1 Department of Cell Biology, Yale University School of Medicine, I-354 SHM PO
Box 208005, New Haven, CT 06520-8005, USA
2 Department of Genetics, Yale University School of Medicine, I-354 SHM PO Box
208005, New Haven, CT 06520-8005, USA
Author for correspondence (e-mail:
michael.stern{at}yale.edu)
Accepted 12 May 2003
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SUMMARY |
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Key words: FGF receptor, EGL-15, Alternative splicing, Chemoattraction, Achondroplasia, Sex myoblast
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INTRODUCTION |
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The domain structure of FGF receptors comprises three extracellular
immunoglobulin (IG) domains, a small extracellular region of predominantly
acidic amino acids (the acid box), a single transmembrane domain and an
intracellular tyrosine kinase domain
(Dionne et al., 1991). Binding
of an FGF ligand to IG domains 2 and 3 (IG2 and IG3) induces receptor
dimerization and upregulates the intracellular tyrosine kinase activity, which
in turn modulates the activity of downstream signaling pathways
(Boilly et al., 2000
;
Green et al., 1996
;
Klint and Claesson-Welsh,
1999
).
Understanding the factors that underlie the specific processes mediated by
FGF signaling in vertebrates is complicated by the tremendous diversity among
the signaling complexes. At least 22 FGF ligands and five receptors have been
identified in humans (Kim et al.,
2001; Ornitz and Itoh,
2001
). Receptor activity can also be modified through extensive
alternate splicing that generates a diverse range of FGF receptor structures
(McKeehan et al., 1998
). The
functional consequences and physiological relevance of only a few of these are
known. One important alternate splicing event lies within the second half of
IG3, which can be encoded by one of two alternate exons (IIIb or IIIc)
(reviewed by Green et al.,
1996
). In vertebrate FGFR2, the use of exon IIIc confers the
ability to bind FGF1 and FGF2 with high affinity but dramatically reduces the
interaction of this receptor with FGF7. By contrast, use of exon IIIb enables
robust interaction with both FGF7 and FGF1, but generates a receptor with very
low affinity for FGF2 (Ornitz et al.,
1996
). The differential ligand responsiveness this alternate
splicing creates confers mesenchymal/epithelial specificity to different forms
of the same receptor. Finally, various heparan sulfate proteoglycans (HSPGs)
participate in FGF signaling as part of the ligand/receptor/HSPG ternary
complex, adding to the molecular complexity of FGF signaling
(Chang et al., 2000
;
McKeehan et al., 1998
;
Perrimon and Bernfield,
2000
).
The analysis of FGF signaling in model organisms such as Caenorhabditis
elegans and Drosophila melanogaster has served as a paradigm for
understanding many aspects of FGF signal transduction, in part due to the
reduced molecular complexity of these systems. The C. elegans genome
contains a single FGF receptor gene and two genes that encode FGF ligands,
representing a relatively simple system to dissect the molecular mechanisms of
FGF signaling (Borland et al.,
2001). Similar to its vertebrate counterparts, EGL-15 plays a
crucial role in multiple types of biological processes. Two major events
mediated by EGL-15 control are the migrations of the hermaphrodite sex
myoblasts (SMs) and an early essential function
(DeVore et al., 1995
).
Proper migration of the hermaphrodite SMs plays an important role in
ensuring egg-laying proficiency. The SMs are a pair of muscle precursor cells
that migrate anteriorly to functional positions flanking the precise center of
the developing gonad. After migrating, each SM divides three times to generate
a set of 16 cells that differentiate to generate the muscles required for egg
laying (Sulston and Horvitz,
1977). Improper SM migration can result in the generation of
egg-laying muscles in non-functional positions and, consequently, an inability
to lay eggs (the Egl phenotype).
Several mechanisms cooperate to guide SM migration. Multiple central
gonadal cells express EGL-17/FGF, which serves to attract the SMs to their
precise final positions (Branda and Stern,
2000; Burdine et al.,
1998
; Thomas et al.,
1990
). This guidance mechanism has been termed the gonad-dependent
attraction. In the absence of the EGL-17 chemoattractant, the SMs remain
significantly posterior of normal due to a gonad-dependent repulsion
(Stern and Horvitz, 1991
).
This dramatic posterior displacement of the SMs is also observed in a special
class of mutations in egl-15 termed the egl-15(Egl) alleles
(DeVore et al., 1995
;
Stern and Horvitz, 1991
).
A large number of egl-15 alleles affect its essential function to
varying degrees; these alleles can be ordered in an allelic series based on
the degree of their defect in this function
(Borland et al., 2001;
DeVore et al., 1995
). Complete
loss of EGL-15 activity results in an early developmental arrest and larval
lethality (Let) (DeVore et al.,
1995
). Severe hypomorphic alleles can confer a scrawny (Scr) body
morphology. Certain EGL-15 mutations that cause a milder decrease in the
activity of this receptor display only a Soc phenotype, named for their
suppression of the Clear phenotype caused by mutations in clr-1.
clr-1 encodes a receptor tyrosine phosphatase that acts as a negative
regulator of EGL-15 function (Kokel et
al., 1998
). Compromised function of CLR-1 results in the
hyperactivation of the EGL-15 signaling pathway. egl-15(Soc)
mutations compromise the level of EGL-15 activity sufficiently to bring
signaling levels back to within the normal range even in the absence of the
CLR-1 negative regulator.
The two C. elegans FGFs appear to be responsible for the two,
distinct EGL-15-mediated processes. Loss of EGL-17/FGF results in migration
defects very similar to those seen in the egl-15(Egl) mutants
(Stern and Horvitz, 1991).
Conversely, complete loss of LET-756/FGF function confers a larval arrest
lethality similar to that observed in egl-15 null animals
(Roubin et al., 1999
). This
separation of ligand function is similar to that observed for ligands of the
Drosophila EGF receptor, where the spitz, gurken and
vein ligands are each responsible for eliciting a subset of the
functions of the receptor (Freeman,
1998
; Schweitzer and Shilo,
1997
). Functional differences of the Drosophila EGF
receptor arise in part from temporal and spatial regulation of ligand
expression. We report the identification of a novel EGL-15 receptor isoform
that is generated by alternate splicing, and present evidence that the two
different forms of EGL-15 are responsible for its two distinct functions.
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MATERIALS AND METHODS |
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Determination and representation of sex myoblast position
The final positions of sex myoblasts were determined with respect to the
underlying hypodermal Pn.p cells as previously described
(Thomas et al., 1990). SM
distributions for each strain are depicted using box-and-whisker plots
(Moore and McCabe, 1993
)
aligned to a schematic representation of the Pn.p cell metric. In brief, each
set of SMs is ordered according to anteroposterior position and divided into
quartiles. The `box' includes the positions of SMs within the two central
quartiles. An additional vertical line within the box indicates the median SM
position at the boundary between the second and third quartiles. Overlap of
the line representing median position with a right or left border of the box
is depicted as a thickening of that line. A quartile length (1Q) is determined
based on the range of positions covered by the 2Q box length. Bars
(`whiskers') of up to 1.5 Q length extend from the edges of the box to
additional data points. As whisker length does not extend beyond the range of
data points, these bars may be shorter than a 1.5 Q length, or even absent.
Data points beyond the edge of the bars (`outliers') are indicated by
individual hash marks. This representation of SMs therefore depicts the
overall range of SMs as well as their general distribution and median
position. Sex myoblasts positioned dorsally are represented by asterisks and
were treated outside of this data set (Fig.
5).
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Sequences of newly discovered egl-15 cDNAs are available with the following GenBank Accession Numbers: the complete EGL-15(5A)/type I CTD cDNA (AY288941); type 2 CTD (AY268435); type 3 CTD (AY288942); type 4 CTD (AY268436); type 5 CTD (AY292532). Amino acids within the 5A domain are numbered D128(5A)-G193(5A), linking directly to T246, at which point the common sequences continue. 5B domain amino acids will continue to be numbered as previously, from D128-R245.
PCR analysis establishing exon 5A
Exon 5A was originally detected by nested PCR using a pool of
single-stranded cDNA. The product of a first round PCR reaction using a primer
within the putative beginning of exon 5A
(5'-CCACTTAAACTGTTCGATTGGC-3') and exon 7
(5'-GAATTCGTATCCGCCGGACCGAGCAC-3') was used as the template for a
second round of PCR using nested primers
(5'-AGATCTCGGAAATGAGGAGAGTGAAAAGC-3' and
5'-GGTA-CCTTTGCACACACCATAAACAAAATTCC-3') that generated a product
of 450 bp. The sequence of this product revealed the existence of a
transcript containing 112 bp of exon 5A spliced to the beginning of exon
6.
Analysis of cDNA splicing patterns
RT-PCR of egl-15 was performed on RNA isolated from L2-stage and
mixed-stage populations of wild-type C. elegans. Reactions were
carried out in duplicate using a common downstream primer located in exon 21
(5'-CTGGTCAAAAATGACTAGATC-3') together with either a non-specific
primer located in exon 1 (5'-TGGTCAAGAATGACTAGATC-3') or an exon
5A-specific primer (5'-CCTTTACTCCTCTCACTTTTCGG-3'). These products
were subsequently PCR amplified, in combination with the above primers, using
either a nested exon 5A-specific primer
(5'-GCTCTCGAGCCACTTAAACTGTTCGATTGGC-3') or a nested non-specific
primer (5'-CAGTAGATCTGATGAGTTATTTCCTT-GCATCCTGCC-3'). This step
yielded separate pools of non-specifically amplified L2-stage or mixed-stage
cDNAs, as well as exon 5A-specific L2-stage or mixed stage cDNAs. Diagnostic
PCR was used to determine whether transcripts encoding the exon 5A and exon 5B
forms were represented in the cDNA pools.
5A-specific cDNAs from both the L2-derived cDNA pool as well as the mixed stage-derived cDNA pool were cloned using the TA Cloning Kit (Invitrogen) and characterized for their splicing pattern. In addition, all of the 5A-containing cDNAs from the L2-derived non-specific cDNA pool, a matching number of 5B-containing cDNAs from the same pool, and nine additional 5B-containing cDNAs from the mixed stage-derived non-specific cDNA pool were similarly cloned.
cDNA clones were subjected to a BamHI/NdeI restriction digest to determine their 3' splicing patterns; PCR amplification of the 3' ends was also used to aid in the characterization of splicing patterns. Transcripts were categorized based on restriction patterns and the size of the PCR products; a single transcript from each class was then sequenced to determine the precise sites of the splice junctions. Several cDNAs were clearly splicing intermediates since the open reading frame did not extend much beyond the novel splicing pattern. These are not included in the list of alternatively spliced forms in Fig. 3.
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The egl-17 genomic rescuing fragment was previously described
(Burdine et al., 1997). A
let-756 genomic rescuing fragment was cloned from the cosmid CO5D11.
To make the Plet-756::egl-17 chimera, a 0.8 kb
PstI-NcoI fragment was PCR generated and used to replace the
full 4.1 kb egl-17 promoter fragment of NH#354. Subsequent insertion
of a 1.2 kb NcoI PCR product completed the downstream region of the
let-756 promoter. To make the Pegl-17::let-756
chimera a BamHI-NcoI egl-17 promoter fragment
wherein the start methionine was altered to contain a NcoI
restriction site was ligated with a NcoI-XbaI
let-756 coding sequence fragment (NcoI restriction site also
at the start methionine) into Bluescript KSII vector (Stratagene) using
BamHI and XbaI.
Transgenic rescue assays
Transgenic arrays were generated using standard germline transformation
techniques (Mello et al.,
1991).
egl-15 essential function
egl-15 tester DNA at 20 ng/µl was introduced into
hermaphrodites of genotype +/szT1; dpy-20(e1282ts); unc-115(e2225)
egl-15(n1456)/szT1 along with the dpy-20(+)-containing
co-transformation marker plasmid pMH86 (50 ng/µl). Stable transgenic lines
were established in the balanced heterozygous strain. Rescue of the
egl-15 Let defect was assessed by the segregation of viable Unc lines
of genotype dpy-20(e1282ts); unc-115(e2225) egl-15(n1456); ayEx[dpy-20(+);
egl-15(tester)].
Egl rescue assay
To quantitate the Egl penetrance in transgenic lines, a minimum of 30 L4
transformants were placed on a seeded plate at 20°C. Animals were scored
36 hours and 48 hours later and categorized as non-Egl, semi-Egl, Egl or
Bag-of-worms (Bag). The percent non-Egl animals was calculated from the
proportion of animals that failed to become Egl or Bag. A line was considered
to rescue if at least 60% of the transgenic animals were non-Egl. Data from
the 48 hour time-point are represented throughout this manuscript. Transgenic
rescue of the SM migration defect of egl-15(Egl) animals was assayed
in a dpy-20(e1282ts); egl-15(n1458) background.
let-756 essential function
Plasmids encoding either let-756 or egl-17 were
introduced into dpy-17(e164) let-756(s2887) unc-32(e189); sDp3
hermaphrodites. The sDp3 duplication covers both the dpy-17
and let-756 genes. The
Pmyo-2::GFP co-transformation marker
plasmid was included at 5 ng/µl. Stable transgenic lines were established
in the duplication-bearing strain. Rescue of the let-756 Let defect
was assessed by the segregation of viable Dpy animals of genotype
dpy-17(e164) let-756(s2887) unc-32(e189); ayEx[let-756 tester].
Brood size assay
The average brood size of all let-756(null) rescued lines was
determined. Ten L4-stage rescued animals per line were picked to an individual
plate. Adults were transferred to new plates every 24 hours. The number of
viable progeny (L4 stage or older) was determined for each plate at 48 and 72
hours post-egg lay. The average brood size per transgenic line was determined.
Data from the rescued lines for each transgene were then averaged together,
generating the data portrayed in Table
1.
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RESULTS |
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The C-terminal domain (CTD) of EGL-15 appears to be required specifically
for SM chemoattraction. The egl-15(Egl) allele n1457 is a
nonsense mutation (Q948ochre) predicted to remove the CTD; its only apparent
defect is posteriorly displaced SMs and the resulting Egl phenotype
(Fig. 2A). Animals bearing this
mutation fail to exhibit either a scrawny body morphology or the Soc
phenotype, even though minor perturbations of general EGL-15 function can
confer a Soc phenotype with no effect on egg-laying competence [for example,
the Soc allele egl-15(n1783)]. The behavior of the
temperature-sensitive allele n1477ts is also consistent with the CTD
being required for SM chemoattraction. This mutation, like n1457,
corresponds to a nonsense mutation at the 3' end of the
egl-15-coding region, but truncates the very C terminus of the kinase
domain as well as the CTD (Fig.
1). n1477ts animals are Egl and have SM positioning
defects at all temperatures, but display an increasingly severe scrawny body
morphology only at higher temperatures. The truncation of the amino acids at
the very end of the final -helix of the kinase domain has been
postulated to destabilize EGL-15, accounting for the temperature-sensitive
effect on body morphology (Mohammadi et
al., 1996
). However, the elimination of the CTD can account for
the SM migration defects that are observed at all temperatures. The phenotypic
characteristics of these two mutants suggest a specific requirement of the CTD
for SM chemoattraction.
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We tested for the presence of an egl-15 transcript containing this sequence using RT-PCR. This yielded a product whose sequence corresponded to a portion of the `intronic' region between exons 5 and 6 spliced to the known splice acceptor site of exon 6. The presence of a splice junction confirmed that we had identified a spliced transcript rather than a genomic DNA contaminant. Similar analysis of the splicing pattern upstream of this new exon revealed a junction between the normal exon 4 splice donor site and the predicted splice acceptor site of the new exon. This new exon is therefore spliced between exons 4 and 6 as an alternative exon 5, known as exon 5A (Fig. 2B). The originally identified exon 5 is now termed exon 5B. Use of the alternative exon 5A maintains a continuous open reading frame, resulting in a putative new isoform of EGL-15, which we refer to as EGL-15(5A) (Fig. 2C).
Besides egl-15(n1457), the other egl-15(Egl) alleles all have lesions affecting this newly identified exon 5A (Fig. 2B). The base change associated with n1458 results in a nonsense mutation (Fig. 2B). A distinct lesion, also resulting in a nonsense mutation, was found in n484 animals (Fig. 2B). The remaining egl-15(Egl) allele, ay1, is a base change that alters the splice acceptor site at the beginning of this alternate exon (Fig. 2B). These mutations are predicted to truncate or severely reduce the product of egl-15 transcripts containing this exon. As the truncation would occur in the extracellular region of EGL-15, these mutations are likely to destroy the function of the EGL-15(5A) isoform. Consistent with these mutations being null alleles for EGL-15(5A), the SM distribution in n484/nDf19 animals resembles that of n484 homozygotes (Fig. 2A). Because these egl-15(Egl) mutants only have SM migration defects, these data demonstrate that EGL-15(5A) is specifically required for SM chemoattraction (5A, for attraction). The original EGL-15 isoform is now referred to as EGL-15(5B).
EGL-15 isoform structure
To understand the structural differences between the various isoforms of
EGL-15, we characterized a large number of individual egl-15 cDNAs.
These cDNAs were characterized by restriction enzyme digestion, diagnostic PCR
and/or sequence determination. Eleven independent egl-15 cDNAs were
available from an EST database
(http://www.ddbj.nig.ac.jp/celegans/html/CE_INDEX.html).
Analysis of these cDNAs revealed that two of these eleven cDNAs extend to exon
5 and contain exon 5B; the remaining cDNAs end prior to this exon. To
characterize transcripts containing exon 5A, four sets of RTPCR pools were
used to generate cDNAs containing exon 5 sequences. These were generated from
RNA preparations either from mixed-stage populations or from L2
hermaphrodites, the stage during which the SMs migrate
(Sulston and Horvitz, 1977).
From each of these RNA preparations, both `5A-specific' and exon 5
`non-specific' RTPCR cDNA pools were generated.
Analysis of the `non-specific' exon 5-containing cDNAs revealed that egl-15(5A) is enriched at the L2 stage (Fig. 3). None of the cDNA clones (0/25) generated from the mixed-stage population were found to contain exon 5A. By contrast, 8/40 cDNA clones generated nonspecifically from a staged L2 hermaphrodite population contained exon 5A; the remaining clones all used exon 5B. Thus, exons 5A and 5B are used in a mutually exclusive manner, and the EGL-15 isoform required specifically for SM migration is enriched at the stage when SM migration is occurring. Interestingly, although removal of all exon 5 sequences entirely would result in normal FGF receptor architecture, no such transcripts were isolated.
Besides the alternative splicing of exon 5, the only additional alternative splicing detected was in the region between exons 17 and 19. Exon 18 encodes the extreme C terminus of the CTD. A characterization of the 3' splicing pattern for 23 5A-containing, 17 5B-containing and the Kohara EST cDNA clones (YK ESTs) showed that there is one major variant in this region (type 1) and four minor variants (types 2-5) (Fig. 3). These variant transcripts encode a total of four different peptides at the extreme C terminus of EGL-15. Nine of the ESTs and one cDNA of each type were fully sequenced; no splice variation in other regions of egl-15 was observed. Although five types of C termini were found, their distribution is roughly equivalent in the 5A- and 5B-containing cDNAs (Fig. 3).
EGL-15(5A) and EGL-15(5B) are required for distinct EGL-15
functions
To assess the importance of the 5A and 5B isoforms for the two distinct
EGL-15 activities, we created constructs that lacked the ability to produce
either EGL-15(5A) or EGL-15(5B). Nonsense mutations in either the 5A exon
[egl-15(5A-B+)] or the 5B exon
[egl-15(5A+B-)] were introduced into a genomic
rescuing fragment. These were tested using transgenic rescue assays.
To test for the ability to carry out the essential function, these plasmids were assayed for rescue of the viability defect of an egl-15(null) mutant. If the construct rescued and generated homozygous viable transgenic lines, the lines were also tested for rescue of the SM migration defect. As shown in Fig. 4A, the Let defect of egl-15(n1456) was rescued in transgenic lines expressing EGL-15(5B) alone (2/2 lines) but not those expressing only EGL-15(5A) (0/7 lines). Combined with the fact that egl-15 mutants that lack EGL-15(5A) are viable, these data indicate that EGL-15(5B) is both necessary and sufficient for the essential function of EGL-15.
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Specificity of ligand function
The endogenous functions of the two C. elegans FGFs precisely
match the normal specificity of the two EGL-15 isoforms: EGL-17 and EGL-15(5A)
are required for SM chemoattraction, and LET-756 and EGL-15(5B) are required
for the essential function. This ligand-receptor match suggests a simple model
in which EGL-17 interacts specifically with EGL-15(5A) to trigger SM
chemoattraction, while LET-756 interacts specifically with EGL-15(5B) to
trigger the essential function (Fig.
2C). The response elicited by each ligand could be specified by
the ligand itself, tissue-specific patterns of expression or something
inherent in the different receptor isoforms.
To begin to test the molecular basis of this signaling specificity, we assessed whether the roles of the two ligands could be switched by altering their sites of expression. For each ligand, we used genomic constructs containing either the endogenous 5' flanking region or the heterologous 5' flanking region from the other gene; these constructs were tested in germline transformation rescue assays (Table 1 and Fig. 5). We assumed, but did not rigorously establish, that this exchange of the 5' flanking regions for the two C. elegans FGF genes would switch their sites of expression. To test for rescue of egl-17, transgenic lines were scored for rescue of both the Egl phenotype and the SM migration defect. To test for rescue of let-756, transgenic lines were qualitatively scored for rescue of the larval lethality, and the brood size of rescued animals was determined to assess the extent of rescue.
When expressed under the control of the heterologous promoter, each of the ligands could rescue the defect of the heterologous gene (Table 1). Thus, LET-756 can function as an SM chemoattractant and EGL-17 can trigger the essential function of EGL-15. However, the extent of rescue was not as robust as that observed with the ligand that normally mediates these activities (Table 1 and Fig. 5). The decreased rescue was manifested in any of a number of ways: rescue could require higher concentrations of the transgenic construct, the proportion of rescued lines could be significantly lower, or the extent of rescue could be less complete. For example, let-756, when expressed under the control of the egl-17 promoter, could carry out the chemoattractive function of egl-17 (Fig. 5). Despite its ability to carry out the heterologous function, a higher concentration of rescuing DNA was necessary and the proportion of rescued lines was reduced (Fig. 5). Similarly, although egl-17 could rescue the lethality of let-756 mutants when expressed under the control of the let-756 promoter, the brood size of the viable transgenic animals was significantly decreased (Table 1). Nevertheless, despite differences in efficiency, these two FGFs can stimulate either process.
When carrying out a heterologous function, the ligand might use either the receptor isoform normally associated with that function or the isoform normally associated with that ligand. To determine whether LET-756 functions as an SM chemoattractant via EGL-15(5A) or EGL-15(5B), we introduced the rescuing Pegl-17::let-756 transgenes into a background lacking EGL-15(5A) (Fig. 6). For both lines tested, rescuing activity of the let-756 chimeric transgene was completely lost in the absence of EGL-15(5A) (Fig. 6). Thus, LET-756 can function as an SM chemoattractant, but requires EGL-15(5A), the EGL-15 isoform that normally functions in chemoattraction.
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DISCUSSION |
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Exon 5 of the egl-15 gene encodes an additional FGF receptor domain that is inserted between the first and second IG domains within its extracellular region. A theoretical transcript in which exon 4 is spliced directly to exon 6 would encode a full-length FGF receptor that is structurally identical to vertebrate FGF receptors. Nevertheless, a comprehensive egl-15 cDNA analysis did not reveal the existence of any egl-15 transcripts encoding an FGF receptor architecture without one of the two EGL-15-specific inserts. The conservation of both exons 5A and 5B in the related nematode Caenorhabditis briggsae (http://www.sanger.ac.uk/projects/c_briggsae/BLAST_server.shtml) suggests an important biological function for this novel domain and its alternative splicing. Interestingly, the 5A and 5B domains are unique to EGL-15, with no significant homology to regions of any other protein in public databases or even within the genomic sequences of human FGFRs.
The C-terminal domain (CTD) is the only other site at which alternate
splicing in egl-15 was observed. This splicing variation generates
four different C-terminal protein variants, an abundant Type 1 variant and
three less abundant minor variants. In all cases, the first 91 amino acids of
the CTD are common, each variant differing only at the extreme C terminus of
the CTD. For comparison, the n1457 truncation occurs after the first
15 amino acids of the CTD. Each CTD splice variant appears to be equivalently
represented in both 5A- and 5B-containing cDNAs. The lack of a requirement for
this domain in the essential function suggests that alternate splicing of this
region cannot account for the functional differences observed among receptor
isoforms. Nevertheless, our data reveal the crucial role the C-terminal domain
serves during SM chemoattraction, a function that has precedent with other FGF
receptors. Previous work has identified a 14 amino acid segment of the hFGFR1
CTD necessary for the chemotaxis function of this receptor
(Landgren et al., 1998). This
motif is not conserved in the EGL-15 CTD, although the function of this domain
may be conserved. Analysis of components that interact with this portion of
EGL-15 might provide important insights into the links between FGF receptors
and the downstream signaling pathways that drive chemotactic movement.
The distinct functions of the two EGL-15 receptor isoforms raises the question of the roles of the two EGL-15-specific inserts in generating the specific in vivo responses to EGL-15 activation. The observed signaling specificity of the two modules of ligand/receptor pairs can derive from many sources, including the specific ligand used, the nature of the receptor mediating the response, and tissue-specific expression of either of these components or other factors that participate in signal transduction. The discovery of two EGL-15 isoforms and the signaling modules they are associated with has allowed us to test signaling specificity in this system more rigorously.
We began this investigation by testing the interchangeability of the two FGF ligands and their dependence on the specific receptor isoforms. Our data demonstrate that the ligands exhibit a significant degree of functional equivalence; either ligand can be used to stimulate either EGL-15-mediated process. By contrast, we observed a specific functional requirement for each receptor isoform. EGL-15(5A) is required to mediate chemoattraction of the sex myoblasts, and EGL-15(5B) is required to elicit the essential function of this receptor. These data demonstrate that signaling specificity is determined in significant part by the specific receptor isoform, independent of the stimulating ligand.
It is likely that multiple mechanisms contribute to generate the specific
cellular responses to EGL-15 activation. The sites of ligand expression are
important for defining which of the two ligands triggers the two major EGL-15
activities, as swapping their promoters reveals a large degree of functional
interchangeability. However, in addition to this mechanism, differences in
their affinities for the two EGL-15 isoforms might also contribute to the
observed signaling specificity. Although the major ligand-binding determinant
for FGF receptors comprises the IG2 and IG3 domains, the alternative
EGL-15-specific inserts are situated in a region that could easily affect
ligand-binding affinity. In fact, other regions of the extracellular domain
are known to modify ligand-binding affinity
(Shimizu et al., 2001;
Wang et al., 1995
).
Considering the decreased efficiency of heterologous ligand rescue, it remains
possible that differences in ligand-receptor affinities contribute to the
mechanism by which the different ligands stimulate different biological
events. A detailed biochemical analysis of ligand binding affinities will be
necessary to resolve this issue.
In addition to a region of signaling specificity arising from tissue-specific ligand expression, some aspect of the isoforms helps determine their functional specificity. At the moment, the mechanistic basis of this specificity is not clear, but several possibilities are likely. One possibility is that the normal specificity observed for the isoforms is due to tissue-specific alternative splicing. Modifying the expression of these isoforms could test this hypothesis. Alternatively, the structure of these domains might confer functional specificity. This could be effected by providing an alternative conformation that might alter intracellular signaling potentials, by allowing alternative modifications, or by permitting interactions with different co-receptors.
The set of missense mutations in the extracellular domain of EGL-15 bears
an interesting relation to a specific achondroplasia (dwarfism) mutation in
human FGFR3. Within the collection of egl-15 alleles characterized,
only three missense mutations were identified in the extracellular domain.
n2205 (G492E) is the identical alteration detected in a case of
achondroplasia, affecting the analogous residue G346 at the C terminus of IG3
of human FGFR3 (Prinos et al.,
1995). Interestingly, the other extracellular missense mutations
in EGL-15 are related to this mutant allele. n2202 (G492R) changes
the same residue to a different, and oppositely charged, amino acid, while
n2210 (G374E) is the identical mutational alteration at the
corresponding position within IG2 rather than IG3. These mutations were
isolated as soc alleles of egl-15, indicating that they have
a net effect of reduced EGL-15 signaling. By contrast, achondroplasia
mutations are thought to result in elevated FGF receptor activity
(Webster and Donoghue, 1997
).
As these mutations affect the core of the ligand-binding region of FGF
receptors, it is possible that their effects on activity are extremely context
dependent. The net effect these mutations have on receptor function may
reflect a balance between the impairment of ligand binding and an ability of
the mutation to constitutively activate the receptor.
The discovery of the EGL-15(5A) isoform has provided a clear model for how
EGL-15 contributes to the gonad-dependent attraction, and thereby clarified
the contribution of FGF signaling to the mechanisms involved in SM migration
guidance. The simplicity of this migration makes it ideal to probe the complex
series of events that propel migrating cells: a bilaterally symmetric pair of
cells move in a single anterior direction, at a fairly uniform rate from
well-defined initial positions to precise final positions. Nonetheless, a
complex series of guidance mechanisms is used to accomplish this apparently
simple migration (Branda and Stern,
2000; Chen and Stern,
1998
). The results of our current study present an important
foundation from which to explore fully the mechanisms by which EGL-15 assists
in the integration of these multiple migratory cues, as well as the mechanism
by which FGF receptors achieve signaling specificity.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Genetics, Harvard Medical School, Goldenson
Building, Boston, MA 02115, USA
Present address: Fox Chase Cancer Center, Medical Oncology, 7701 Burholme
Avenue, Room W364, Philadelphia, PA 19111, USA
Present address: Department of Molecular Biology, Princeton University,
Princeton, NJ 08544, USA
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