From the Program in Molecular Neurobiology, Institute
of Biotechnology, Viikki Biocenter, University of Helsinki, FIN-00014
Helsinki, Finland, the ¶ Division of Experimental Oncology and the
Department of Pathology, Istituto Nazionale Tumori,
20133 Milan, Italy
Received for publication, September 11, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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Glial cell line-derived neurotrophic factor
(GDNF) family ligands signal through receptor complex consisting
of a glycosylphosphatidylinositol-linked GDNF family receptor
(GFR) The glial cell line-derived neurotrophic factor
(GDNF)1 family ligands GDNF,
neurturin (NRTN), artemin (ARTN), and persephin (PSPN) are structurally
related neurotrophic factors that signal through a multicomponent
receptor composed of the transmembrane receptor tyrosine kinase RET and
high affinity glycosylphosphatidylinositol (GPI)-anchored proteins, the
GDNF family PSPN mRNA is expressed at low levels in many rat tissues, where two
transcripts, an unspliced and a functional spliced form are produced
(6, 7). PSPN promotes the survival of embryonic motor neurons in
vitro and rescues nigral dopamine neurons following neurotoxic
injury in vivo but does not support the survival of any
peripheral neurons tested (6). However, unlike GDNF and NRTN, PSPN does
not induce neurite outgrowth of rat P8 motor axons in organotypic
cultures (8).
Somatic rearrangements caused by chromosomal inversions activate the
oncogenic potential of RET in human papillary thyroid carcinomas,
whereas germline point mutations are responsible for multiple endocrine
neoplasia type 2 (MEN2; reviewed in Refs. 9-11). The malignancies
associated with these syndromes arise in several RET-expressing neural
crest-derived cell populations. However, RET is expressed at high
levels in many other cell types which do not show malignant changes in
MEN2 (12). MEN2A is characterized by medullary thyroid carcinoma (MTC),
pheochromocytomas and parathyroid hyperplasia, whereas MEN2B is
characterized by MTC associated with pheochromocytomas, enteric
ganglioneuromas, skeletal abnormalities, and mucosal neuromas. MTC is
the only phenotype in familial medullary thyroid carcinoma. In
MEN2A, cysteine substitutions in the extracellular domain of RET cause
aberrant homodimerization and ligand-independent constitutive
activation of RET (13, 14), which alone or together with unknown
factors cause the malignant proliferation of cells. Familial medullary
thyroid carcinoma mutations are more evenly distributed among the
extracellular cysteines but are also found in the intracellular part of
RET. Most cases of MEN2B contain a specific mutation in the tyrosine
kinase domain, which is also frequently found in sporadic MTC. This
leads to constitutive activation of RET with altered substrate
specificity in downstream signaling pathway (13, 14). However, in
MEN2B-RET transfected cells, ligand binding has been reported to
increase the intensity of RET signaling (15, 16). Transgenic mouse
models of these mutations have shown to cause some of the malignant
phenotypes found in human MEN2 syndromes (17-19). The tissue-specific
tumorigenesis and phenotypic variability within a MEN2 family and
between individuals carrying the same mutation in RET, suggests that
further genetic events or modifier genes are required to induce the
tumor phenotype.
In this paper, we have characterized the human GFR RNA Isolation--
RNA from different human tissues was
purchased from CLONTECH (Human Total RNA Panels
I-V). Total RNA from primary MTC tumors was isolated using
UltraspecTM-II RNA isolation system (Biotecx Laboratories,
Inc.). Total RNA from the TT cell line (ATCC number CRL1803) was
isolated with Trizol reagent (Life Technologies). RNA from human fetal
thyroid and adrenal tissue (17- and 18-week-old, respectively) was
isolated from autopsy samples (Hospital for Children and Adolescents,
University of Helsinki, Finland), by RNAwizTM isolation
reagent (Ambion). Total RNA from human blood cells was isolated using
the QIAampTM RNA blood mini kit (Qiagen) according to the
manufacturer's instructions. The RET mutation of five MTC patients had
been analyzed: one from a MEN2A family member contained the germline
C634R mutation, one patient was positive for the M918T but the germline
situation was unknown, and three were sporadic MTCs of which one was
positive for the M918T mutation.
RACE Cloning and RT-PCR--
Human GFRA4 cDNAs
were identified from a human thyroid cDNA
(CLONTECH Marathon Ready cDNA) by the 3'- and
5'-RACE method using the GC-rich PCR kit (Roche Molecular Biochemicals)
according to the manufacturer's instructions. Reverse transcription
reactions were performed using Superscript II (Life Technologies). PCR
systems were GC-rich PCR kit (Roche Molecular Biochemicals) for
GFRA4 and PSPN, Expand Long Template PCR system
(Roche Molecular Biochemicals) for GFRA1, GFRA2,
GFRA3, and RET, and Dynazyme II (Finnzymes) for
PPIA (alias cyclophilin). PCR was run 40 cycles for
GFRA4, 35 cycles for PSPN, GFRA1, and
GFRA2, and 30 cycles for GFRA3, RET, and
PPIA using annealing temperatures of 55-60 °C.
Full-length human GFRA4 cDNA was obtained using Expand
High Fidelity system PCR enzyme (Roche Molecular Biochemicals) together
with GC-rich PCR system buffers (Roche Molecular Biochemicals). PCR
products were cloned into mammalian expression vector pCR3.1 (Invitrogen).
The antisense primers used in 5'-RACE were the mouse-specific
Gfra4 primer 5'-CACGTTGTCCAGGTAGTTGG-3' and a second
human-specific GFRA4 nested primer
5'-GCACTGCGCCACATACTCGGA-3'. The 3' region of GFRA4
was cloned in two steps, first using a GFRA4-specific sense
primer P2 (see Fig. 1A) 5'-GCTCCGAGTATGTGGCGCAGT-3' and a
nested primer 5'-GCTCACCCACGCACTGCTCTTCTG-3'. The 3'-region containing
the stop codon and 3'-untranslated region sequence was cloned using a
sense primer 5'-CCTAACTACGTGGACAACGTGAGC-3' and a second nested primer
5'-ATGGTGCCATTCAGGCCTTTGCCAG-3' or 5'-GCAGGTGTCCTCCACAGGCAG-3'. RT-PCR
of genomic human DNA was used to locate the intronic nucleotide
sequences between exons 4 and 5, and between exons 5 and 6, using a
sense primer 5'-CCTAACTACGTGGACAACGTGAGC-3' and an antisense primer
5'-GAAGTATGGAGAGCAGGGAGCGTC-3'. Full-length human GFRA4
cDNAs were obtained using a sense primer P1 (see Fig. 1A), 5'-CCACCATGGTCCGCTGCCTGG-3' and an antisense primer P3,
5'-GAGGTCGCTGTCCTAATCAGAG-3'.
Primers used in RT-PCR for human, mouse, and rat PSPN were as described
in Ref. 6. Primers used in RT-PCR for human GFRA1 flanked
nucleotides 491-870 of GenBankTM sequence AF042080, for
GFRA2 flanked nucleotides 148-427 of GenBankTM
sequence U93703, for GFRA3 flanked nucleotides 574-1203 of GenBankTM sequence NM001496, and for RET flanked
nucleotides 833-1114 of GenBankTM sequence X12949. The
amounts of total RNA in samples were normalized by amplification of a
PPIA fragment.
125I-Labeled PSPN Binding--
Murine neuroblastoma
Neuro-2a cells were transfected (FuGene6, Roche Molecular Biochemicals)
with full-length GFRA4a cDNA in pCR3.1 vector
(Invitrogen), and bulk selected with 400 µg/ml G418. PSPN was
enzymatically iodinated by lactoperoxidase, to a specific activity of
100,000 cpm/ng. Binding assays were performed essentially as described
(20) with 0.9 nM 125I-PSPN in binding buffer
(Dulbecco's modified Eagle's medium containing 0.2% bovine serum
albumin and 15 mM Hepes, pH 7.5) for 4 h on ice,
either in the presence or absence of different concentrations of
unlabeled human PSPN (PeproTech EC Ltd.), rat GDNF (Cephalon, Inc.),
human NRTN (PeproTech EC Ltd.), or human ARTN (a gift from Drs. J. Milbrandt and E. M. Johnson, Jr.). The amount of
125I-PSPN bound to mock-transfected cells was at the
background level. Kd was calculated using
Cheng-Prusoff equation (21).
Chemical Cross-linking and Immunoprecipitation--
Chinese
hamster ovary cells were transfected (FuGene 6) with full-length human
GFRA4a cDNA, grown for 2 days, washed and incubated with
0.9 nM 125I-PSPN in binding buffer (see above)
on ice for 4 h. After washing the cells were incubated with 1 mM bis-suberate (Pierce) in phosphate-buffered saline at
room temperature for 25 min. Following washes, some samples were
treated with 0.5 unit/ml phosphoinositide-phospholipase C
(Sigma) at +37 °C for 30 min. Cells were lysed in Laemmli buffer containing
The cross-linking reaction for human GFRA4a and mock
transfected (pcDNA3; Invitrogen) Neuro-2a cells was done as
described above but the cells were lysed in Nonidet P-40 lysis buffer
(1 × Tris-buffered saline, 2 mM EDTA, 1% Nonidet
P-40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
CompleteTM protease inhibitor mixture, Roche Molecular
Biochemicals), clarified by centrifugation at 14,000 rpm for 20 min,
immunoprecipitated with 1 µg/ml Ret antibodies (Santa Cruz
Biotechnology), and collected with Protein A-Sepharose. The
immunocomplexes were washed with Nonidet P-40 buffer, and separated by
7.5% SDS-polyacrylamide gel electrophoresis.
Ret Phosphorylation Assay--
Semiconfluent human
GFRA4a-, rat Gfra1-, or vector-expressing
Neuro-2a cells were maintained with 0.5% serum for 24 h and then
in serum-free medium for 4 h prior to stimulation. After stimulation with PSPN (0.01-100 ng/ml) or GDNF (100 ng/ml), cells were
lysed and precipitated with agarose-conjugated anti-phosphotyrosine antibodies (4G10, Upstate Biotechnology) overnight at +4 °C on ice.
The membranes were probed with 60 ng/ml anti-Ret antibodies (Santa Cruz Biotechnology).
Neuronal Microinjections--
Superior cervical ganglion (SCG)
neurons from postnatal day 1-2 FVB or NMRI strain mice were grown for
5-6 days on polyornithine/laminin-coated dishes with NGF (30 ng/ml).
50:50 ng/µl cDNA mixtures of short isoform of human
RET (gift from Dr. M. Billaud) and either mouse Gfra4a1 (in pcDNA3, Invitrogen) or human
GFRA4a (in pCR3.1, not shown) or empty pcDNA3 vector
were pressure-microinjected into nuclei. To find successfully injected
neurons later, a plasmid encoding enhanced green fluorescent protein
(10 ng/µl) was included in every injection mixture. Neurons were
grown overnight with NGF and thereafter in NGF-free medium containing
blocking anti-NGF antibodies (Roche Molecular Biochemicals) and PSPN,
GDNF, NRTN, or ARTN, all at 100 ng/ml. Number of living fluorescent
neurons or uninjected control neurons was then counted (initial
neurons). 30-80 initial neurons were successfully injected for every
treatment group. Healthy fluorescent neurons with intact nuclei and
phase-bright cytoplasm were counted 70-75 h later and expressed as
percentage of initial neurons. Experiments were repeated on independent
cultures: n = 7 for PSPN, n = 4 for
GDNF and NRTN, n = 3 for ARTN. In each repeat, all
treatment groups (plasmid combinations and controls) for a given
factor, shown as a separate group of bars on Fig. 4, were assayed
collectively in the same experiment. Experiments with GFR In Situ Hybridization--
In situ hybridizations for
RET, GFRA1, GFRA2, GFRA3,
and GFRA4 were performed as described (22) on cryosections
of the same thyroid tumor samples analyzed by RT-PCR. A 206-bp cDNA
fragment of the 5'-end of human GFRA4 was used as template
for sense and antisense RNA probes. PCR fragments of GFRA1,
GFRA2, GFRA3, and RET generated with the same primers
as in RT-PCR were cloned and sequenced and used as templates for
antisense and sense RNA probes. Control sections hybridized with sense
probe did not show labeling above background (not shown). Dark-field
and corresponding bright-field images of Nissl counterstained sections
were digitized and processed using Adobe PhotoShop software.
Characterization of Human GFRA4--
Sequence information from the
mouse Gfra4 cDNA and gene (5) was used to identify human
GFRA4 cDNA and genomic clones. First, the genomic region
covering putative exon 2 to exon 4 was cloned by PCR from human genomic
DNA. Next, the structure of GFRA4 cDNAs and gene were
characterized by 5'- and 3'-RACE of adult human thyroid cDNA and
PCR of genomic DNA using primers corresponding to different regions of
the putative exons of human GFRA4 (see "Materials and
Methods"). The sequence of human GFRA4 exon 1 encoding the
signal sequence showed high homology to the mouse Gfra4 exon 1a (5). cDNAs encoding proteins with an alternative signal sequence
homologous to the 1b signal sequence found in mouse, or putative
transmembrane and soluble isoforms found in mouse (5), were not
identified from human thyroid. Instead, three different alternatively
spliced GFRA4 cDNAs were identified (GFRA4a, GFRA4b, and GFRA4c, Fig.
1B): (i) GFRA4a (810 bp)
corresponds to the mouse Gfra4 transcript a1 (5). The
predicted protein (GFR
The other identified alternative human GFRA4 transcripts
are: (ii) GFRA4b (900 bp), in which the small intron (79 bp)
separating exons 2 and 3 (Fig. 1A) is included in the
transcript, and the 3'-splice site of exon 4 is located 11 bp upstream
of the respective splice site used in the GFRA4a transcript.
The small intron between exons 2 and 3 is also inefficiently spliced in
the majority of mouse tissues. Inclusion of this intron in mouse
Gfra4 transcripts with exon 1a or exon 1b leads to a
putative soluble protein isoform (5). In contrast, translation of the
transcript GFRA4b in human would lead to a GPI-linked
protein of 299 amino acids. In this protein isoform, the N- and
C-terminal ends are identical to the respective regions of
GFRA4a, but the middle region consists of a stretch of 66 amino acids translated in different frame from intron 2 and exon 3. This sequence is not homologous to any protein in public data bases.
(iii) GFRA4c (867 bp), in which the introns between exons 2 and 3, and between exons 3 and 4 are included in the transcript. These
introns would lead to a frameshift with a stop codon located inside
exon 5 and production of a putative soluble isoform of 236 amino acids.
It is interesting to note that the sequences of the 3'-splice sites of
both mouse and human exon 3 and exon 4 contain a short polypyrimidine
tract interrupted with purines, which is characteristic to
alternatively spliced exons (24).
Expression of GFRA4, PSPN, and RET in Different Human
Tissues--
Expression of GFRA4, PSPN, and
RET was analyzed by RT-PCR in 27 different adult and 4 fetal
tissues (Fig. 2 and not shown). Using
primers P2 and P3, expression of GFRA4 was detected at high levels in the adult thyroid gland (Fig. 2) and, at lower levels, in the
fetal adrenal and fetal thyroid gland (not shown), whereas all other
tissues analyzed did not express detectable levels of GFRA4.
Similar results were obtained with other combinations of primers P1,
P2, and P3 (data not shown). On Fig. 2, the smallest fragment of 699 bp
corresponds to the GFRA4a cDNA where all introns are
spliced out, whereas the 789-bp fragment corresponds to the intron
2-containing GFRA4b transcript. The 863-bp PCR fragment corresponds to the cDNA containing both introns 2 and 3 encoding the putative soluble isoform (GFRA4c, Fig. 1B).
The unspliced transcript (560 bp) of PSPN was expressed in all human
tissues examined using primers detecting the full-length
PSPN transcript (Fig. 2, second row). No
transcripts were observed when RNA was used as a template for PCR,
which shows that the cDNA samples did not contain chromosomal DNA.
Low levels of spliced PSPN transcripts (476 bp) encoding the
functional protein were present in adult human adrenal gland,
cerebellum, spinal cord, and testis, and were not detected in the
thyroid gland. The nucleotide sequences for the spliced and unspliced
PSPN transcripts were verified by sequencing. RET mRNA
was expressed at variable levels in most tissues examined (Fig. 2,
third row).
PSPN, but not Other GDNF Family Ligands Binds and Activates Human
GFR
Next we studied the binding of PSPN to GFRA4a-transfected
Chinese hamster ovary cells, which do not express Ret. Treatment of the
cells with 125I-PSPN, followed by chemical cross-linking
and analysis by SDS-polyacrylamide gel electrophoresis, resulted in a
major band of ~46 kDa and minor bands of ~62 and 92 kDa (Fig.
3B). The ~46-kDa band corresponds a PSPN monomer (~16
kDa) cross-linked to monomeric GFR
To study the interaction of PSPN with Ret, proteins cross-linked to
125I-PSPN in hGFR
To determine whether PSPN binding to human GFR PSPN Specifically Promotes Survival of Neurons Ectopically
Expressing GFR Selective Expression of GFRA4 and RET mRNAs in Medullary but
not Other Primary Thyroid Tumors--
Co-localization of
Gfra4 and Ret in developing and mature mouse
thyroid C-cells (5), and their coexpression in adult human thyroid
tissue (Fig. 2), prompted us to study the expression of GFRA4,
RET, and PSPN mRNAs in human primary thyroid
tumors. RT-PCR analysis of GFRA4 expression, using primers
generating full-length transcript, showed that similar levels of
GFRA4 mRNAs encoding the two GPI-linked isoforms and
lower levels of transcripts encoding the soluble isoform, are expressed
in all eight MTC samples analyzed (Fig.
5, first row).
GFRA4 mRNA was not detected in any of the follicular
thyroid adenomas (FTA), follicular thyroid carcinomas (FTC), or
papillary thyroid carcinomas (PTC) analyzed. Cellular localization of
GFRA4 mRNA was studied by in situ
hybridization in the same thyroid tumor samples (Fig.
6, A and B).
GFRA4 was highly and evenly expressed by virtually all the
malignant C-cells in the MTC samples analyzed, but not in the
accompanying connective tissue and blood vessels. No expression of
GFRA4 was detected in the adjacent apparently normal
follicle cells found in some samples. Other thyroid tumors, including
FTA, FTC, and PTC did not express GFRA4. None of the thyroid
tumors expressed detectable levels of spliced PSPN mRNA, whereas
the unspliced form of PSPN transcript was present in all tumor samples
analyzed (Fig. 5, second row). High levels of RET
expression were seen in all MTC tumors (Fig. 5, third row,
and Fig. 6).
Tumor samples were further analyzed for GFRA1,
GFRA2, and GFRA3 mRNA expression, by RT-PCR
and in situ hybridization (Fig. 6A and Table
I). In some tumor samples, low levels of
GFRA1, GFRA2, and GFRA3 mRNAs were present in
subsets of the tumor cells. To study if the GFRA4 gene is
mutated in the MTC tumors, full-length GFRA4 cDNA clones
from eight MTC samples, the TT cell line, and normal thyroid were
sequenced. Except for a few polymorphisms in the coding region, which
do not change the amino acid composition of GFR Structure and Expression of Human GFR
Of 27 different adult and 4 fetal tissues studied, GFRA4
transcripts were mainly detected in the adult thyroid gland, but also
at low levels in the fetal adrenal and thyroid glands. However, although high levels of Gfra4 are present in the
intermediate lobe of the mouse pituitary (5), we did not find
GFRA4 mRNA in the adult human pituitary, possibly due to
the rudimentary nature of the pituitary intermediate lobe in human. Low
levels of GFRA4 transcripts were also detected in fetal but
not in the adult human adrenal gland, whereas high Gfra4
levels are found in corresponding mouse tissue (5). This apparently
predominant expression of human GFRA4 in thyroid and adrenal
glands suggests a more tissue-specific transcriptional regulation and
possibly a more restricted role for GFR
In agreement with previous results (12, 25, 26), RET
mRNA was expressed at variable levels in many adult human tissues, including the thyroid and adrenal glands. The functional spliced PSPN mRNA has previously not been identified in human
(6). In this study, we found the unspliced transcript of
PSPN in all human tissues examined. However, we could also
detect low levels of the spliced PSPN mRNA in human
adrenal gland, cerebellum, spinal cord, and testis. In contrast,
similar levels of both PSPN transcripts are present in most
tissues in rat (6, 7). Although the spliced transcript encoding
functional PSPN is expressed in the rat thyroid (not shown), we could
not detect its expression in either adult human (Fig. 2) or postnatal
mouse (not shown) thyroid gland. Adrenal gland was the only tissue
where the spliced PSPN transcript was detected in all three
species analyzed (mouse, rat and human). Thus, the source of ligand for
GFR
After completion of this study, a Gfra4 cDNA encoding a
different GFR
Masure et al. (27) identified only the exon IB containing
rat Gfra4 transcript and showed that high levels of
Gfra4 mRNA are present in the brain using cRNA probe
specific to the region that is present in all Gfra4
alternatively spliced transcripts. They did not study if the
Gfra4 transcripts expressed in brain encode functional
protein isoforms with signal sequence. We have shown that in mouse the
transcripts containing exon Ia or exon Ib are expressed at extremely
low levels in the nervous system. Furthermore, we have shown that
although high levels of Gfra4 mRNA are present in the
mouse and rat nervous system, RACE, RT-PCR, and RNase protection
analyses of Gfra4 transcripts showed that majority of these
transcripts encode truncated protein isoforms without a functional
signal sequence (5) (data not shown). Taken together, our results
strongly suggest that the Gfra4 transcripts seen by Masure
et al. (27) in the rat brain encode unfunctional protein isoforms.
GFR
The cross-linking studies show that PSPN is able to bind GFR
We also demonstrate that PSPN but not other GDNF family ligands, can
promote the survival of cultured SCG neurons expressing Gfra4 and RET. In our assay, neurons coexpressing
RET plus GFR Putative Role for GFR
Strong RET expression was also localized to the malignant
C-cells consistent with previous reports (25, 34). Low levels of
GFRA1, GFRA2, and GFRA3 transcripts
were present in subsets of tumor cells, indicating that their
expression is not specific to particular thyroid tumor cell type. This
result is in line with a recent report showing GFRA1 and
GFRA2 expression in some MTC tumor cells (35). However, our
results showing low GFRA1 and GFRA2 expression
also in normal thyroid, in other thyroid tumor types, and not in all
MTC samples, does not support the idea that GFR subunit and the transmembrane receptor tyrosine kinase
RET. The inherited cancer syndrome multiple endocrine neoplasia
type 2 (MEN2), associated with different mutations in RET,
is characterized by medullary thyroid carcinoma. GDNF signals via
GFR
1, neurturin via GFR
2, artemin via GFR
3, whereas the mammalian GFR
receptor for persephin (PSPN) is unknown. Here we
characterize the human GFR
4 as the ligand-binding subunit required
together with RET for PSPN signaling. Human and mouse GFR
4 lack the
first Cys-rich domain characteristic of other GFR
receptors.
Unlabeled PSPN displaces 125I-PSPN from
GFRA4-transfected cells, which express endogenous Ret. PSPN
can be specifically cross-linked to mammalian GFR
4 and Ret, and is
able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family
ligands, promotes the survival of cultured sympathetic neurons
microinjected with GFRA4. We identified different splice
forms of human GFRA4 mRNA encoding for two
glycosylphosphatidylinositol-linked and one putative soluble isoform
that were predominantly expressed in the thyroid gland. Overlapping
expression of RET and GFRA4 but not other
GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFR
4 may restrict the MEN2 syndrome to these cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
receptors 1-4 (GFR
1-4, reviewed in Refs. 1 and 2).
GFR
4 was first described from chicken and shown to be the
preferential receptor for PSPN (3, 4). Recently, we characterized a
mouse GFR
4 receptor (5). It differs from all other GFR
receptors,
including chicken GFR
4, being smaller in size and lacking the first
Cys-rich domain. Mouse Gfra4 transcripts are expressed in
many embryonic and adult tissues but efficient splicing leading to a
functional GPI-linked isoform, as well as putative transmembrane and
soluble isoforms, occurs only in thyroid and adrenal medulla and in
pituitary intermediate lobe (5). In mouse, Gfra4 and
Ret are coexpressed only in the thyroid C-cells and adrenal
chromaffin cells. In chicken, Gfra4 mRNA is broadly
expressed during embryonic development, including the spinal
motoneurons and kidney (4). Chicken GFR
4 also binds mouse PSPN and
confers survival response to PSPN in the presence of Ret (3). However,
due to different structures of chicken and mammalian GFR
4, as well
as the lack of information about the existence of chicken GFR
3,
ligand specificity of mammalian GFR
4 cannot be directly extrapolated
from experiments with chicken GFR
4.
4 receptor and
show that it is a functional co-receptor for PSPN, which mediates RET
activation. Selective coexpression of GFRA4 and
RET in normal and malignant C-cells suggests that
GFRA4 is a candidate modifier gene in MTC.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol.
4 alone
(with and without PSPN) were performed separately (n = 4) and combined with other PSPN data. Significance of the differences
between means was estimated by one-way ANOVA followed by Tuckey's
post-hoc test at the significance level of
= 0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4a, 290 amino acids, Fig. 1C)
contains a putative N-terminal hydrophobic signal, one
N-linked glycosylation site (NVSA) at position 178, and a
hydrophobic stretch of amino acids in the C terminus, preceded by a
hydrophilic linker region, consistent with a GPI-anchor signal sequence
(23). The amino acid identity between mouse and human GFR
4a is 76%,
whereas the identity between human and chicken GFR
4 is 54%
(covering amino acids 143-340 in chicken GFR
4).
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Fig. 1.
Structure of the human GFRA4
gene compared with mouse Gfra4 and different
splice forms of human GFRA4 mRNA.
A, structure of the human GFRA4 gene
(top) compared with mouse Gfra4 gene
(bottom). Both contain at least 6 exons but no alternative
exon 1b has been found in human. Also the putative transmembrane form
encoded by the alternatively spliced exon 6 found in mouse is absent in
human. Primers P1-P3, used in RT-PCR in Figs. 2 and 5 are marked by
arrowheads. B, splicing of the GFRA4 gene in
human thyroid gland. Two GPI-linked isoforms (GFRA4a and
GFRA4b), and one putative soluble isoform
(GFRA4c) are produced by alternative splicing. In transcript
encoding for GFR 4a all the introns are spliced, whereas GFR
4b
contains the intron between exons 2 and 3. Transcript encoding GFR
4c
contain introns between exons 2 and 3 and between exons 3 and 4. Asterisk marks the stop codon. C, proposed
schematic domain structure of human GFR
4 isoforms.
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Fig. 2.
RT-PCR analysis of GFRA4, PSPN,
and RET transcripts in various human
tissues. Top row, GFRA4 expression is
detected only in thyroid gland using primers P2 and P3 (see Fig.
1A). All the introns in the transcript corresponding to 699 bp are spliced. The 789-bp transcript contains a small intron between
exons 2 and 3 and corresponds to the GFR 4b form. The 863-bp fragment
contains introns between exons 2 and 3 and between exons 3 and 4, and
corresponds to the soluble GFR
4c form. Second row, an
unspliced transcript of the human PSPN is expressed in all
tissues (560 bp), whereas the spliced transcript (476 bp), which
corresponds to the functional PSPN, is weakly expressed in cerebellum,
adrenal gland, and testis and barely detectable in spinal cord. To
exclude DNA contamination in RT-PCR reactions, controls containing RNA
were also subjected to PCR (not shown). Third row, RET
expression (282 bp) is detectable in almost all human tissues examined
but the expression levels are strongest in adult brain, cerebellum,
spinal cord, salivary gland, adrenal gland, and prostate. Bottom
row, control RT-PCR with PPIA transcript
showing equal loading of cDNA. In all PCR experiments, negative
water controls were included (not shown).
4-Ret Receptor Complex--
To study the binding of human PSPN
to human GFR
4, we used GFRA4a-transfected mouse
neuroblastoma Neuro-2a cells (hGFR
4/Neuro-2a), which endogenously
express Ret. These cells strongly bound 125I-PSPN, while
mock-transfected Neuro-2a cells did not (Fig.
3C). Low concentrations (~1
nM) of unlabeled PSPN effectively displaced 125I-PSPN from the GFRA4-expressing cells,
whereas GDNF (up to 300 nM), NRTN (up to 200 nM), and ARTN (up to 200 nM) were ineffective (Fig. 3A). Thus, in the presence of Ret, human GFR
4 binds
specifically PSPN, but not the other GDNF family ligands. PSPN binds to
human GFR
4 with a dissociation constant (Kd) of
~100 pM (Fig. 3A).
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Fig. 3.
Binding and chemical cross-linking of
125I-PSPN to human GFR 4 and
Ret. A, displacement binding of 125I-PSPN
by PSPN, NRTN, GDNF, and ARTN from human GFR
4a-expressing Neuro-2a
cells, shown as percentage of 125I-PSPN bound in the
absence of a cold ligand (100%). One representative experiment out of
four is shown. B, chemical cross-linking of
125I-PSPN to vector- (lanes 1 and 2)
or hGFRA4a transfected (lanes 3-6) Chinese
hamster ovary (CHO) cells, in the presence (lanes 2, 3, and 5) or absence (lanes 1, 4, and
6) of unlabeled PSPN, treated (lanes 3 and
4) or not treated (lanes 1, 2, 5, and
6) with phosphoinositide-phospholipase C. Bands are
designated as follows: arrowhead, ~46 kDa;
arrow, ~62 kDa; asterisk, ~92 kDa.
C, proteins cross-linked to 125I-PSPN in the
presence (lanes 1 and 3) or absence (lanes
2 and 4) of unlabeled PSPN in vector- (lanes
1 and 2) or GFRA4a- (lanes 3 and
4) transfected Neuro-2a cells. Bands are designated as in
B. D, immunoprecipitation of Ret from vector- (lanes
1 and 3) or GFRA4a-transfected (lanes
2 and 4) Neuro-2a cells after chemical cross-linking of
125I-PSPN, separated in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of
-mercaptoethanol (
-Me). Bands are designated as
follows: arrowhead, ~46 kDa; arrow, ~190 kDa;
diamond, ~220 kDa; asterisk, ~400 kDa.
E, phosphorylation of Ret in mock- (lanes 1 and
2), GFRA4a- (lanes 3-5) or
Gfra1-transfected Neuro-2a cells upon stimulation with 100 ng/ml PSPN (lanes 2, 4, and 5) or 100 ng/ml GDNF
(lane 7). WB, Western blot; IP,
immunoprecipitation. F, dose-dependent
phosphorylation of Ret with PSPN in hGFRA4a-transfected
Neuro-2a cells.
4 (30 kDa). The ~62- and 92-kDa
bands most probably correspond to PSPN dimer cross-linked to GFR
4
monomer and dimer, respectively. Additional 16- and 32-kDa bands
represent PSPN monomer and dimer, respectively (Fig. 3B, lane
6). No specific bands were detected from mock-transfected cells
(Fig. 3B, lanes 1 and 2). The amount of
cell-bound complexes was greatly reduced by adding unlabeled PSPN or by
removal of the GPI-anchored proteins by phosphoinositide-specific
phospholipase C after cross-linking (Fig. 3B, lanes 4 and
5). Combination of these two treatments further reduced the
yield of cross-linked products to undetectable levels (Fig. 3B,
lane 3). Thus, the human GFR
4 protein, encoded by the
GFRA4a transcript indeed contains a GPI anchor. Cross-linked
complexes of the same sizes were also identified from hGFR
4/Neuro-2a
cells (Fig. 3C). In addition, a minor band of about 200 kDa
was observed (Fig. 3C) that could be a complex of
PSPN-GFR
4 with Ret.
4/Neuro-2a cells were precipitated with
Ret antibodies (Fig. 3D, lanes 2 and 4). The
major cross-linked complexes of ~190 and 220 kDa, as well as minor
complexes of about 62 and 92 kDa (same size as bands in Fig.
3C), and also of ~400 kDa were obtained under reducing
conditions (Fig. 3D, lane 2). Under nonreducing conditions,
the ~400-kDa band was greatly intensified, which indicates the
presence of S-S-bound complexes. No cross-linked cell-bound products
were detected using parental Neuro-2a cells (Fig. 3D, lanes
1 and 3). The bands of about 190 and 220 kDa correspond to the complexes of PSPN-Ret (186 kDa) and PSPN-GFR
4-Ret (216 kDa),
respectively. The components of the complex of about 400 kDa could be a
dimer of the 186-kDa PSPN-Ret complex.
4 mediates Ret
autophosphorylation, we used hGFR
4/Neuro-2a cells treated with PSPN.
Gfra1 expressing Neuro-2a cells treated with GDNF served as
a positive control. PSPN induced Ret tyrosine autophosphorylation in
hGFR
4/Neuro-2a cells, which showed a phosphorylated band of 170 kDa,
corresponding to the active form of Ret (Fig. 3E). No phosphorylation of Ret was observed in vector-transfected Neuro-2a cells (Fig. 3E, mock). Stimulation of Ret phosphorylation
was dose-dependent starting at 0.1 ng/ml PSPN (Fig.
3F).
4 and RET--
We further studied whether binding of
PSPN to mammalian GFR
4-Ret complex triggers functional cellular
programs, as described for chick GFR
4 (3). We expressed the
GPI-linked mouse Gfra4 or human GFRA4a together
with human RET in neonatal mouse SCG neurons and maintained
the neurons further with PSPN for 3 days. SCG neurons are trophically
dependent on NGF but not on PSPN (3, 5, 6, Fig.
4). As verified by RT-PCR, neonatal mouse
SCG expressed transcripts for Ret but not for
Gfra4 (data not shown). Significant portion of neurons
overexpressing mouse Gfra4 and RET was maintained by PSPN (60 ± 5, 1% versus 16 ± 1, 2% without
PSPN, p < 0.001), whereas omission of Gfra4
abolished this trophic effect (Fig. 4). Similar results were obtained
with human GFRA4a (not shown). Moreover, introduction of
Gfra4 alone was already sufficient to confer PSPN dependence
to the SCG neurons (Fig. 4), obviously via endogenous Ret, whereas
without PSPN, the survival rate of Gfra4-injected neurons
did not differ from that of uninjected or vector-injected neurons (Fig.
4). GDNF and NRTN also maintained part of neurons overexpressing
RET and Gfra4, but these effects were not reduced
by omission of Gfra4 (Fig. 4). Moreover, both factors
maintained also uninjected neurons similarly to receptor-injected neurons (Fig. 4). ARTN promoted survival of neither GFR
4/RET injected nor uninjected mouse SCG neurons (Fig. 4). Thus, GFR
4-RET complex requires PSPN, but not other GDNF family members, to activate a
survival-promoting program in SCG neurons.
View larger version (28K):
[in a new window]
Fig. 4.
PSPN specifically promotes survival of
RET/Gfra4-injected SCG neurons. Neonatal mouse
SCG neurons were grown 5 days with NGF, injected with indicated plasmid
mixtures (all 50:50 ng/µl), grown overnight with NGF, then NGF was
changed for indicated factors (all 100 ng/ml). Numbers of viable
neurons after a 3-day treatment period are expressed relative to the
number of initial neurons. Mean ± S.E of three to seven
independent repeats for each factor is shown. *, p < 0.05; ***, p < 0.001.
View larger version (37K):
[in a new window]
Fig. 5.
Semiquantitative RT-PCR analysis of
GFRA4, PSPN, and RET in different
human thyroid tumors. Top row, GFRA4
mRNA is selectively expressed in MTC. No GFRA4
transcripts are detectable in FTA, FTC, or PTC. Each lane represents
tumor cDNA from different patient. Normal thyroid gland, including
medullary tissue (NT) also expresses GFRA4. Two
major transcripts of 831 and 910 bp seen in MTC and TT cells correspond
to GFR 4a and GFR
4b, respectively (primers P1 and P3, see Fig.
1 A). The minor transcript of 999 bp corresponds to the
soluble GFR
4c. Second row, no spliced form of the
PSPN transcript is detectable using primers covering the
initiation and the stop codons. Third row, RET
mRNA is expressed in all MTC samples. Bottom row,
control RT-PCR with PPIA transcript showing equal loading of
cDNA. All experiments contained negative water controls (not
shown).
View larger version (132K):
[in a new window]
Fig. 6.
Localization of GFRA1, GFRA2, GFRA3,
GFRA4, and RET mRNAs in thyroid tumors
by in situ hybridization. A, typical
representative examples of autoradiography films showing
GFRA1-GFRA4 and RET cRNA hybridization to
adjacent frozen sections of different thyroid tumors. Of the four types
of tumors represented, only the MTC sections were strongly stained for
GFRA4 and RET expression. GFRA1,
GFRA2, and GFRA3 mRNA levels were very low or
undetectable in all samples including MTC. B,
GFRA4 and RET probes were hybridized to frozen
sections representing FTA, FTC, PTC, and MTC samples and sections were
counterstained by Nissl substance. GFRA4 and RET
is highly expressed in all malignant cells of the MTC sample but not in
the surrounding connective tissue. Bar in A = 0.5 cm, in B = 100 µm.
4, no mutations were
found in the MTC samples or in the TT cell line. Thus, the coding
region of GFRA4 gene is not mutated in MTC tumors.
Expression of human GFRA1, GFRA2, GFRA3, GFRA4, and RET in thyroid
tumors and normal thyroid
, no labeling above background;
to +++, highest detected expression level based on visual inspection.
The numbers below the tissue gradings denote the number of samples
positive/total number of samples analyzed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4--
In this study, we
describe the cloning of human GFR
4 cDNAs
encoding three different isoforms of human GFR
4, two GPI-linked and
one putative soluble isoform, from adult human thyroid gland. We have
earlier described the cloning and expression of various mouse GFR
4
cDNAs encoding different protein isoforms (5). As in mouse, all
human GFR
4 isoforms lack the first Cys-rich domain (D1) common for
all known GFR
receptors, including chicken GFR
4 (4).
4 in human, than in chicken
and rodents, where Gfra4 transcripts are expressed in many
tissues (4, 5). Consistent with this, no human ESTs for
GFRA4 were present in public data bases in contrast to
several mouse ESTs for Gfra4 (5).
4 in human thyroid remains elusive. It is possible that
functional PSPN is expressed in the thyroid region at developmental
stages not analyzed here due to the lack of commercially available RNAs.
4 isoform was cloned from rat brain and reported as a
GPI-linked receptor for PSPN (27). However, the signal sequence encoded
by the rat Gfra4 cDNA in that report (27) is very weak and differs from the GFR
4 signal sequence we have identified by
5'-RACE cloning from rat thyroid
(MACCLESALLLLLLLLLLGSASS).2
The rat GFR
4 signal sequence identified by us is strong and shows
high homology to the mouse (76%) and human (67%) signal sequences
encoded by exon 1a in mouse and by exon 1 in human, suggesting that
this rat GFR
4 signal sequence is also functional. The putative rat
GFR
4 signal sequence identified by Masure et al. (27)
shows 59% amino acid identity with the putative signal sequence
encoded by the alternatively spliced exon 1b of mouse Gfra4
(5). Our results suggest that both mouse and rat Gfra4 genes
have two 5'-exons encoding alternative N termini of the GFR
4
protein. In human, however, GFRA4 cDNAs encoding
alternative signal sequences were not identified.
4 Is the Functional Receptor for PSPN--
The binding and
cross-linking studies demonstrated that association of PSPN with the
Ret receptor protein-tyrosine kinase is mediated by GFR
4. Ligand
displacement binding showed that in the presence of Ret, only PSPN but
not GDNF, NRTN, or ARTN was effective in displacing
125I-PSPN from GFRA4-expressing Neuro-2a cells.
The dissociation constant (Kd) of about 100 pM found here is 10 times lower than the
Kd of about 1 nM reported for mouse
PSPN, binding to chicken GFR
4 (3), and 60 times lower than the about 6 nM reported for rat PSPN binding to immobilized rat
GFR
4 fusion protein (27), but similar to those reported for GFR
1,
GFR
2, and GFR
3 and their cognate ligands (28-30). The lower
binding of mammalian PSPN to chicken GFR
4 probably reflects the
species difference in GFR
4 structure. PSPN has not yet been
characterized from chicken and, if it exists, might differ
significantly from the mammalian PSPN.
4 also
in the absence of Ret. This is consistent with the model that a GDNF
family ligand first binds to the corresponding GFR
receptor and
subsequently the ligand-GFR
complex binds to Ret (31). However, our
results also agree with the alternative model in which the ligand binds
a preformed GFR
-Ret complex (32). Although PSPN binding to chicken
GFR
4 has been shown earlier (3), we demonstrate here for the first
time that the association of PSPN with GFR
4 results in activation of
the Ret tyrosine kinase. PSPN treatment of cells expressing GFR
4
rapidly induced Ret autophosphorylation in a dose-dependent
manner. PSPN is unable to stimulate Ret autophosphorylation in cells
that do not express GFR
4. Thus, the GFR
4a isoform characterized in this report is a functional receptor for PSPN in triggering Ret
activation. In contrast to this, the rat GFR
4 isoform with a poor
signal sequence described by Masure et al. (27) bound PSPN
only as soluble fusion protein and did not lead to Ret activation.
4 showed an elevated response to GDNF and NRTN but not
to ARTN, however, also the uninjected neurons responded to GDNF and
NRTN. We could detect Ret, as well as Gfra1 and
Gfra2, but not Gfra4 transcripts in mouse SCG
(not shown). Therefore, it seems clear that GDNF and NRTN maintain
these neurons via activation of endogenous receptors but not via
GFR
4. Absence of Ret in mouse SCG, reported in a previous
study (33), probably results from different mouse strains used. Taken
together, the binding of PSPN to GFR
4-Ret complex triggers
functional cellular responses.
4 in Medullary Thyroid Carcinoma--
As
the RET proto-oncogene plays an important role in the oncogenesis of
MTC, a logical step was to examine GFR
4 expression in these tumors.
GFRA4 transcripts encoding the two GPI-anchored isoforms
were expressed at high level in all MTC samples. In addition, moderate
levels of GFRA4c encoding the putative soluble form of GFR
4 were found in all MTC samples. In contrast, no GFRA4
expression was detected in any other type of thyroid tumor analyzed.
Taken together, our data suggest that the expression of
GFRA4 in thyroid tumors is C-cell specific, and therefore
antibodies against GFR
4 could be useful in tumor diagnostics. It
will be important to study whether metastases derived from MTC tumors
and other endocrine tumors such as pheochromocytomas express
GFRA4.
1 or GFR
2 play a
role in the primary pathogenesis of MTC. We have proposed that
coexpression of GFR
4 with mutated RET may specify tissues affected
by MEN2 syndromes (5), and thus GFR
4 be necessary or at least
permissive for the initial hyperplasia of C-cells occurring in MTC. On
the other hand, as suggested in a recent report, other GFR
receptors
could interfere with the dimerization of mutant RET receptors, and
therefore inhibit tumor development in tissues not involved in MEN2
syndromes (36). It should be possible to test these hypotheses by
examining whether the tumor development is suppressed or enhanced in
different GFR
-deficient mice crossed with MEN2 transgenic mice
(17-19). Further in vitro studies are also needed to reveal
the roles for PSPN signaling via different GFR
4 isoforms through
normal and mutant RET.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Eila Kujamäki, Maarit Ohranen, and Satu Åkerberg for excellent technical assistance, Drs. J. Milbrandt and E. M. Johnson, Jr., for ARTN, and Dr. M. Billaud for the human RET construct.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Academy of Finland, Biocentrum Helsinki, Cephalon, Inc., EU Biomed II Grant BMH4-97-2157, and the Sigrid Juselius 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) AJ291673, AJ291674, and AJ291675.
§ Contributed equally to the results of this article.
** Biocentrum Helsinki fellow. To whom correspondence should be addressed. Tel: 358-9-191-59-359; Fax: 358-9-191-59-366; E-mail: Saarma@operoni.helsinki.fi.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008279200
2 M. Lindahl and T. Timmusk, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GDNF, glial cell line-derived neurotrophic factor; ARTN, artemin; FTA, follicular thyroid adenoma; FTC, follicular thyroid carcinoma; GFR, glial cell line-derived neurotrophic factor family receptor; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; NRTN, neurturin; PSPN, persephin; PTC, papillary thyroid carcinoma, SCG, superior cervical ganglion; PSPN, persephin; GPI, glycosylphosphatidylinositol; RT-PCR, reverse transcriptase-polymerase chain reaction; RACE, rapid amplification of cDNA ends; NGF, nerve growth factor; bp, base pair(s).
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