours or longer), vulval induction was insensitive to
starvation. For example, in let-60(n1046) L1 larvae that were fed for
3 to 7 hours before starvation, the average number of induced VPCs per animal
ranged from 3.5 to 3.6, but in larvae fed for 9 hours or longer before
starvation, the average number of induced VPCs was between 4.5 and 4.4.
(Induction in unstarved let-60(n1046) larvae was 4.4 induced VPCs per
animal.)
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RESULTS |
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To test if endogenous SRA-13 inhibits RAS signalling, we isolated the sra-13 loss-of-function mutation zh13 (see Materials and Methods). sra-13(zh13) animals carry a deletion of 1756 bp that removes 396 bp of 5' promoter sequences and all but the last exon of SRA-13 (Fig. 1B). Thus, the zh13 mutation is most probably a null allele. Henceforth, we refer to this allele as sra-13(0). sra-13(0) single mutant animals are healthy and exhibit no obvious morphological defects. In particular, vulval development in sra-13(0) animals appeared to occur normally (Table 1, row 2). However, this does not exclude the possibility that sra-13(0) may negatively regulate RAS-mediated signalling (see below).
SRA-13 is expressed in the AWA and AWC chemosensory neurones
The SRA proteins are expressed in chemosensory neurones, interneurones and
various other tissues, including muscle and hypodermal cells
(Troemel et al., 1995). To
examine the expression pattern of SRA-13, we built translational and
transcriptional GFP reporter constructs. The translational
sra-13::gfp reporter was generated by fusing a 2.4 kb genomic
fragment containing 900 bp of 5' promoter sequences reaching up to the
3' end of the neighbouring gene (F49E12.4) and the entire open reading
frame of sra-13 to a gfp cassette. The C-terminal fusion to
GFP is likely to inactivate SRA-13, as animals carrying the
sra-13::gfp reporter transgene did not exhibit any of the phenotypes
caused by SRA-13 overexpression (see below). For the transcriptional reporter,
the 900 bp of 5' promoter sequences were fused to a gfp::lacZ
fusion gene (a gift from A. Fire).
Both gfp reporter transgenes showed strong expression in two
bilaterally symmetric pairs of neurones in the head of the animal
(Fig. 2A). We identified these
neurones as the AWAL/R and AWCL/R chemosensory neurones by comparing the
sra- 13::gfp expression pattern with the expression pattern
of the gcy-10::gfp reporter, which is expressed in the AWB and AWC
chemosensory neurones and interneurone I1
(Yu et al., 1997), and by
staining sra-13::gfp worms with DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate), which stains the ASK, ADL, ASI, AWB, ASH and ASJ, but not the
AWA, neurones. In the AWA and AWC neurones, the translational SRA-13::GFP
fusion protein was present in the cell body, the axons and the dendrites
(Fig. 2A). In addition to the
four chemosensory neurones, the transcriptional fusion was expressed in muscle
and hypodermal cells throughout the animal
(Fig. 2B-D). The expression of
SRA-13 in the chemosensory neurones suggested that, like the other SRA
proteins, SRA-13 might be involved in sensing the environment.
SRA-13 negatively regulates olfaction
The AWA and the AWC neurones are required for sensing a variety of volatile
attractants present in the environment
(Bargmann et al., 1993). Upon
exposure to a volatile attractant, the RAS/MAPK pathway is rapidly activated
in the chemosensory neurone that is used to detect the attractant
(Hirotsu et al., 2000
).
Mutations that reduce the activity of the RAS/MAPK pathway severely compromise
the ability of the animals to respond to a volatile attractant, resulting in a
reduced chemotaxis towards the source of the attractant. For example, animals
carrying reduction-of-function mutations in let-60 ras, lin-45 raf or
mek-2 mapkk exhibit a reduced chemotaxis index to the volatile
attractants isoamylalcohol and diacetyl, which are sensed by AWC and AWA,
respectively (Hirotsu et al.,
2000
). We thus speculated that SRA-13 in the AWA and AWC neurones
might act antagonistically to the RAS/MAPK pathway during olfaction. Moreover,
sra-13(0) animals were not defective for chemotaxis to isoamylalcohol
or diacetyl. On the contrary, they displayed stronger chemotaxis to limiting
concentrations of isoamylalcohol and diacetyl than did wild-type animals (Fig.
3A,3B),
suggesting that SRA-13 negatively regulates olfaction. Furthermore, loss of
sra-13 function partially suppressed the chemotaxis defects caused by
reduction-of-function mutations in components of the RAS/MAPK pathway. For
example, in let-60 ras(n2021rf) animals olfaction is severely
compromised, causing an approximately threefold reduction in the chemotaxis
index towards isoamylalcohol or diacetyl (Fig.
3C,3D)
(Hirotsu et al., 2000
). By
contrast, sra-13(0); let-60(rf) double mutants exhibited a twofold
higher chemotaxis index to isoamylalcohol than let-60(rf) single
mutants and nearly wild-type chemotaxis to diacetyl. Similar results were
obtained using reduction-of-function mutations in lin-45 raf and
mek-2 mapkk (Fig.
3C,3D).
Overexpression of SRA-13 caused a phenotype opposite to that observed in sra-13(0) animals. To increase SRA-13 activity, we used the sra-13(xs) strain that carries multiple copies of the sra-13 genomic region on an extrachromosomal array. sra-13(xs) animals were defective in chemotaxis towards isoamylalcohol and diacetyl (Fig. 3E,3F), while sra-13(xs) animals could detect NaCl as well as wild-type animals, indicating that their chemotaxis defect was specific for volatile chemicals and that the general locomotion was not compromised (data not shown). Animals carrying a sra-13 transgene with a mutation truncating the protein after the first 21 amino acids (sra-13(mut)) did not exhibit a significant decrease in chemotaxis to isaomylalcohol and diacetyl (Fig. 3E,3F).
Olfaction seems to require just the right amount of RAS/MAPK signalling, as
mutations that decrease or increase the activity of the LET-60 RAS protein
both affect the ability of the animals to sense volatile attractants
(Hirotsu et al., 2000). For
example, in let-60(n1046gf) animals, chemotaxis to isoamylalcohol is
severely compromised (Fig. 3E),
possibly because perpetual RAS/MAPK signalling might trigger a desensitizing
mechanism. We next tested if overexpression of SRA-13 overcomes the chemotaxis
defect of let-60(gf) mutants. The sra-13(xs) transgene
partially rescued the strong chemotaxis defect of let-60(gf) animals
to isoamylalcohol (Fig. 3E). By
contrast, chemotaxis to diacetyl was only moderately affected by the
let-60(gf) mutation, and let-60(gf); sra-13(xs) animals
showed a further reduction in the chemotaxis index to diacetyl
(Fig. 3F)
(Hirotsu et al., 2000
). It
thus appears that the olfaction of diacetyl tolerates higher levels of
RAS/MAPK activation, while the sra-13(xs) transgene may reduce RAS
pathway activity below wild-type levels.
To exclude the possibility that SRA-13 is required for the development of the chemosensory circuit we expressed SRA-13 under control a heat-shock inducible promoter (hs::sra-13) (Fig. 3I,3K). hs::sra-13 animals that were grown at the permissive temperature (20°C) until they reached adulthood and then subjected to heat-shocks to induce SRA-13 expression were severely defective in chemotaxis to both isoamylalcohol and diacetyl. By contrast, wild-type animals that were subjected to the same heat-shock regime and hs::sra-13 control animals that were kept at 20°C showed no significant reduction in chemotaxis.
The experiments presented so far suggested that SRA-13 antagonizes RAS-mediated signalling during olfaction.
However, these results did not allow us to distinguish whether SRA-13 signalling directly inhibits the activity of the RAS/MAPK pathway or if SRA-13 acts in a parallel pathway that antagonizes the response to the RAS/MAPK signal.
SRA-13 inhibits MAPK activation during olfaction
To address this point, we tested if the sra-13(0) mutation causes
an increased activity
of the RAS/MAPK pathway in the chemosensory neurones. For this purpose, we
determined the levels of activated MPK-1 MAPK in the AWC neurones after
stimulation with a volatile attractant
(Hirotsu et al., 2000).
Animals carrying the sra-13::gfp reporter to label the AWA and AWC
neurones were briefly stimulated with isoamylalcohol and then stained with an
antibody specific for the diphosphorylated, activated form of MPK-1 (DP-MPK-1)
and an anti-GFP antibody to visualize the AWA and AWC neurones (see Materials
and Methods). In most wild-type animals, a sharp increase in DP-MPK-1 staining
in the AWC neurones could be observed after stimulation
(Fig. 4A), while in most
let-60(rf) animals no or very little of DP-MPK-1 staining was
detected in AWC (Fig. 4D).
However, in isoamylalcohol stimulated sra-13(0); let-60(rf) double
mutants we found an increased frequency of strong DP-MPK-1 staining in the AWC
neurones, indicating that a loss of sra-13 function partially
restores MPK-1 activation in let-60(rf) mutants
(Fig. 4G). Thus, SRA-13
negatively regulates the activity of the RAS/MAPK pathway at the level or
upstream of MPK-1.
SRA-13 signals via the GPA-5 G subunit
To examine whether SRA-13 signals through a specific G protein
subunit, we tested if loss-of-function mutations in different G
genes
suppressed the sra-13(xs) phenotype. We tested gpa-2, gpa-3,
gpa-5 and gpa-7 because these four G
genes had previously
been shown to inhibit chemotaxis to volatile attractants
(Jansen et al., 1999
). Of the
four gpa mutants tested, only the gpa-5(0) mutation
suppressed the chemotaxis defect of sra-13(xs) animals (Fig.
3G,3H).
Similar to sra-13(0), the gpa-5(0) mutation enhanced
chemotaxis towards isoamylalcohol and diacetyl (data not shown), and
suppressed the chemotaxis defect of let-60(rf) mutants (Fig.
3G,3H).
From these experiments, we conclude that GPA-5 probably acts downstream of
SRA-13 to transduce an inhibitory signal. However, GPA-5 may not be the only
G
protein acting downstream of SRA-13, as chemotaxis was not restored
to wild-type levels in gpa-5(0); sra-13(xs) animals. We also noticed
that the gpa-5(0) mutation suppressed the chemotaxis defect of
let-60(rf) mutants to isoamylalcohol more efficiently than the
sra-13(0) mutation, suggesting that GPA-5 may transduce signals from
additional GPCRs (Fig. 3G).
SRA-13 and GPA-5 inhibit vulval induction
We had originally identified SRA-13 because its overexpression partially
suppressed the Muv phenotype of mutants that exhibit a hyperactivation of the
RAS/MAPK pathway. To investigate if endogenous SRA-13 and GPA-5 inhibit vulval
induction, we performed epistasis analysis by combining the sra-13(0)
and gpa-5(0) mutations with reduction-of-function mutations in the
RTK/RAS/MAPK pathway that cause a partial Vul phenotype. Vulval induction in
sra-13(0) single mutants was normal
(Table 1, row 2), but loss of
sra-13 function significantly suppressed the Vul phenotype caused by
the let-60 ras(rf) (Table
1, rows 6,7) or the dominant negative let-60 ras(n2031dn)
mutation (Table 1, rows 9,10).
However, the sra-13(0) mutation did not suppress the stronger Vul
phenotype caused by the sem-5 grb2(n2019), let-23 egfr(sy1) and
lin-45 raf(sy96) mutations or the let-60(n1876) mutation
that completely blocks the RAS signalling pathway in the VPCs and causes a
100% penetrant Vul phenotype (Table
1, rows 3,4 and data not shown). Loss of gpa-5 function
efficiently suppressed the Vul phenotype of let-60(rf) mutants as
well as the stronger Vul phenotype caused by the sem-5(rf) mutation,
suggesting that also during vulval induction GPA-5 may transduce inhibitory
signals from additional GPCRs (Table
1, rows 5,8).
Next, we tested if excess SRA-13 inhibits vulval induction. Overexpression of SRA-13 under control of its own promoter (sra-13(xs), Table 1, rows 11,12) as well as from a heat-shock promoter (hs::sra-13, Table 1, rows 18-20) reduced the penetrance of the Muv phenotype caused by the let-60 ras gain-of-function mutation, whereas the sra-13(mut) transgene had no effect (Table 1, row 13). Moreover, hs::sra-13 animals exhibited a weak Vul phenotype in a wild-type background (Table 1, rows 14,15). In addition, in sra-13(0); let-60(rf); hs::sra-13 animals, the levels of vulval induction were comparable with those observed in let-60(rf) single mutants (Table 1, compare rows 6, 7 and 17). Taken together, these results indicated that SRA-13 and GPA-5 negatively regulate vulval induction.
SRA-13 links vulval induction to growth conditions
sra-13(xs) animals exhibited pleiotropic phenotypes besides the
chemotaxis defect described above. Fourteen percent of sra-13(xs)
animals (n=169) developed into dauer larvae when grown in the
presence of abundant food at 25°C, whereas wild-type animals did not form
any dauer larvae under the same conditions (n=423). Moreover, adult
sra-13(xs) animals displayed a reduced rate of egg laying. They
contained on the average 13±11 (n=19) fertilized eggs in the
uterus while wild-type animals contained 8±5 (n=25) eggs. As
enhanced dauer formation and a reduced egg-laying rate are usually observed
when wild-type animals are deprived of food (C. Trent, PhD thesis, MIT, 1982),
the hyperactivation of the SRA-13 pathway appeared to mimic the effects of
food starvation.
We also noticed that vulval induction is sensitive to food levels. The
effect of food on vulval induction becomes apparent in mutants that exhibit a
hyperactivation of the RAS/MAPK pathway. For example, when let-60
ras(gf) animals were grown in the absence of food during the L2 and L3
stages, which is the period when vulval induction occurs, the penetrance of
the Muv phenotype was decreased compared with non-starved control animals
(Table 2, rows 3,4, for the
starvation protocol see Materials and Methods). A similar reduction of vulval
induction by food starvation was observed in ga89, a
temperature-sensitive let-60 ras gain-of-function allele, and in
hs::mpk-1 animals (Table
2, rows 11,12,13,14). However, lin-3 egf(xs) and
let-23 egfr(gf) animals were insensitive to food starvation,
suggesting that the overproduction of the inductive anchor cell signal or the
constitutive activation of LET-23 EGFR can overcome the inhibitory starvation
signal (Table 2, rows 7-10).
Furthermore, the e1370ts mutation that reduces the activity of the
DAF-2 insulin receptor without causing entry into the dauer stage at 20°C
(Dorman et al., 1995) caused a
reduction in the penetrance of the let-60(gf) Muv phenotype
comparable to food starvation (Table
2, row 15). By contrast, in sra-13(0); let-60(gf) double
mutants, vulval induction remained unchanged after starvation during the L2
and L3 stages (Table 2, rows
16,17), but dropped to the levels observed in food-starved let-60(gf)
single mutants when the sra-13(xs) transgene was introduced
(Table 2, rows 18,19). Thus,
loss of sra-13 function appears to uncouple vulval induction from
environmental conditions. To test if the reduction in vulval induction by
starvation requires the input from the sensory system, we used the
che-3(e1124) and osm-5(p813) mutations that disrupt the
structure and activity of the sensory neurones, respectively, and hence make
the animals insensitive to the environment
(Perkins et al., 1986
). As in
sra-13(0) animals, no drop in vulval induction could be observed in
che-3(e1124); let-60(n1046gf) and let-60(n1046gf);
osm-5(p813) double mutants when the animals were deprived of food during
vulval induction (Table 2, rows
20-23). Thus, under starvation conditions, SRA-13 is required to transmit a
starvation signal from the sensory system to the VPCs or to produce an
inhibitory signal in the VPCs.
|
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DISCUSSION |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SRA-13 and GPA-5 negatively regulate olfaction in the AWA and AWC
chemosensory neurones
RAS-mediated signalling in the chemosensory AWC and AWA neurones is
necessary for the response to volatile attractants
(Hirotsu et al., 2000).
RAS/MAPK signalling may be directly involved in controlling synaptic
transmission, for example by regulating synaptic vesicle supply
(Hirotsu et al., 2000
), or the
MAPK signal could be required to elicit a prolonged response by inducing the
expression of specific genes that mediate the long-term effects necessary for
chemotaxis. It is not currently known how RAS is activated in chemosensory
neurones when an odorant binds to a GPCR. In particular, RAS activation in the
chemosensory neurones does not appear to involve an RTK or an adaptor protein
like SEM-5 GRB2 (Hirotsu et al.,
2000
). Instead, GPCRs may trigger through the action of
heterotrimeric G proteins the influx of Ca2+ ions that stimulate a
Ca2+-activated guanine nucleotide release factors similar to
mammalian RAS-GRF (Farnsworth et al.,
1995
).
To our knowledge, SRA-13 is the first member of the SRA family of
chemosensory receptors for which an inhibitory function during olfaction has
been demonstrated. The SRA-13 signal attenuates the response to volatile
attractants, probably by activating a heterotrimeric G protein consisting of
the GPA-5 G subunit. Accordingly, GPA-5 has previously been found to
negatively regulate olfaction of volatile attractants
(Jansen et al., 1999
), while
other G
subunits, such as ODR-3, positively regulate the response to
volatile attractants (Roayaie et al.,
1998
). Achieving the right balance between stimulatory and
inhibitory G proteins may be important to set a threshold level for the
detection of odorants and allow the animals to detect odorant concentration
gradients. Moreover, negative regulation of RAS/MAPK signalling during
olfaction may be necessary to discriminate between different odorants
(Wes and Bargmann, 2001
) or to
adapt olfaction to changes in the environment. We do not know if SRA-13
functions as a chemosensory receptor because we have not identified its
ligand(s). SRA-13 could function as a constitutive, ligand-independent
inhibitor of olfaction. However, overexpression of SRA-13 induces phenotypes
reminiscent of food starvation, suggesting that SRA-13 is activated when food
becomes limiting. Thus, SRA-13 may suppress the olfaction of certain odorants
under adverse growth conditions.
SRA-13 and GPA-5 negatively regulate vulval induction
During vulval induction, the activity of the RTK/RAS/MAPK pathway is
tightly controlled to ensure that only one VPC (P6.p) adopts the 1° cell
fate (Kornfeld, 1997;
Sternberg and Han, 1998
;
Wang and Sternberg, 2001
). For
this purpose, several negative and positive regulators act at different steps
of the pathway. In particular, the activity of the RAS protein LET-60 appears
to be controlled by a number of factors in addition to the guanine nucleotide
exchange factor SOS-1 that transduces the signal from the LET-23 RTK
(Belcheva and Coscia, 2002
;
Chang et al., 2000
;
Gutkind, 2000
;
Lowes et al., 2002
). Our
observation that the GPCR SRA-13 negatively regulates RAS/MAPK signalling via
a heterotrimeric G protein containing GPA-5 fits well into this picture.
Moreover, a gain-of-function mutation in the Gq
protein
EGL-30 causes excess vulval induction (N. Moghal, L. R. Garcia, L. Khan, K.
Iwasaki and P. W. Sternberg, unpublished). Thus, the balance between the
inhibitory SRA-13/GPA-5 signal and a stimulatory signal that is received by an
as yet unidentified GPCR and transduced by EGL-30 may control RAS-mediated
signalling in the VPCs.
In contrast to RAS/MAPK signalling during olfaction, the RAS/MAPK pathway
in the VPCs evokes a binary all-or-none cell fate choice with a relatively
high threshold level. Under normal growth conditions when food is abundant,
the sra-13(0) mutation slightly increases the activity of the
RAS/MAPK pathway, but not above the threshold required to change the normal
pattern of vulval induction. For this reason, the inhibitory activity of
SRA-13 and GPA-5 manifests only in a sensitized genetic background. The same
observation has been made for other inhibitors of the RTK/RAS/MAPK pathway,
such as sli-1 (Jongeward et al.,
1995), gap-1 (Hajnal
et al., 1997
) or lip-1
(Berset et al., 2001
).
Cell-autonomous or non-autonomous function of SRA-13 during food
starvation
The overexpression phenotype has suggested that SRA-13 may send an
inhibitory signal during food starvation or under other unfavourable
conditions. Interestingly, vulval induction is sensitive to food starvation in
sra-13(+) animals but becomes insensitive to starvation in
sra-13(0) animals. One model predicts that during food starvation,
SRA-13 acts cell-autonomously in the VPCs to send an inhibitory signal. It is
possible that SRA-13 is expressed in the VPCs at relatively low levels that
were not detected with the translational sra-13::gfp reporter because
the SRA-13 protein may be unstable in non-neuronal cells and the
transcriptional reporter is expressed in muscle and hypodermal cells.
Experiments using ectopic expression of SRA-13 in the VPCs (data not shown) do
not prove a cell-autonomous function of sra-13 as RAS signalling may
be repressed when artificially high levels of SRA-13 are produced in the VPCs,
even though endogenous SRA-13 may not act in these cells. Another equally
likely possibility is that the activation of SRA-13 in the sensory neurones
leads to the production of a secondary signal that globally downregulates
RAS/MAPK signalling in the animal. The observation that mutations in
osm-5 and che-3, which perturb the function of the sensory
neurones, uncouple vulval induction from food starvation similar to loss of
sra-13 function is consistent with such a cell non-autonomous model.
However, the close lineage relationship between the VPCs and the chemosensory
neurones make it difficult to perform a genetic mosaic analysis that could
determine the cellular site of SRA-13 action during vulval development.
Finally, it should be noted that GPCRs are the targets for many (up to 60%) of the drugs currently used in medicine. Therefore, by controlling GPCR signalling it may be possible modulate the activity of the RAS/MAPK signalling pathway in order to control cellular proliferation and differentiation.
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ACKNOWLEDGMENTS |
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