From the Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden
Received for publication, July 12, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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The glial cell line-derived neurotrophic factor
(GDNF) family comprise a subclass of cystine-knot superfamily ligands
that interact with a multisubunit receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich glycosyl
phosphatidylinositol-anchored binding subunit called GDNF family
receptor Binding of polypeptide growth factors to cell surface receptors is
the first event in the series of protein-protein interactions leading
to the distinct biological responses characteristic of a given factor.
Understanding the molecular interactions underlying the affinity and
selectivity of ligand-receptor complexes is of fundamental importance
if we are to comprehend fully the molecular basis of signal
transduction by polypeptide growth factors.
A large group of growth factors contain a distinctive structural motif,
the "cystine knot," formed by a cluster of three disulfide bridges,
with two disulfide bridges and their connecting residues forming a ring
structure through which the third disulfide bridge passes (1, 2). The
protomer of cystine-knot superfamily factors is typically elongated and
formed primarily by GDNF1 is the prototype member
of a new subfamily of cystine-knot ligands with important roles in the
control of neuron survival and differentiation (5), kidney
morphogenesis (6), and sperm cell development (7). In vertebrates, the
GDNF family is known to comprise the following four distinct proteins:
GDNF (8), neurturin (NTN) (9), persephin (PSP) (10), and artemin (ART) (11), also identified as enovin (12) or neublastin (13). An unusual
feature of the receptor complex for GDNF family ligands is the
requirement of two receptor subunits, one specialized in ligand binding
and another in transmembrane signaling. All four members of the GDNF
family signal through activation of the c-Ret receptor tyrosine kinase
(14-17). c-Ret, however, can not bind ligand on its own but requires
the presence of a glycosyl phosphatidylinositol (GPI)-anchored
co-receptor known as GDNF family receptor Experiments using purified proteins (20) and overexpression in cell
lines (21) have demonstrated that c-Ret and GFR In the work presented here, we have explored the molecular basis of
ligand recognition by GFR Reagents--
The cDNAs for rat GFR Construction of Chimeric Receptors and Deletions--
An
hemagglutinin (HA) epitope (YPYDVPDYA) was inserted after the putative
signal peptide sequence of each GFR Cross-linking Assay--
Ligands were radioiodinated to specific
activities of about 0.5 to 2 × 108 cpm/µg by the
lactoperoxidase method as described previously (28). COS cells were
transfected using DEAE-dextran. 48 h after transfection, cells
were rinsed once with chilled binding buffer (1 mg/ml bovine serum
albumin, 1 mg/ml glucose, 0.1 mM CaCl2, 0.1 mM MgCl2 in phosphate-buffered saline) and
equilibrated with binding buffer containing 50-100 ng/ml
radioiodinated ligand for 4 h at 4 °C. Subsequently,
bis(succinimidyl) suberate (BS3) cross-linker was added to
a final concentration of 0.8 mM and incubated for an
additional 45 min at 4 °C. The reaction was quenched by adding 50 mM glycine. Cells were rinsed twice with chilled 50 mM glycine in phosphate-buffered saline and lysed in RIPA
buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 50 mM NaF, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, 10%
glycerol, 1 mM EDTA, 10 mM 2-glycerolphosphate, 2 mM Na3VO4). To assess expression
of the various GFR Phosphorylation and GFR
To assess ligand-independent association of different GFR Specificity and Promiscuity in the Interaction of GFR Domain Boundaries and Chimeric Receptors--
Secondary structure
predictions indicated that GFR The N-terminal Domain Is Not a Determinant of Ligand Binding
Specificity--
We constructed chimeras between two relatively
distant members of the GFR
The results from chimeras between GFR The Central Domain Is a Crucial Determinant of Ligand Binding
Specificity--
We then analyzed chimeras between more closely
related receptors, i.e. GFR
The results of c-Ret phosphorylation assays in Neuro2A cells were again
in general agreement with the binding data, i.e. the chimeras with the highest activity were the ones that retained the
central domain from the cognate receptor (Fig. 4, C and
D). A number of GFR Binding Determinants for GDNF and NTN Reside in Discrete Subcentral
Domains of GFR Truncated GFR
To investigate whether portions of the C-terminal tail of GFR The N-terminal Domain of GFR
We also determined whether the N- and C-terminal regions of GFR Distinct Hydrophobic and Positively Charged Residues in the Central
Domain of GFR In this study, we have investigated the structural determinants
required for ligand binding specificity in the family of GFR The major finding of our study is the localization of the ligand
binding specificity domain of GFR It is possible that the N-terminal domain of GFR Two sets of residues were identified as critical for the interaction of
GFR Interestingly, several GFR Another interesting observation made in the present study concerns the
two deletion constructs of GFR In conclusion, GFR (GFR
). All four GDNF family ligands utilize c-Ret as a
common signaling receptor, whereas specificity is conferred by
differential binding to four distinct GFR
homologues. To understand
how the different GFR
s discriminate ligands, we have constructed a
large set of chimeric and truncated receptors and analyzed their ligand
binding and signaling capabilities. The major determinant of ligand
binding was found in the most conserved region of the molecule, a
central domain predicted to contain four conserved
helices and two
strands. Distinct hydrophobic and positively charged residues in
this central region were required for binding of GFR
1 to GDNF. Interaction of GFR
1 and GFR
2 with GDNF and neurturin required distinct subsegments within this central domain, which allowed the
construction of chimeric receptors that responded equally well to both
ligands. C-terminal segments adjacent to the central domain are
necessary and have modulatory function in ligand binding. In contrast,
the N-terminal domain was dispensable without compromising ligand
binding specificity. Ligand-independent interaction with c-Ret also
resides in the central domain of GFR
1, albeit within a distinct and
smaller region than that required for ligand binding. Our results
indicate that the central region of this class of receptors constitutes
a novel binding domain for cystine-knot superfamily ligands.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strands connected by highly flexible hairpin
loops, where most of the sequence variability among paralog factors is
located. Different configurations of dimer assembly lead to various
shapes, elongated either along or across the 2-fold symmetry axis,
which are characteristic of the different subfamilies. Two
high-resolution structures of the complex of a cystine-knot factor with
its corresponding receptor have been obtained recently, that of
vascular endothelial growth factor with the ligand binding domain of
Flt-1 (3) and the one of nerve growth factor with the ligand binding
domain of TrkA (4). In both cases, distinct immunoglobulin-like
domains in the receptors are both necessary and sufficient for ligand
binding. However, not all receptors of cystine-knot superfamily ligands contain immunoglobulin-like domains, suggesting the existence of novel
binding domains for this class of ligands.
(GFR
) (18, 19).
Interestingly, four different GFR
receptors have been identified
(GFR
s 1 to 4), each selective, although not totally specific, for
one member of the GDNF ligand family (reviewed in Ref. 5).
receptors can also
associate with low-affinity in the absence of ligand. In the presence
of c-Ret, the ligand binding specificity of GFR
receptors broadens,
allowing them to interact with different GDNF family ligands, as well
as several mutants with defective GFR
binding (11, 20-22). In
vivo, GFR
receptors are more widely expressed than c-Ret,
suggesting additional roles for these "ectopic" sites of GFR
expression. Both cell-autonomous and non-cell-autonomous functions have
been proposed for GFR
receptors expressed in the absence of c-Ret.
Recent evidence obtained in c-Ret-deficient cell lines and primary
sensory neurons isolated from c-Ret knock-out mice indicates the
existence of alternative signaling mechanisms mediated by GFR
receptors acting in a cell-autonomous manner independently of c-Ret
(23, 24). GFR
receptors may also function in a non-cell-autonomous
manner to capture and concentrate diffusible GDNF family ligands from
the extracellular space and then present these factors in
trans to afferent c-Ret-expressing cells (25, 26).
receptors. A large collection of chimeric
and truncated receptors was generated and tested in binding and c-Ret
autophosphorylation assays. The results of these experiments allowed us
to identify a determinant of ligand binding specificity in a central
region containing four predicted
helices and two short
strands.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, rat GFR
2, and
mouse GFR
3 subcloned in the pCDNA3 vector (Invitrogen) were
obtained as described previously (21). The cDNA for chicken GFR
4
(27), a gift from Alun Davies (University of Edinburgh, Edinburgh,
United Kingdom), was also subcloned in pCDNA3. Monoclonal IgG
against hemagglutinin was from BabCO; anti-phosphotyrosine monoclonal
IgG, PY99, and goat polyclonal antibodies, C-20 and T-20, against c-Ret
were from Santa Cruz Biotechnology. GDNF was prepared in Sf21
cells as described previously (28). NTN was from Peprotech. Recombinant ART and PSP were generous gifts from Jannssen Research Foundation (Belgium). GFR
1-Fc was from R&D Systems. Reagents for
radioiodination and cross-linkings were purchased from Amersham
Pharmacia Biotech, Sigma, and Pierce.
construct by Kunkel mutagenesis
(29). Both Kunkel method and QuickChange mutagenesis (Stratagene) were
used to incorporate novel restriction sites at selected conserved
strings of residues PYE (BsiWI), RRR (NarI), and SGN (BspEI). Binary chimeras were
prepared using these new restriction sites for segmental exchanges.
Each exchange site was located in predicted loop regions in the
different receptor constructs. Secondary structure predictions were
done using PHD Predict (30). Alignments were done with Clustal
X. N-terminal deletions for GFR
1 (
N57,
N84,
N113, and
N144) and for GFR
2 (
N126) were obtained by polymerase chain
reaction using upstream primers containing the HA epitope sequence
facilitating the grafting of the appropriate signal peptide using
NdeI sites within the epitope and the cytomegalovirus
promoter region of pCDNA3 (see Fig. 2). C-terminal deletions in
GFR
1 (
C31,
C55, and
C79) upstream of Gly-421 (to retain the
GPI anchor) were made sequentially from the smallest to the largest
truncation using QuickChange mutagenesis.
constructs, an aliquot of each lysate was used
for immunoblots on polyvinylidene difluoride (Hybond P) membrane
processed for detection of the HA tag. The rest of the lysates were
immunoprecipitated using the anti-HA antibody and run on a 10% SDS
polyacrylamide gel. Immunoblot analysis was done using enhanced
chemifluorescence (Amersham Pharmacia Biotech). Both immunoblot and
autoradiographic exposures were detected and quantified using a
Storm840 phosphor/fluorimager (Molecular Dynamics).
-Ret Interaction Assays--
Neuro2A
cells were transfected with the different GFR
constructs by the
calcium phosphate precipitation method. The day after transfection,
cells were switched to serum-free medium containing 5 µM
all-trans retinoic acid, 0.1% bovine serum albumin, and N2 supplements and were incubated for 16-20 h. An hour before
stimulation, cells were incubated with fresh serum-free medium.
Stimulations were done by adding ligands to a final concentration of 50 ng/ml for 12 min, after which cells are lysed in RIPA buffer. Aliquots were taken from each lysate for HA immunoblot analysis, whereas the
rest of the lysates were used for c-Ret immunoprecipitations. c-Ret
phosphorylation was assessed by immunoblotting using an anti-phosphotyrosine antibody.
constructs
with c-Ret, co-transfections were done in COS cells using DEAE-dextran.
Transfected cells were lysed 48 h later in RIPA buffer containing
60 mM
-octylglucoside. The presence of both sodium
deoxycholate and
-octylglucoside in the cell lysis buffer assured
complete solubilization of membrane lipid rafts, which was required to
establish the co-existence of both GFR
and c-Ret receptors in the
same molecular complex, as opposed to the same subcellular compartment.
Cell lysates were immunoprecipitated for c-Ret and analyzed by Western
blotting with anti-HA antibodies, recognizing epitope-tagged GFR
constructs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Receptors
with GDNF Family Ligands--
No previous study had examined the
binding specificity of all four GDNF family ligands to all four members
of the GFR
receptor family in the same experiment. We used
125I-labeled GDNF family ligands in chemical cross-linking
assays to COS fibroblasts transiently expressing different GFR
receptors carrying an HA tag at the N terminus (Fig.
1A). In all cases, affinity
labeling of the receptor band could be prevented by addition of an
excess (up to 100-fold) of the corresponding unlabeled ligand (data not
shown), indicating specific binding. Western blotting of the same cell
lysates with anti-HA antibodies (Fig. 1A, lower panels) was used to normalize the binding to the levels of
expression of the different GFR
receptors for quantification. These
experiments confirmed the main established interactions between GDNF
family ligands and GFR
receptors (Fig. 1B, solid
arrows) and revealed a number of additional interactions of lower
affinity between non-cognate pairs (Fig. 1B, dotted
arrows). Interestingly, although GFR
3 was only able to bind
ART, this ligand was capable of interacting, to varying degrees, with
all GFR
receptors (Fig. 1A). Conversely, although PSP
binding could only be detected to GFR
4, this receptor was able to
interact with lower affinity with all members of the GDNF ligand family
(Fig. 1A).
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Fig. 1.
Specificity and promiscuity in the
interaction of GFR receptors with GDNF family
ligands. A, affinity labeling of GFR
receptors
expressed in COS cells with iodinated GDNF family ligands. The
lower panels show aliquots of cell lysates analyzed with HA
antibodies by Western blotting. Numbers below the
lanes indicate relative binding normalized to expression
levels. Control (no GFR
receptor) was set to zero, whereas the
cognate interaction was set to one. IB,
immunoblot. B, the diagram shows the main
(cognate) interactions between GDNF ligands and GFR
receptors
(thick arrows), as well as different cross-reactivities
(dashed arrows) revealed by affinity labeling
experiments.
receptors contain primarily
helices connected by shorter segments of undefined structure,
presumably representing loops (Fig. 2).
Two predicted short
strands are also conserved in the central
region of all four GFR
s (Fig. 2). The remarkable conservation of the predicted pattern of secondary structure elements among different GFR
receptors suggests that these may represent true structural elements in this class of receptors. For the first set of chimeric receptors, we initially defined two internal boundaries corresponding to two highly conserved triplets in the primary sequence of GFR
receptors, i.e. PYE and SGN (Fig. 2). These boundaries
coincide with interhelical regions in secondary structure predictions
and divide the GFR
molecule into N-terminal (three predicted
helices), central (four predicted helices and two
strands), and
C-terminal (two predicted helices) domains of roughly 100, 200, and 100 residues, respectively. Similar domain boundaries have also been
defined by others (5). The percentage similarity of each of the
domains in GFR
2, -3, and -4 with respect to GFR
1 is shown in
Table I. Unique restriction sites,
BsiWI and BspEI, were created by silent mutagenesis at the conserved PYE and SGN triplets, respectively, in all
four GFR
receptors. Chimeric molecules were then constructed by
exchanging homologous domains using these two restriction sites.
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Fig. 2.
Conservation of predicted secondary structure
elements and domain boundaries in GFR
receptors. The aligned amino acid sequences of rat
GFR
1, rat GFR
2, mouse GFR
3, and chicken GFR
4 are shown.
Sequences predicted to correspond to
helices are bolded
in red, and those to
strands in green. Signal
peptide sequences are underlined at the N termini. Conserved
triplets used for silent mutagenesis (PYE, RRR, and SGN) are
bolded in black, and the introduced restriction
sites are indicated with open arrows. Solid curved
arrows denote the boundaries of the N- and C-terminal truncations
made in GFR
1. All C-terminal truncations of GFR
1 retain the GPI
signal peptide (underlined at the C termini) and the
preceding 9 residues as indicated by the last solid curved
arrow on the GFR
1 sequence. The boundaries of the
C21
deletion in GFR
1 are indicated with open curved arrows.
Putative GPI cleavage sites are double underlined.
Percentage similarity of N-terminal (less signal sequence), central
(PYE to SGN), and C-terminal (until GPI consensus sequence) domains of
GFR2 (rat), GFR
3 (mouse), and GFR
4 (chicken) receptors
relative to GFR
1 (rat)
receptor family, GFR
1 and GFR
3, and
tested their ability to bind 125I-labeled GDNF and ART and
to activate the c-Ret receptor. These experiments indicated that the
N-terminal domain is dispensable for ligand binding specificity. A
receptor molecule containing the N terminus of GFR
3 and the central
and C-terminal domains of GFR
1 (termed 3-1-1) was able to bind GDNF
as efficiently as wild type GFR
1 (Fig.
3A). Similarly, the 1-3-3
chimera was able to bind ART, albeit somewhat less efficiently than
wild type GFR
3 (Fig. 3A). On the other hand, exchanges
involving the central and C-terminal domains disrupted ligand binding
(Fig. 3A), suggesting the requirement of these domains for
ligand interaction. The ability of the receptor chimeras to induce
ligand-dependent autophosphorylation of c-Ret was examined
after transient transfection into the neuroblastoma line Neuro2A, which
expresses c-Ret endogenously. In agreement with the results from
cross-linking experiments, only GFR
molecules retaining the central
and C-terminal domains of GFR
1 were able to support
GDNF-dependent stimulation of c-Ret tyrosine
phosphorylation (Fig. 3B).
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Fig. 3.
The N-terminal domain is not a determinant of
ligand binding specificity. A, affinity labeling of
chimeras between GFR 1 and GFR
3 receptors with
125I-GDNF and 125I-ART as indicated. The
lower panels show aliquots of cell lysates analyzed with HA
antibodies by Western blotting. Numbers below the
lanes indicate relative binding normalized to expression
levels. Control (no GFR
receptor) was set to zero, whereas the
interaction with the preferred wild type receptor (i.e.
1-1-1 for GDNF and 3-3-3 for ART) was set to one. IB,
immunoblot. B, phosphorylation of c-Ret induced by GDNF in
Neuro2A cells expressing chimeras between GFR
1 and GFR
3
receptors. The middle panel shows reprobing of the same
filter with anti-c-Ret antibodies. The lower panel shows
aliquots of cell lysates analyzed with HA antibodies. C,
affinity labeling of chimeras between GFR
2 and GFR
3 receptors
with 125I-NTN. D, phosphorylation of c-Ret
induced by NTN in Neuro2A cells expressing chimeras between GFR
2 and
GFR
3 receptors. E, summary of results.
1 and GFR
3 receptors
suggested that the N-terminal domain was not essential for ligand recognition and indicated the importance of the central and C-terminal segments of the GFR
molecule. To determine the generality of these
observations for other members of the GFR
family, we constructed chimeras between GFR
2 and GFR
3 and tested their ability to bind 125I-labeled NTN and to activate the c-Ret receptor. The
only molecule capable of significant NTN binding, in addition to wild
type GFR
2, was the 3-2-2 chimera (Fig. 3C). This result
indicated that, similar to GFR
1, the N terminus of GFR
2 is
dispensable for ligand binding specificity, and both the central and
C-terminal domains are required for interaction with NTN. In agreement
with the cross-linking data, the 3-2-2 chimera also supported
NTN-dependent c-Ret phosphorylation (Fig. 3D).
In Neuro2A cells, however, the 2-2-3 chimera was also able to mediate
ligand-dependent c-Ret phosphorylation, despite its
undetectable binding to 125I-NTN in COS cells (Fig. 3,
C and D). These data are in accordance with
previous observations indicating that c-Ret and GFR
receptors collaborate in the binding of suboptimal ligands and suggested that,
under certain circumstances and in the presence of c-Ret, the
C-terminal domain of GFR
receptors may also be exchanged without
compromising ligand binding specificity (see below).
1 and -2, GFR
1 and -4, and
GFR
2 and -4. The chimeras between GFR
1 and GFR
2 revealed a
reciprocal pattern of binding to 125I-GDNF and
125I-NTN (Fig.
4A). In all cases, the
receptor chimeras showing the strongest binding to a given ligand were
those retaining the central domain from the cognate receptor,
i.e. X-1-X for GDNF and X-2-X for NTN. A similar
pattern was observed in chimeras between GFR
1 and -4 analyzed by
affinity labeling with 125I-GDNF (Fig. 4B) and
in chimeras between GFR
2 and -4 analyzed with 125I-NTN
and 125I-PSP (data not shown). Together, these data
demonstrate the importance of the central domain of GFR
receptors
for ligand recognition. In contrast to the chimeras between GFR
1 and
-3, the C-terminal domains of GFR
1, -2, and -4 could be exchanged
without loss of binding (Fig. 4, A and B),
probably because of their closer relative similarity (Table I). This
indicates that the C-terminal domain can modulate ligand binding but is
not an essential determinant of specificity.
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Fig. 4.
The central domain is a crucial determinant
of ligand binding specificity. A, affinity labeling of
chimeras between GFR 1 and GFR
2 receptors with
125I-GDNF and 125I-NTN as indicated. The
lower panels show aliquots of cell lysates analyzed with HA
antibodies by Western blotting. Numbers below the
lanes indicate relative binding normalized to expression
levels. Control (no GFR
receptor) was set to zero, whereas the
interaction with the preferred wild type receptor (i.e.
1-1-1 for GDNF and 2-2-2 for NTN) was set to one. IB,
immunoblot. B, affinity labeling of chimeras between GFR
1
and GFR
4 receptors with 125I-GDNF. C,
phosphorylation of c-Ret induced by GDNF and NTN in Neuro2A cells
expressing chimeras between GFR
1 and GFR
2 receptors. The
middle panel in each set shows reprobing of the
corresponding filter with anti-c-Ret antibodies. The lower
panels show aliquots of cell lysates analyzed with HA antibodies.
D, phosphorylation of c-Ret induced by GDNF in Neuro2A cells
expressing chimeras between GFR
1 and GFR
4 receptors.
E, summary of results.
molecules that showed low or
undetectable binding, however, did mediate significant levels of c-Ret
phosphorylation after GDNF stimulation, such as the 2-2-1 and the
2-2-2 molecules (Fig. 4C). Interestingly, all chimeras
between GFR
1 and GFR
2, as well as wild type GFR
1, supported
some degree of c-Ret activation after NTN stimulation (Fig.
4C, lower panels). These data are in agreement
with results from previous studies demonstrating significant
cross-reactivity between the GFR
1-GDNF and GFR
2-NTN systems in
the presence of c-Ret (20, 21, 31) and support a role for c-Ret in
modulating the interaction of the receptor complex with suboptimal ligands.
1 and GFR
2--
To further investigate
structure-function relationships in the GFR
receptor family, we
constructed chimeras involving subsegments of the central domain of
GFR
1 and GFR
2 receptors. We took advantage of the conserved RRR
triplet sequence located in the middle of the central domain, between
the two predicted
strands (Fig. 2), to introduce a unique
NarI site by silent mutagenesis. This new boundary
subdivides the central domain into two roughly equal halves of
approximately 100 residues, each predicted to contain two
helices
and a
strand. Cross-linking studies with 125I-GDNF
indicated that the chimeras that retained binding were the ones that
contained the second central subsegment from GFR
1, i.e.
corresponding to the formula X-2'-1'-X (Fig.
5A). Conversely, the chimeras
that retained binding to 125I-NTN contained the first
central subsegment from GFR
2 (Fig. 5A), suggesting
different structural requirements for ligand binding specificity within
the central domains of GFR
1 and GFR
2. Thus, the chimeric receptor
2-2'-1'-1, which combined both elements, had a broader specificity and
was able to bind 125I-GDNF and 125I-NTN with
comparable efficiency (Fig. 5A). As with the previous chimeras between these two receptors, all constructs supported some
level of c-Ret phosphorylation over control when introduced into
Neuro2A cells (Fig. 5B), although the molecules that bound ligand more efficiently were still the most active. This again confirms
the contribution of c-Ret in ligand recognition and demonstrates that
the low ligand binding efficiency of some of the chimeras was not
because of structural problems in these molecules, as they were still
able to support some degree of ligand-dependent c-Ret
activation.
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Fig. 5.
Binding determinants for GDNF and NTN reside
in discrete subcentral domains of GFR 1 and
GFR
2. A, affinity labeling of
chimeras between the subcentral domains of GFR
1 and GFR
2
receptors with 125I-GDNF and 125I-NTN as
indicated. The lower panels show aliquots of cell lysates
analyzed with HA antibodies by Western blotting. Numbers
below the lanes indicate relative binding normalized to
expression levels. Control (no GFR
receptor) was set to zero,
whereas the interaction with the preferred wild type receptor
(i.e. 1-1'-1'-1 for GDNF and 2-2'-2'-2 for NTN) was set to
one. IB, immunoblot. B, phosphorylation of c-Ret
induced by GDNF and NTN in Neuro2A cells expressing chimeras between
the subcentral domains of GFR
1 and GFR
2 receptors. The
middle panel in each set shows reprobing of the
corresponding filter with anti-c-Ret antibodies. The lower
panels show aliquots of cell lysates analyzed with HA antibodies.
C, summary of results.
Receptors Lacking the N-terminal Domain Retain
Ligand Binding--
Although the N-terminal domain of GFR
receptors
did not appear to play a role in ligand specificity, it was unclear
whether it was at all necessary for ligand binding or it had some other function, such as contacting the c-Ret receptor. This question became
all the more important in view of the proposed existence of a natural
splice variant of GFR
2 lacking the first 146 residues (termed
GFR
2c) (32). We therefore generated truncated versions of GFR
1
lacking the first (GFR
1-
N57), the first and the second (GFR
1-
N87), or all three (GFR
1-
N113) predicted
helices
of the N-terminal domain (see Fig. 2). In addition, we also generated a
GFR
2 construct corresponding to the reported GFR
2c splice variant, an analogous construct based on the GFR
1 receptor
(GFR
1-
N144), and a GFR
1 construct lacking all first four
predicted
helices, including predicted helix 4 in the first portion
of the central domain that was essential for ligand binding
(GFR
1-
N161) (see Fig. 2). All constructs carried an HA tag at the
N terminus and were analyzed in cross-linking and c-Ret phosphorylation
assays as above. The GFR
1-
N87 variant that lacked the first two
predicted helices of the N terminus was not produced in COS cells,
presumably because the deletion removed an odd number of cystines,
which could have resulted in misfolding of the protein. On the other hand, all remaining N-terminally truncated variants of GFR
1
(i.e.
N57,
N113,
N144, and
N161) were produced
at normal levels (Fig. 6, A
and B). The GFR
1 deletion mutants
N57,
N113, and
N144 were all able to bind GDNF, albeit with lower efficiency than
the wild type receptor, particularly the
N57 deletion (Fig. 6A). The
N161 deletion construct was however unable to
bind GDNF (Fig. 6B), in agreement with the importance of the
central region of GFR
receptors for ligand binding. The GFR
2c
splice variant (GFR
2-
N146) was able to bind 125I-NTN
at a level comparable with that of wild type GFR
2 (Fig. 6C). Together, these data demonstrate that the N-terminal
domain of GFR
receptors is not absolutely required for ligand
binding, and, in the case of GFR
2, an equally efficient interaction
can also take place in its absence.
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Fig. 6.
Truncated GFR
receptors lacking the N-terminal domain and part of the
C-terminal domain retain ligand binding. A, affinity
labeling of N-terminal deletion constructs of GFR
1 with
125I-GDNF. The lower panels show aliquots of
cell lysates analyzed with HA antibodies by Western blotting.
Numbers below the lanes indicate relative binding
normalized to expression levels. Control (no GFR
receptor) was set
to zero, whereas the interaction with wild type GFR
1 was set to one.
IB, immunoblot. B, affinity labeling of N- and
C-terminal deletion constructs of GFR
1 with 125I-GDNF.
C, affinity labeling of the GFR
2 N-terminal deletion
construct
N146 (GFR
2c) with 125I-NTN. D,
linear diagrams of N- and C-terminal deletion constructs of GFR
1 and
GFR
2. The central domain between PYE and SGN is shaded,
and the C-terminal region is hatched. A qualitative summary
of the results is shown to the right.
1 may
also be dispensable, we constructed a series of C-terminal truncations
(see Fig. 2) upstream of Gly-421 and the putative GPI anchor signal
sequence (last black arrow in Fig. 2). All three GFR
1
C-terminal deletion constructs were produced in COS cells, and both
C31 and
C55 retained their ability to bind 125I-GDNF
(Fig. 6B). A compound deletion mutant lacking 144 residues in the N terminus and 31 residues in the C terminus
(GFR
1-
N144
C31) was also able to bind GDNF (Fig.
6B). However, the
C79 deletion, which disrupts a
predicted helix in the C-terminal domain of GFR
1 (see Fig. 2), was
unable to bind GDNF (Fig. 6B). The region immediately downstream of this predicted helix does not appear to be necessary for
GDNF binding, as indicated by the activity of the
C21 deletion (Fig.
6D). Together, these data indicate that, in the C-terminal region of GFR
receptors, the integrity of the predicted helices is
required for ligand binding.
Receptors Is Dispensable for
Ligand-dependent or -independent Interactions with
c-Ret--
The question whether the N- and C-terminal domains of
GFR
receptors play any role in the interaction with c-Ret was
addressed in several ways. To investigate ligand-dependent
interactions, c-Ret autophosphorylation assays were performed in
Neuro2A cells carrying truncated GFR
receptors. In agreement with
the binding data, the
N57,
N113, and
N144 GFR
1 truncated
receptors were all able to induce GDNF-dependent
stimulation of c-Ret phosphorylation (Fig.
7, A and B),
suggesting that the N-terminal domain of GFR
1 is not crucial for the
contact of the GFR
1-GDNF complex with c-Ret. Also in agreement with
their ability to cross-link 125I-GDNF, the
C31 and
C55 C-terminal deletion mutants, as well as the
N144
C31
compound mutant, also mediated ligand-dependent stimulation
of c-Ret phosphorylation (Fig. 7, B and C).
Likewise, the GFR
2c splice variant, lacking the first 146 N-terminal
residues, was also able to mediate NTN-dependent c-Ret
activation (Fig. 7D). As expected, the truncated GFR
1
receptors that did not bind GDNF (i.e.
N161 and
C79)
were not able to stimulate c-Ret phosphorylation (Fig.
7C).
View larger version (50K):
[in a new window]
Fig. 7.
The N-terminal domain of
GFR receptors is dispensable for
ligand-dependent activation of c-Ret. A-C,
tyrosine phosphorylation of c-Ret stimulated by GDNF in Neuro2A cells
expressing N- and C-terminal deletion constructs of GFR
1. The
middle panel shows reprobing of the same filter with
anti-c-Ret antibodies. The lower panel shows aliquots of
cell lysates analyzed with HA antibodies. IB, immunoblot.
D, tyrosine phosphorylation of c-Ret stimulated by NTN in
Neuro2A cells expressing the GFR
2 N-terminal deletion construct
N146 (GFR
2c).
1
were required for ligand-independent interaction with the c-Ret
receptor using a co-immunoprecipitation assay in transiently transfected COS cells, which do not produce detectable levels of GDNF.
Care was taken to solubilize membrane lipid rafts so as to assure that
co-immunoprecipitation of GFR
1 and c-Ret reflected direct
interaction and not co-existence in the same membrane compartment (see
"Experimental Procedures"). Immunoprecipitation of c-Ret brought
down HA-tagged wild type GFR
1 receptors only in cells that received
the c-Ret expression plasmid, indicating a direct receptor-receptor
interaction in the absence of ligand (data not shown). All N- and
C-terminal deletions of GFR
1 were able to interact with c-Ret in
this assay, suggesting that these regions are not involved in the
interaction of this receptor with c-Ret (data not shown). Intriguingly,
also the GFR
1-
N161 and
C79 deletion mutants, which were unable
to bind GDNF and to activate c-Ret, were still capable of interacting
with the c-Ret receptor in a ligand-independent manner (data not
shown), indicating that distinct structural determinants in GFR
1 may
be required for ligand binding and association with c-Ret, respectively.
1 Mediate Binding to GDNF--
To begin to identify
individual amino acid residues in GFR
1 involved in the binding of
GDNF, we searched for short stretches of residues in the central domain
(predicted helices 4 to 7) with chemical properties complementary to
the GDNF binding surface. Previous site-directed mutagenesis studies
have identified sets of hydrophobic and negatively charged residues in
exposed loop regions of GDNF that are required for efficient binding to
GFR
1 (22). Two motifs in the center of the GFR
1 molecule, the
hydrophobic triplet MLF in the first predicted
strand (
1 in Fig.
2) and the basic triplet RRR (NarI site in Fig. 2), were
selected for site-directed mutagenesis and functional analysis.
Replacement of MLF into an alanine triplet (
1 MLF-A3)
abolished binding of GFR
1 to GDNF (Fig.
8A). Moreover, replacement of
RRR into an alanine triplet (
1 R3-A3) also
impaired the ability of GFR
1 to interact with GDNF (Fig. 8A). Thus, distinct hydrophobic and positively charged
residues in the central domain of GFR
1 mediate binding to GDNF.
Despite their inability to bind GDNF, however, both the
MLF-A3 and R3-A3 GFR
1 mutants
retained the capacity of mediating substantial levels of c-Ret
phosphorylation in the presence of GDNF (Fig. 8B),
indicating that they do not form part of the interaction site to
c-Ret.
View larger version (28K):
[in a new window]
Fig. 8.
Distinct hydrophobic and positively charged
residues in the central domain of GFR 1 mediate
binding to GDNF. A, affinity labeling of wild type
GFR
1 (
1) and MLF and RRR mutants with 125I-GDNF. The
lower panel shows aliquots of cell lysates analyzed with HA
antibodies by Western blotting and confirms equal levels of expression
among the different constructs. IB, immunoblot.
B, phosphorylation of c-Ret induced by GDNF in Neuro2A cells
expressing MLF and RRR GFR
1 mutants. The middle panel
shows reprobing of the corresponding filter with anti-c-Ret antibodies.
The lower panels show aliquots of cell lysates analyzed with
HA antibodies. C, summary scheme with salient features of
the predicted secondary structure of GFR
receptors. Solid
black dots denote cystine residues. Predicted
helices are
represented as cylinders, and
strands as flat
arrows. Amino acid sequences used for domain boundaries (PYE, RRR,
and SGN) are indicated. The positions of N- and C-terminal deletions
are indicated. Ligand binding specificity was found to reside in the
central domain (dark gray helices), with contribution from
immediately adjacent sequences in the C-terminal domain (gray
helices). The N-terminal domain of GFR
s (light gray
helices) does not play a major role in ligand binding or c-Ret
interactions. Distinct residues involved in the binding of GFR
1 to
GDNF include the RRR triplet in the center of the molecule and the MLF
sequence in the first predicted
strand.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors. This class of receptors has a unique pattern of cystine residues, predicted
helices,
strands, and loops spread over approximately 400 amino acid residues and lack many of the domains most
commonly present in other receptors, such as leucine repeats, immunoglobulin, and fibronectin-like domains. Thus, the GFR
receptor family likely represents a structurally novel receptor class. Among the
four members of this family, the central region of the molecule between
the PYE and SGN motifs shows the highest conservation, whereas the
N-terminal and, in particular, the C-terminal regions are much less conserved.
receptors to the central region of
the molecule containing four predicted
helices and two short
strands (Fig. 8C). The localization of the determinant of
ligand specificity in GFR
receptors to the most conserved part of
the molecule, as opposed to the most variable N- and C termini, was
unexpected. The fact that the C-terminal domains of GFR
1, -2, or -4 could not be substituted for that of GFR
3 without loss of ligand
binding is in agreement with the latter being the most divergent member
of the GFR
family. Although a portion of this domain could be
deleted without loss of ligand binding, the integrity of the two
predicted
helices present in this region could not be compromised.
Together, these data suggest that some determinants in the C-terminal
part of the GFR
molecule may also contribute to ligand binding. In
contrast, the N-terminal region could be exchanged among all receptors
without loss of binding. In GFR
1 and -2, this region could be
deleted without abolishing ligand interaction, indicating that it does not significantly contribute to ligand binding. Our results also indicate that the N-terminal domain of GFR
receptors does not participate in the recruitment and activation of the c-Ret molecule, nor in ligand-independent interactions between the two receptors. Preliminary structural analysis of GFR
1 by cyanogen bromide
digestion and Edman microsequencing indicates that the central region
that was most relevant to ligand binding in GFR
1 forms a core
structural unit reinforced by disulfide
bridges.2 No disulfide
bridges appear to link this core with the N-terminal region, suggesting
that the latter forms a distinct structural domain, separated from the
rest of the GFR
molecule. Interestingly, the putative mammalian
homologue of the chicken GFR
4 has recently been isolated and shown
to lack the N-terminal domain altogether (33, 34). In agreement with
our findings, this molecule is still capable of binding PSP (34). Thus,
at least two GFR
receptor variants, the GFR
2c splice variant and
the mammalian GFR
4 homologue, have lost the N-terminal domain
without any apparent loss of function, suggesting that this region of
the molecule may not be under a strong evolutionary pressure.
receptors plays
other roles distinct from ligand binding. Several
ligand-dependent signaling events have been described in
cells expressing GFR
receptors in the absence of c-Ret (23, 24),
presumably mediated by the collaboration of GFR
receptors with other
transmembrane proteins. Thus, it is possible that the N-terminal domain
of GFR
receptors plays a role in the interaction with transmembrane
molecules other than c-Ret. Another possibility is suggested by a
recent study indicating that N-glycans, as opposed to the
GPI anchor itself, mediate the apical sorting of GPI-anchored receptors
in epithelial cells (35). It is possible that the N-terminal domain of
GFR
s participates in the polarized sorting of the receptor in
neurons and that alternative splicing of this domain regulates the
targeting of receptor molecules to different subcellular compartments.
1 with GDNF. The MLF and RRR triplets in the central region of
GFR
1 have complementary properties to the receptor binding surface
identified in GDNF, characterized by hydrophobic and negatively charged
residues (22). In GDNF, Ile-64, Leu-114, Leu-118, Tyr-120, and Ile-122
form a hydrophobic patch, whereas Asp-52, Glu-61, Glu-62, and Asp-116
form a negatively charged patch, which are both required for binding to
GFR
1, suggesting that they could be interacting with the MLF and RRR
triplets, respectively, that we identified here in GFR
1.
Interestingly, the MLF and RRR sequences are highly conserved among
members of the GFR
family (see Fig. 2), indicating that these
residues do not represent specificity determinants but rather form part
of a common epitope in GFR
receptors for binding to GDNF family ligands. In the neurotrophin family, the crystal structures of the
ligand binding domains of Trk receptors (36) and of the TrkA-nerve
growth factor complex (4), as well as extensive site-directed
mutagenesis studies (37-40), bring support to the idea that
specificity among related members of families of cognate ligands and
receptors is provided by variable residues that modulate the affinity
of a core binding interface that is common to all family members.
chimeras, including the three point
mutants made in GFR
1 that showed no or little ligand binding, were
still able to mediate ligand-dependent activation of c-Ret, indicating that suboptimal GFR
receptors can still contribute to the
formation of a functional receptor complex in the presence of c-Ret.
These data are in agreement with reciprocal observations made with
several GDNF mutants and non-cognate ligand-receptor pairs showing that
some ligands with low or negligible affinity for individual GFR
receptors may still be able to activate c-Ret in a
GFR
-dependent manner (11, 20-22). Thus, either a
residual affinity between suboptimal pairs of GDNF ligands and GFR
s
is still capable of recruiting c-Ret to stabilize the complex, or, as
previously proposed, GFR
s and c-Ret exist to some extent in a
pre-formed complex that allows the interaction of suboptimal ligands or
suboptimal GFR
molecules. In either case, our results indicate that
c-Ret facilitates suboptimal interactions between GDNF family ligands
and GFR
receptors and confirm the role of c-Ret in ligand
recognition. These observations also indicate that structure-function
studies of GDNF ligands and GFR
s based only on functional responses
without direct assessment of ligand-receptor binding are likely to miss
the actual determinants directly involved in these interactions (see
for example Ref. 41).
1, i.e.
N161 and
C79, that were still capable of forming a complex with c-Ret in the absence
of ligand, despite their complete inability to bind GDNF or mediate
GDNF-dependent c-Ret phosphorylation. In fact, nearly half
of the GFR
1 molecule (161 residues from the N terminus and 79 residues from the C terminus) may be deleted without affecting its
ability to interact with the c-Ret receptor. This result suggests that,
although ligand and c-Ret binding require the same central domain in
GFR
receptors, distinct structural determinants within this domain
participate in its interaction with GDNF and c-Ret.
receptors utilize a relatively conserved, central
region of the molecule for both ligand binding and interaction with
c-Ret. Other parts of the GFR
molecule appear to play modulatory roles in ligand binding and c-Ret activation. The N terminus in particular may have novel functions, possibly in subcellular sorting or
in the interaction of GFR
receptors with other transmembrane molecules.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Bob Gordon, Stefan Massuré, and Miroslav Cik for providing PSP and ART ligands, Ella Cederlund and Thomas Bergman for help with HPLC and amino acid sequencing, Ann-Sophie Nilsson and Annika Ahlsén for technical assistance, and Xiaoli Li-Ellström for secretarial help.
![]() |
FOOTNOTES |
---|
* Financial support was obtained from the Swedish Medical Research Council (K99-33X-10908-06C), the European Commission (BMH4-97-2157), the Göran Gustafssons Stiftelse, and the Karolinska Institute.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.
To whom correspondence should be addressed: Div. of Molecular
Neurobiology, Dept. of Neuroscience, Karolinska Inst., Berzelius väg 1, 17177 Stockholm, Sweden. Tel.: 46-8-728-7660; Fax:
46-8-33-9548; E-mail: carlos@cajal.mbb.ki.se.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M006157200
2 Unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GDNF, glial cell
line-derived neurotrophic factor;
NTN, neurturin;
PSP, persephin;
ART, artemin;
GPI, glycosyl phosphatidylinositol;
GFR, GDNF family
receptor
;
HA, hemagglutinin.
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