From the Departments of Biological Chemistry and the
** Reproductive Sciences Program, University of Michigan,
Ann Arbor, Michigan 48109-0606
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ABSTRACT |
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A new family of neuronal survival factors
comprised of glial cell line-derived neurotrophic factor (GDNF) and
neurturin has recently been described (Kotzbauer, P. T., Lampe,
P. A., Heuckeroth, R. O., Golden, J. P., Creedon,
D. J., Johnson, E. M., Jr., and Milbrandt, J. (1997)
Nature 384, 467-470). These molecules, which are related
to transforming growth factor-, are important in embryogenesis and
in the survival of distinct neuronal populations. These molecules
signal through a novel receptor system that includes the Ret receptor
tyrosine kinase, a ligand (i.e. GDNF or neurturin), and an
accessory glycosyl-phosphatidylinositol-linked molecule that is
responsible for high affinity binding of the ligand. Two accessory
molecules denoted GDNF family receptor 1 and 2 (GFR
-1 and GFR
-2)
have been described that function in GDNF and neurturin signaling
complexes. We have identified a novel co-receptor belonging to this
family based on similarity to GFR
-1, which we have named GFR
-3.
GFR
-3 displays 33% amino acid identity with GFR
-1 and 36%
identity with GFR
-2. Despite the similarity of GFR
-3 to GFR
-1
and GFR
-2, it is unable to activate Ret in conjunction with GDNF,
suggesting that there are likely additional undiscovered ligands and/or
Ret-like receptors to be identified. GFR
-3 is anchored to the cell
membrane by a phosphatidylinositol-specific phospholipase C-resistant
glycosyl-phosphatidylinositol linkage. GFR
-3 is highly expressed by
embryonic day 11 but is not appreciably expressed in the adult mouse.
In situ hybridization analyses demonstrate that GFR
-3 is
located in dorsal root ganglia and the superior cervical sympathetic
ganglion. Comparison of the expression patterns of GFR
-3 and Ret
suggests that these molecules could form a receptor pair and interact
with GDNF family members to play unique roles in development.
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INTRODUCTION |
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Glial cell line-derived neurotrophic factor
(GDNF)1 was initially
discovered because of its ability to promote the survival of embryonic
ventral midbrain dopaminergic neurons in culture (1). It is also
capable of supporting the survival of rat superior cervical and dorsal
root ganglion neurons in vitro as well as a variety of other
neuronal populations in the central and peripheral nervous systems
(2-6). Recently, a related molecule, neurturin (NTN), was purified
based on its ability to maintain rat superior cervical ganglion neurons
in culture (7). Together these molecules have established the existence
of a new family of neuronal survival factors displaying similarity to
transforming growth factor- (20% sequence identity). Structurally,
GDNF and NTN contain a Cys knot motif that is also present in nerve
growth factor, platelet-derived growth factor, and transforming growth
factor-
family members. This motif consists of six Cys residues that
form three intramolecular disulfide linkages between parallel
-sheets (8). The high degree of sequence conservation between GDNF
and NTN (42% sequence identity) as well as the conservation of the Cys
knot motif suggests that these molecules would serve as ligands for
related receptors. In fact, it was demonstrated that both GDNF (9-12)
and NTN (12-15) can activate the Ret receptor utilizing different
co-receptors for high affinity binding.
The c-ret proto-oncogene (16) codes for a receptor tyrosine
kinase with a Cys-rich extracellular domain, a single transmembrane domain, and a cytoplasmic tyrosine kinase domain (17). Mutations in the
Cys-rich region of the extracellular domain can lead to familial
medullary thyroid carcinoma or multiple endocrine neoplasia 2A (18,
19), whereas a mutation at amino acid 918 in the kinase domain of the
receptor causes multiple endocrine neoplasia 2B, a more aggressive form
of the disease (20). Inactivating mutations throughout the gene lead to
Hirschsprung's disease, which is characterized by loss of innervation
of the lower intestinal tract (21). In the developing mouse, RET is
specifically expressed in a number of tissues including the ureteric
bud of the developing kidney and the neural crest cells that innervate
the lower intestine. Mice that lack functional RET receptors have a
Hirschsprung's like phenotype and fail to develop kidneys (22).
Although it was surprising that a tyrosine kinase could be activated by
a member of the transforming growth factor- family, mice that lack GDNF displayed a phenotype similar to the Ret knockout and provided the
first clue that GDNF and Ret were a receptor/ligand pair (23).
Interaction of GDNF with Ret requires an additional protein, GFR-1.
GFR
-1 was the first co-receptor isolated using an expression cloning
strategy to identify high affinity binding proteins for GDNF (24, 25).
This molecule is attached to the cell surface via a
glycosyl-phosphatidyl-inositol (GPI) linkage. Recently, an additional
co-receptor, GFR
-2, was isolated by virtue of its sequence
similarity with GFR
-1 (13-15). As mentioned previously, GFR
-1
and GDNF form a high affinity complex that is capable of activating Ret
(24, 25). However, GFR
-1 has also been reported to bind NTN and
activate Ret (14). Similarly, GFR
-2 can bind to both GDNF and NTN
and activate Ret but appears to bind with higher affinity to NTN (14,
15). This is unique in that the specificity of the tripartite signaling
complex can be controlled either by the choice of co-receptor or by the
local concentration of a particular ligand.
Here we report the identification and characterization of a new GFR
family member that we have named GFR
-3. Expression profiles of this
molecule suggest that it is very important in embryogenesis, particularly in the nervous system. The existence of several family members and their differential expression patterns raises the possibility that this tripartite receptor/ligand system is a paradigm utilized in many tissues during development.
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MATERIALS AND METHODS |
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Sequence of GFR-3--
A mouse expressed sequence tag data
base clone encoding GFR
-3 (Image consortium; accession number
AA050083) was sequenced and analyzed. A mistake in the image consortium
clone that generated a premature stop codon in the coding sequence was
identified by sequencing multiple polymerase chain reaction products
obtained from a mouse fetal brain library using primers based on the
expressed sequence tag sequence. This base was corrected using the
QuikChange site-directed mutagenesis kit (Stratagene). Alignment was
performed using the Clustal W program (MacVector).
Cell Culture and Transient Transfection-- COS and 293 cells were grown in a 5% CO2 environment using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, glutamine (293 cells only), penicillin, and streptomycin. Subconfluent 293 or COS cells in 10-cm dishes were transfected with 8 µg of the DNA construct of interest using LipofectAMINE reagent (50 µl) as described by the manufacturer (Life Technologies, Inc.).
NB41A3 cells were grown in a 6% CO2 environment using Ham's F-10 supplemented with 15% heat-inactivated horse serum and 2.5% fetal bovine serum, penicillin, and streptomycin. Subconfluent NB41A3 cells in 35-mm dishes were transfected using a total of 2 µg of DNA and 4 µl of LipofectAMINE reagent for 10 h according to the manufacturer's instructions.Luciferase Assays-- 48 h post-transfection, cells were harvested by scraping, pelleted at 250 × g for 10 min, and resuspended in 400 µl of 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol, and 0.1% Triton X-100. The extracts were cleared of cell debris by centrifugation at 12,000 × g for 5 min. The luciferase assay was performed as described by Brasier et al. (26) using the same amount of protein for each sample.
Western Analyses--
NB41A3 cells were grown in 60-mm dishes
and transfected with 6 µg of vector (pcDNA3) or GFR-1 and
GFR
-3 pcDNA3 expression constructs using 12 µl of
LipofectAMINE as described above. Samples were treated with 100 ng/ml
GDNF for 5 min at room temperature prior to harvesting. Immune
precipitation with the Ret polyclonal antibody was performed as
described (29). Samples were run on a 10% SDS-PAGE gel, transferred to
Immobilon (Millipore), and immunoblotted with phosphotyrosine antibody
(1:10,000; Upstate Biotechnology, Inc.) or Ret antibody (1:500) as
described (29).
Cleavage of the GPI linkage by Phosphatidylinositol-specific
Phospholipase C--
293 cells were transiently transfected with
Flag-tagged GFR-1 and GFR
-3 expression constructions as described
above. Cells recovered for 48 h, were harvested in Dulbecco's
modified Eagle's medium containing 2% bovine serum albumin, 20 mM Hepes, pH 7.6, and treated with 1 unit of phospholipase
C (Boehringer Mannheim) for 30 min at 37 °C. After pelleting, the
medium was collected (1 ml) for immune precipitation, and the cells
were lysed in Dulbecco's phosphate-buffered saline (Life Technologies,
Inc.), 1% Triton X-100, 0.1 mg/ml phenylmethlysulfonyl fluoride. Both
were subjected to immune precipitation as described using Flag M2
affinity gel (Eastman Kodak Company) (11). The resulting samples were
analyzed by 10% SDS-PAGE and transferred to Immobilon. Western
analysis was performed with Flag M2 monoclonal antibody (1:2500;
Eastman Kodak Company) in Tris-buffered saline (10 mM Tris,
pH 8.0, 150 mM NaCl, 5 mM MgCl2),
0.1% Tween 20, and 5% nonfat dry milk. Horseradish peroxidase protein
A (Amersham Corp.) was used at a 1:5000 dilution in the above buffer,
and the antibody complexes were visualized by enhanced
chemiluminescence (National Diagnostics).
Biotinylation of GFR-3--
COS cells were transfected with
the Flag-tagged GFR
-3 construct as described above. Two days
post-transfection, cells were washed three times with Ringer's
solution (10 mM Hepes, pH 7.4, 154 mM NaCl, 7.2 mM KCl and 1.8 mM CaCl2) and
incubated with 2 ml of 200 µg/ml EZ-Link sulfo-NHS-Biotin (Pierce)
for 30 min at 4 °C. Following the biotin incubation, the cells were
washed five times with Tris saline (10 mM Tris, pH 7.4, 120 mM NaCl), and the cells were lysed in phosphate-buffered
saline, 1% Triton X-100, and 0.1 mg/ml phenylmethlysulfonyl fluoride.
Immunoprecipitations were performed using the Flag M2 antibody (4 µg)
followed by incubation with protein A-agarose (Life Technologies,
Inc.). The resulting samples were analyzed by 10% SDS-PAGE and
transferred to Immobilon (Millipore). Western analysis was performed
using horseradish peroxidase-conjugated streptavidin (1:500; Upstate
Biotechnology Inc.) in Tris-buffered saline, 0.1% Tween 20, and 2%
bovine serum albumin. Horseradish peroxidase protein A (Amersham Corp.)
was used at a 1:5,000 dilution in the above buffer, and the antibody complexes were visualized by enhanced chemiluminescence (National Diagnostics).
Radioisotopic Labeling Procedure--
293 cells (2 × 106 cells) were transfected as described above with
constructs encoding either Flag-tagged GFR-1, a transmembrane version of Flag-tagged GFR
-1, or Flag-tagged GFR
-3. After an overnight recovery period, cells were incubated with
[2-14C]ethan-1-ol-2-amine HCl (100 µCi/4 ml, 50-62
mCi/mmol, CFA 329, Amersham Corp.) for 18 h in Dulbecco's
modified Eagle's medium and 10% dialyzed fetal bovine serum. The
cells were harvested and subjected to immune precipitation as described
above. A portion of the samples was used for Western analysis as
described previously, and the remainder was analyzed by SDS-PAGE
followed by fixing in 10% acetic acid and fluorography with AmplifyTM
(Amersham Corp.). The film was exposed at
80 °C for 24 h.
Northern Analyses-- For the cell lines, 20 µg of total RNA was run on a 1% denaturing agarose gel, transferred to Hybond N (Amersham Corp.) and probed with the appropriately labeled cDNA. Both the multi-tissue Northern and developmental Northern were purchased (CLONTECH) and probed with the appropriately labeled DNA.
In Situ Hybridization Analysis--
A partial cDNA fragment
of the mouse RET proto-oncogene was cloned by reverse
transcription-polymerase chain reaction from Neuro 2A RNA using
oligonucleotides corresponding to the murine cDNA sequence between
nucleotide 361 and nucleotide 690. The fragment was subcloned into the
pGEM-T vector (Promega), and the plasmid was linearized with
NcoI. Mouse GFR-3 was obtained from the image consortium,
and a PstI fragment was cloned into pJCC (modified pBluescript, Stratagene). The construct was linearized with
NotI. For all of the above constructs, sense and antisense
probes were made using T7 or T3 RNA polymerase (Life Technologies,
Inc.) and [33P]UTP (NEN Life Science Products) (27). For
the hybridization, embryos of E14.5 were frozen in 2-methylbutane and
stored at
80 °C. Frozen embryos were cryostat sectioned (20 µm),
and in situ hybridization was performed as described
previously (27).
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RESULTS AND DISCUSSION |
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GFR-3 Is a Member of the GFR
Family--
GFR
-3 was
identified by a BLAST search (28) of the expressed sequence tag data
base performed using blocks of GFR
-1 amino acid sequences that
contained multiple Cys residues. We reasoned that the placement of
these Cys residues would be highly conserved among GFR
-1-related
molecules. The expressed sequence tag clone (Image consortium;
accession number AA050083) obtained was derived from 13.5-14.5-day-old
mouse embryos and was sequenced in its entirety (Fig.
1). The full-length cDNA contains an
open reading frame of 397 amino acids that contains a hydrophobic
signal peptide as well as a stretch of hydrophobic amino acids at its COOH terminus that comprises a putative GPI linkage sequence or membrane attachment sequence. It also contains three potential N-linked glycosylation sites that are represented by the
sequence Asn-Xaa-(Thr/Ser).
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GFR-3 Cannot Activate Ret Using GDNF as the Ligand--
Because
both GFR
-1 and GFR
-2 can form functional signaling complexes with
Ret upon addition of GDNF, we sought to determine whether GFR
-3 and
GDNF could form a functional signaling complex with Ret. Previously we
determined that activated Ret signals via the mitogen-activated protein
kinase pathway using the Gal-Elk/GAL-Luc reporter system (29). NB41A3
mouse neuroblastoma cells express Ret but require the addition of
either GFR
-1 or GFR
-2 in conjunction with GDNF to activate Ret
(12). When GFR
-1 is transiently introduced into these cells with the
Gal-Elk reporter system, GDNF is able to elicit a 4.5-fold induction of
reporter activity. Neither vector alone (pcDNA3) nor GFR
-3 is
able to increase reporter activity (Fig.
3A). In addition, transient
expression of GFR
-1 in NB41A3 cells treated with GDNF results in
increased tyrosine phosphorylation of Ret, whereas expression of
GFR
-3 does not (Fig. 3B). Therefore, despite the
conservation of this co-receptor family, only GFR
-1 and GFR
-2 are
able to use GDNF to activate Ret. However, GFR
-3 can be
co-immunoprecipitated with Ret when both molecules are transiently
expressed in 293 cells (data not shown). This implies that GFR
-3 and
Ret most likely will form a functional signaling complex in the
presence of the appropriate ligand.
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GFR-3 Membrane Anchor Analysis--
Both GFR
-1 and GFR
-2
possess the characteristic carboxyl-terminal features of GPI-linked
proteins in that they have a COOH-terminal hydrophobic domain separated
by a hydrophilic linker region from a cleavage consensus sequence for
GPI linkage, i.e. Ala-Ser-Ser for GFR
-1 and Gly-Ser-Asn
for GFR
-2 (13, 30). Because the carboxyl terminus of GFR
-3 varies
significantly from the other family members, we sought to determine
whether this molecule is secreted or attached to the extracellular
surface of the plasma membrane via its hydrophobic tail or a GPI
linkage. A common method to demonstrate whether a protein is linked to
the membrane by a GPI linkage is its ability to be cleaved by PIPLC. In
the following experiments, the co-receptors have been tagged with the
Flag epitope on their amino termini for easy detection. As can be seen
in Fig. 4A, GFR
-3 unlike
GFR
-1 cannot be removed from the membrane by treatment with PIPLC.
GFR
-1 undergoes an apparent shift in molecular weight upon cleavage
with PIPLC. This is because GFR
-1 expressed in 293 cells is present
as a doublet that is not well separated in this gel system. The higher
molecular weight species is the molecule that is concentrated in the
medium after PIPLC cleavage. The lower molecular weight species is
presumably in the process of being transported to the cell surface. To
demonstrate that GFR
-3 is attached to the extracellular surface,
cells expressing GFR
-3 were treated with EZ-Link sulfo-NHS-Biotin as
described under "Materials and Methods." The transfected cells
contained a biotinylated protein immunoprecipitated with Flag antibody
corresponding to the expected size of GFR
-3, demonstrating that this
protein is indeed attached to the extracellular surface of the cells. However, these experiments do not allow us to predict whether it is
attached via a GPI linkage or by insertion of its hydrophobic tail
because resistance to PIPLC has been demonstrated for several GPI-linked proteins including erythrocyte acetylcholinesterase. This
protein contains a modified inositol group in its anchor conferring
resistance to PIPLC (31). Chemical analyses of membrane anchors have
determined that they are glycophospholipid units containing
ethanolamine, mannose, glucosamine, phosphatidylinositol, and
occasionally galactose. Fatemi et al. (32) have demonstrated that the GPI-linked protein Thy-1 can be effectively labeled with [3H]ethanolamine. When cells transfected with Flag-tagged
GFR
-1, a transmembrane form of GFR
-1, and GFR
-3 are incubated
in the presence of [14C]ethanolamine, GFR
-1 and
GFR
-3 are both effectively labeled (Fig. 4C). All three
proteins are expressed to equivalent levels as depicted by the Western
analysis of these proteins shown in Fig. 4D. Therefore,
GFR
-3 is a GPI-linked protein that is insensitive to cleavage by
PIPLC. This does not rule out the possibility that other lipases such
as phosphatidylinositol-specific phospholipase D may be used to remove
this molecule from the membrane. Resistance to PIPLC could result from
inaccessibility of the cleavage site in situ as is the case
for the GPI-linked variant surface glycoproteins on intact trypanosomes
(33). Alternatively, as described above, the anchor can be modified to
prevent cleavage. The biological significance of this unusual anchoring
mechanism for this family of co-receptors is not known. However,
because GFR
-1 (25) and GFR
-2 (13) can presumably be severed from
the membrane with PIPLC, the reversibility of this mode of attachment
may be critical for their biological function. Because GFR
-3 cannot
be efficiently removed from the membrane using this enzyme, it may
signal via an alternative mechanism (see Fig. 7).
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Expression of GFR-3 in Embryonic and Adult Mouse--
The
presence of an additional GFR
-1-like receptor raises the question of
whether these molecules are redundant in their role of signaling. To
address this question, we performed Northern analyses on cell lines
known to express the RET receptor. RET is expressed in the
neuroblastoma cell lines LA-N-1, LA-N-2, LA-N-5, SY5Y, and SK-N-SH but
not in SK-N-MC cells (Fig.
5A). Neither GFR
-1 nor
GFR
-3 was found to be expressed in these cell lines either by
Northern analysis (Fig. 5A) or reverse
transcription-polymerase chain reaction (data not shown). In contrast,
GFR
-2 was expressed in LA-N-1 and SK-N-SH cells and was particularly
high in LA-N-2 and LA-N-5 cells (Fig. 5A). Low levels were
expressed in SY5Y cells, which were detectable by reverse
transcription-polymerase chain reaction (data not shown). Because RET
is unable to bind GDNF in the absence of a GFR
-1-like molecule (24,
25), our ability to activate RET upon GDNF treatment in LA-N-1, LA-N-5 and SK-N-SH cells (11, 29) suggests that GFR
-2 acts as a GDNF
receptor and, upon binding GDNF, activates the RET receptor. A recent
publication by Baloh et al. (14) has demonstrated that this
is indeed the case. GFR
-2 functions as a low affinity receptor for
GDNF or as a high affinity receptor for neurturin both signaling in
conjunction with the Ret receptor. Surprisingly none of our Ret
expressing cell lines expressed GFR
-1 or GFR
-3.
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In Situ Hybridization Analysis of GFR-3--
To get a better
understanding of the potential partners for GFR
-3 in the embryo,
this molecule was localized by in situ analysis and compared
with the expression pattern for Ret. Data in Fig. 6A are consistent with
previously reported results demonstrating that at E14 RET is expressed
prominently in cranial ganglia, myenteric ganglia of the gut, motor
neurons in the spinal cord and the hindbrain, the dorsal root ganglia,
and the growing tips of the renal collecting ducts (34, 36).
Interestingly, GFR
-3 is expressed more selectively. At E14 GFR
-3
is expressed in subpopulations of cells in the dorsal root ganglia
(data not shown), selected cranial ganglia, the superior cervical
sympathetic ganglia, and regions in the lower urogenital and digestive
tracts (Fig. 6B). It is not present in detectable levels in
the developing kidney. More similar to Ret, GFR
-1 is expressed
widely in the developing embryo. It is present in the ventral midbrain,
spinal cord motor neurons, subpopulations of the dorsal root ganglia,
the developing kidney, the mesenchyme of the developing gut, the
retina, the pituitary, urogenital tract, and pancreatic primordium
(24). Like GFR
-3, GFR
-2 is expressed less widely being found only
in the developing and adult dorsal root ganglia and the superior
cervical ganglion of the rat (14). Therefore, the co-receptors in the
GFR
family appear to maintain distinct tissue-specific expression
that does not always overlap with Ret expression. This suggests that
other Ret-like receptors or alternative signaling methods may be
involved in GFR
-1-, GFR
-2-, and GFR
-3-dependent
signaling. The ability of GFR
-1 and GFR
-2 but not GFR
-3 to be
removed from the membrane via cleavage of a GPI linkage also implicates
unique signaling mechanisms (Fig. 7). In
cases where Ret and the co-receptor are expressed on the same cell,
soluble ligand can diffuse to the cell, bind to the co-receptor, and
activate Ret (Fig. 7A). Alternatively, the ligand and
co-receptor could be expressed on the same cell, be removed by cleavage
of the GPI linkage, and travel as a pair to activate Ret on a nearby
cell (Fig. 7B). Because GFR
-3 cannot be removed by PIPLC
cleavage, it may be expressed on the same cell as the Ret-like receptor
to act as a co-receptor (Fig. 7A). It will be of interest to
determine if one signaling paradigm prevails over the other in
development as well as in the mature organism.
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ACKNOWLEDGEMENTS |
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We thank Dr. Kim Orth for generously providing Flag-tagged constructs, James Clemens for pJCC, and Zhao-Qin Bao and Chung Lee for excellent technical assistance. Additional thanks to James Clemens and Dr. Kim Orth for helpful comments and for critically reading this manuscript.
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FOOTNOTES |
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* This work was supported by Grant 18024 from the NIDDKD, National Institutes of Health and by grants from the Walther Cancer Institute (to J. E. D.) and from National Alliance for Research on Schizophrenia and Depression and the American Heart Association (to C. A. W.).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.
§ These authors contributed equally to this work.
¶ National Kidney Foundation Fellow.
Supported by the Deutsche Forschungsgemeinschaft.
To whom correspondence should be addressed. Tel.: 313-764-8192;
Fax: 313-763-4581.
1 The abbreviations used are: GDNF, glial cell line-derived neuroptrophic factor; NTN, neurturin; PIPLC, phosphatidylinositol-specific phospholipase C; GPI, glycosyl-phosphatidyl-inositol; PAGE, polyacrylamide gel electrophoresis; E, embryonic day; CNTF, ciliary neurotrophic factor; CNTFR, CNTF receptor.
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REFERENCES |
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