1 Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK
2 Institute of Cell, Animal and Population Biology, King's Buildings, Mayfield
Road, Edinburgh, UK
* Author for correspondence (e-mail: jamie.davies{at}ed.ac.uk)
Accepted 21 August 2002
![]() |
Summary |
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Key words: GDNF, Glycosaminoglycan, Heparan sulphate, c-Ret
![]() |
Introduction |
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The components of the GDNF receptor complex characterized so far comprise a
high-affinity receptor tyrosine kinase, c-Ret, and a
glycosylphosphatidylinositol- (GPI-) linked co-receptor, GFR1
(Jing et al., 1996
). Other
members of the GDNF family, such as neurturin, persephin and artemin, also
signal through c-Ret but do so in association with other co-receptors
(GFR
2-4 respectively) (Airaksinen et
al., 1999
). GFR
1 is associated with membrane rafts, and
promotes translocation of c-Ret to those rafts in the presence of GDNF
(Tansey et al., 2000
).
Interactions between the receptor components are complex. c-Ret, for example,
seems capable of initiating a pro-apoptotic signal in a manner that is
independent of both its kinase activity and ligand binding, though association
between c-Ret and GFR
1 inhibits this pro-apoptotic signal even in the
absence of GDNF (Bordeaux et al.,
2001
).
The interactions between some other growth factors and their (generally
simpler) receptors are facilitated by sulphated glycosaminoglycans. FGF-2, for
example, requires heparan sulphate proteoglycans for activation of its
receptor tyrosine kinase (Rapraeger et
al., 1991; Yayon et al.,
1991
) and HGF requires dermatan sulphates to activate c-Met
(Lyon et al., 2002
). GDNF is
known to bind to heparin, a fact which was used in its original purification
(Lin et al., 1993
), so it is
likely to bind its commoner cell surface relative, heparan sulphate. Heparan
sulphate is known to be required for development of renal collecting ducts in
vivo and in culture (Davies et al.,
1995
; Bullock et al.,
1998
), and the effect of depriving kidneys of heparan sulphate is
very similar to that of depriving them of GDNF or c-Ret. Furthermore, the
development of collecting ducts deprived of heparan sulphates in culture can
be rescued to a large extent by application of supraphysiological
concentrations of exogenous GDNF (Sainio
et al., 1997
). We have therefore tested the hypothesis that
heparan sulphate GAGs play a direct role in GDNF signalling, and find it to be
correct in each of the systems we have examined.
![]() |
Materials and Methods |
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Cell and tissue culture experiments
Cultures of midbrain neurons were made from E15 mouse embryos by the method
of Engele and Franke (Engele and Franke,
1996), and were incubated for 7 days in the absence or presence of
100 ng/ml GDNF and 30 mM sodium chlorate and then stained with antityrosine
hydroxylase to detect dopaminergic neurons. Dorsal root ganglia were dissected
from the thoracic region of E11 mice, plated on poly-ornithine-coated glass
coverslips and cultured in Eagle's MEM (Sigma M5650) with 10% foetal calf
serum and 2 mM glutamate, for 48-72 hours. Hindguts (the distal half of the
length from stomach to cloaca) were isolated from E10 mouse embryos and
cultured in the above medium for 96 hours. PC-12 cells were maintained in RPMI
1640 medium with 10% foetal calf serum, 2 mM glutamate, penicillin and
streptomycin. For neurite outgrowth, they were plated on to 13 mm coverslips
that had been coated for 1 hour in 60 µl of 10 µg/ml EHS laminin (Sigma)
in RPMI 1640 medium with 10% foetal calf serum, 2 mM glutamate, 10 µg/ml
GFR
1-Fc chimeara (R&D systems), penicillin and streptomycin, at the
bottom of 24 well plates (the coating solution was removed before the cells
were added). The cells were then incubated for 3-4 days in the presence or
absence of 100 ng/ml GDNF, 10 ng/ml NGF, 30 mM sodium chlorate, 2 mM sodium
sulphate, 0.3 U/ml heparinase III or 0.3 U/ml chondroitinase ABC (units as
defined by Sigma). Responses were quantitified by counting the total numbers
of cells in at least 12 fields of view, and also the number of cells that bore
neurites at least twice the diameter of the cell body (most were significantly
longer or very much shorter so the judgement of twice the body diameter was
not critical and was done by eye). MDCK cells and RET/GFR
1-MDCK cells
were maintained in the same medium as DRGs, with 0.2 mg/ml G418 in the case of
RET/GFR
1-MDCK cells. Scatter experiments were performed on glass
coverslips in 24-well plates. For staining with anti-phosphotyrosine, cells
were fixed in cold methanol for 5 minutes, washed in PBS, incubated 40 minutes
in 1/100 anti-phosphotyrosine at 4°C, washed in PBS, incubated in 1/100
Sigma FITC-anti mouse IgG for 40 minutes at 4°C, washed again and
photographed on an epifluorescence microscope. For phosphotyrosine
experiments, all photomicrographs were taken and digitised at the same
exposure, and digital adjustments of brightness and contrast were performed on
the grouped images so that the relationships between their relative
brightnesses were maintained.
Immunoprecipitation and blotting
Approximately 2 million RET/GFR1-MDCK cells per 60 mm dish were
cultured overnight and placed in 5 ml serum-free medium for 2 hours and
treated with 0.1-0.5 U/ml heparinase-III for 1 hour. 100 ng/ml GDNF was added
for 30 minutes and cells homogenized in MB buffer (50 ml Tris-HCl, pH 7.4, 150
mM NaCl, 1% NP-40, 10% glycerol, 100 µM sodium orthovanadate, 1% aprotenin,
1 mM PMSF). For immunoprecipitation, goat anti-c-Ret (Santa Cruz) was adsorbed
to rabbit anti-goat IgG agarose beads (Sigma) by incubation on ice for 2
hours. The beads were washed with MB buffer and incubated with RET/GFRa1-MDCK
cell homogenates at 4°C overnight. Beads were washed twice in MB buffer
and bound c-Ret was eluted by boiling in Laemelli buffer containing 5%
ß-mercaptoethanol for 3 minutes. Eluate from each immunoprecipitation was
divided into two equal fractions and subjected to SDS-PAGE analysis and
transferred to Hybond nitrocellulose membranes (Amersham). Membranes were
probed with mouse anti-phosphotyrosine (1/2000) (Upstate Biotechnology, UK) or
with goat anti-c-Ret (1/1000). Secondary antibodies (Sigma) were used at a
1/2000 (anti-mouse-HRP) or at 1/50000 (anti-goat-HRP). Signals were detected
using an ECL plus kit (Amersham).
125I-GDNF binding
RET/GFRa1-MDCK cells were cultured directly on the plastic of 24-well
plates, at approx 60,000 cells/well, and incubated for 20-40 hours in standard
medium (no G418) with or without 30 mM sodium chlorate. They were then washed
in ice cold binding buffer (PBS with 0.5% BSA) and then a range of dilutions
in binding buffer of ice cold stock 125I-GDNF (supplied by
Amersham/Pharmacia 64.1 TBq/mmol, and diluted to 6.41 TBq/mmol using
unlabelled GDNF to form the primary stock) were applied to the wells (total
volume 200 µl/well). After 4 hours on ice, the 125I solution was
removed, the cells were washed twice, quickly, in binding buffer, and were
then lysed in 500 µl 10% SDS. 125I-GDNF was measured using a
Packard-Bell gamma counter. Machine background was assessed using wells to
which no 125I-GDNF had been added, the counter was calibrated using
a known quantity of 125I-GDNF stock diluted in 10% SDS and added
directly to a scintillation vial, and non-specific binding was measured by
applying a 200-fold excess of unlabelled GDNF to some wells. Binding
parameters (Bmax, Kd) were calculated
automatically using built-in functions of Prism (Graphpad software).
![]() |
Results |
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|
Studies of gdnf-/- transgenic mice have shown that
GDNF, expressed naturally by the gut, is required for formation of most of the
enteric nervous system (Pichel et al.,
1996; Moore et al.,
1996
). This observation was the basis of another assay used by us
to explore the requirement of the system for glycosaminoglycans. When hindguts
were isolated from E10 mice and cultured for 96 hours, they developed a
complex network of neurofilament 68-positive nerve fibres in response to their
endogenous GDNF (Fig. 1e), but
those cultured in 20 mM sodium chlorate developed few axons, and those
cultured in 0.1 U/ml heparinase III developed fewer still
(Fig. 1f,g). Early enteric
neurons are not responsive to exogenous NGF, so the control of showing that
cells can still produce axons in response to growth factors that do not
require GAGs is not available in this system. The cultured hindgust could,
however, be protected from the effect of sodium chlorate by the addition of 2
mM sulphate (Fig. 1h). This
common control confirmed that the effects of chlorate were indeed due to it
acting as a competitive inhibitor of GAG sulphation.
The physical geometry of the DRG and gut culture systems described above
made the effect of GDNF difficult to quantify since axons were too tangled to
count and the number of potential axon-forming cells was also uncountable.
PC-12 pheochromocytoma cells will undergo neural differentiation in response
to exogenous GDNF (Chen et al.,
2001), and will do so as scattered single cells that are easily
countable. With no GDNF, PC12 cells attached loosely to their substrate but
remained rounded and extruded only tiny processes
(Fig. 2a). 100 ng/ml GDNF
caused approximately 13% of these cells to produce processes (neurites) at
least twice as long as their cell diameter, and usually much longer
(Fig. 2b). The presence of 30
mM chlorate inhibited this effect strongly
(Fig. 2c) although 2 mM
sulphate again achieved a substantial rescue
(Fig. 2d). Even in the presence
of chlorate, NGF elicited a massive response from PC-12 cells
(Fig. 2e), again showing that
GAGs are not required for neurite formation itself, but rather for cells to be
able to respond to GDNF. The quantitative data from these experiments are
shown in Fig. 2f (except for
the NGF-treated controls, which were impossible to count because the neurite
network was so dense that it was not clear to which cell each neurite
belonged). In contrast to differentiation, total cell numbers were not
affected significantly by the presence of GDNF or chlorate
(Fig. 2g) so, in this system,
the observed rates of axonogenesis are unlikely to reflect differences in
proliferation or survival.
|
Our final morphological assay was based on the behaviour of a cell line
derived from canine collecting ducts and transfected with both c-Ret and
GFR1. Based on the well-known Madin-Darby Canine Kidney (MDCK) cell
line, the transfected cell line is called RET/GFR
1-MDCK. That it does
indeed express both c-Ret and GFR
1 can be seen in the Western blots in
Fig. 5a,b In the absence of
exogenous GDNF, RET/GFR
1-MDCK cells grew as normal MDCK cells; when
grown on glass coverslips in sub-confluent culture, they formed separated
islands with cell-free spaces between them
(Fig. 3; spaces are marked with
arrows). Addition of GDNF caused the cells to scatter so that the formerly
clear inter-island spaces became filled with spindly cells; this response is
similar to that of normal MDCK cells treated with HGF/scatter factor [for a
review of HGF-induced scattering see Balkovetz
(Balkovetz, 1998
)]. Once again,
treatment with 20 mM chlorate or with 0.1 U/ml heparinase III blocked the
effects of exogenous GDNF and the islands of cells remained separated by clear
space.
|
|
GDNF signalling requires heparan sulphates, not chondroitin or
dermatan sulphates
The experiments on enteric neurons and on scatter of RET/GFR1-MDCK
cells reported above suggest that heparan sulphate, the target for the
heparinase III enzyme, is particularly important for GDNF signalling. We
explored this further using both an immunohistochemical assay for GDNF
signalling in RET/GFR
1-MDCK cells and a quantitative neuritogenesis
assay using PC-12 cells. When fixed and stained with anti-phosphotyrosine,
RET/GFR
1-MDCK cells showed only low levels of tyrosine phosphorylation
at their plasma membranes after being cultured for 2 hours in serum-free
medium (Fig. 4a). Tyrosine
phosphorylation at the membrane increased markedly following treatment with
100 ng/ml GDNF (Fig. 4b), but
this rise in tyrosine phosphorylation at the membrane was abolished in cells
whose medium had been supplemented with 30 mM sodium chlorate overnight.
Membrane-associated tyrosine phosphorylation was also abolished by treatment
with 0.1 U/ml heparinase III for 1 hour before GDNF was added
(Fig. 4c). It was not, however,
abolished by treatment with 0.1 U/ml chondroitinase ABC
(Fig. 4d). GDNF was able to
induce significant neuritogenesis in PC12 cells either in the absence of any
exogenous glycansases or in the presence of chondroitinase ABC, but not in the
presence of heparinase III (Fig.
4e). These data strongly suggest that heparan sulphates (targets
of heparinase III) rather than chondroitin/dermatan sulphates (both targets of
chondroitinase ABC) are important to GDNF signalling.
|
Heparan sulphates are required for GDNF to activate c-Ret
In each of the morphological and survival assays above, responses to GDNF
did not take place in the absence of glycosaminoglycans. These assays show
that GAGs are required for a range of GDNF responses, but do not show whether
this requirement is for GDNF to activate its c-Ret receptor tyrosine kinase or
for events subsequent to that activation (such as events in cytoplasmic signal
transduction). In order to establish whether GAGs are needed for GDNF to
activate its c-Ret receptor tyrosine kinase, or merely for events downstream
of Ret activation, we again made use of the RET/GFR1-MDCK cell line.
Phosphorylation of the c-Ret receptor tyrosine kinase was measured by
immunoprecipitation for c-Ret followed by Western Blotting using
anti-phospho-tyrosine as a probe (anti-c-Ret being used as a control probe to
confirm equal recovery from samples during immunoprecipitation).
Phosphorylation of c-Ret was low in cells cultured for 2 hours in serum-free
medium, increased substantially when they were treated with GDNF, but failed
to do so when the cells were treated for the preceding hour with 0.1-0.5 U/ml
heparinase III (Fig. 5a). These
observations were made in four independent runs of the experiment. Heparan
sulphates are therefore required for GDNF to activate its c-Ret receptor
tyrosine kinase.
GAGs are involved in binding GDNF to the cell surface
Now that we have shown GAGs to be required for efficient Ret activation, it
is natural to ask whether GAGs are involved in the binding of GDNF to the cell
surface in the first place. The question is particularly pertinent because
heparan sulphate would be an obvious candidate for being the uncharacterised,
high abundance, low affinity GDNF receptor mentioned by Jing et al.
(Jing et al., 1996) when they
reported the discovery of the high-affinity GPI-linked receptor, GFR1
(as it is now called). If GAGs are involved in binding GDNF to cells, binding
would be expected to be diminished in cells whose GAG synthesis has been
inhibited. This is indeed the case (Fig.
6); while there is no significant difference in the ability of
normal and chlorate-treated RET/GFR
1-MDCK cells to bind GDNF from very
dilute solutions (<300 pM), the binding diverges significantly for higher
concentrations of GDNF. The Bmax (GDNF binding capacity) of the
chlorate treated cells (0.38 pmol/well, s.e. 0.06 pmol/well) is only about a
quarter that of the normal cells (1.52 pmol/well, s.e. 0.82 pmol/well); if
GAGs play a role in binding GDNF to the cell surface, we would expected
Bmax to be reduced when the synthesis of new GAGs has been
inhibited and only a diminishing stock of pre-existing GAGs remains. The
receptors that still persist on the surface of chlorate treated cells retain
an affinity (Kd) of 2.2 nM (s.e. 0.5 nM) similar to that
of controls. This is within an order of magnitude of the
Kd of the uncharacterised abundant low-affinity GDNF
receptor (0.33 nM) described by Jing et al.
(Jing et al., 1996
), but very
different from that of GFR
1 itself (Kd=0.0015
nM).
|
GDNF signalling requires cell surface heparan sulphates
There are several ways in which GAGs might facilitate signalling by growth
factors (discussed in more detail below). One possible role for GAGs, which
are borne by abundant proteoglycans associated with the plasma membrane, is to
bind quantities of growth factor with relatively low affinity and thereby
increase the local concentration of growth factor at the plasma membrane where
its high-affinity receptor tyrosine kinase is situated. This may or may not be
combined with other roles such as stabilisation of the receptor complex. If
GAGs do concentrate GDNF at the plasma membrane, exogenous soluble GAGs added
by an experimenter would be expected to compete with the membrane-located GAGs
and will therefore inhibit signalling, while if GAGs were only required for
other purposes, such as stabilising ligand-receptor complexes, exogenous GAGs
will not be detrimental and may even aid stabilization. We have tested this in
our system by adding exogenous chondroitin, heparan and dermatan sulphates to
the media of RET/GFR1-MDCK cells treated simultaneously with 100 ng/ml
GDNF. As little as 10 ug/ml heparan sulphate inhibited GDNF-induced tyrosine
phosphorylation very effectively (Fig.
7a-i), whereas the same concentration of chondroitin and dermatan
sulphates had no detectable effect and even 100 ug/ml dermatan and chondroitin
sulphates failed to inhibit GDNF signalling completely. A similar effect can
be seen in the response of PC-12 cells to 100 ng/ml GDNF, and as little as 100
ng/ml heparan sulphate is enough to reduce neuritogenesis to control levels
(Fig. 7j, blue bars). Because
these data showed a hint, albeit one not statistically significant at
P=0.05, that low concentrations (10 ng/ml) of exogenous heparan
sulphate may slightly potentiate signalling by GDNF, we tested whether
exogenous heparan sulphate might be able to rescue the response of
chlorate-treated PC-12 cells to GDNF. It was unable to do so at any
concentration examined (Fig.
7j, red bars).
|
In earlier studies on the role of GDNF in kidney development, it was shown
that very high concentrations of GDNF (1000 ng/ml) could begin to rescue renal
morphogenesis even in kidneys deprived of GAGs by chlorate treatment
(Sainio et al., 1997). One
possibility, suggested by us in that paper, was that signalling by
physiological concentrations of GDNF might require GAGs but that
supraphysiological concentrations of GDNF might be able to signal even without
them. We have tested that using RET/GFR
1-MDCK cells, and find that
while GAGs are needed for robust signalling by 100 ng/ml GDNF, GDNF at the
supraphysiological concentration of 1000 ng/ml elicits tyrosine
phosphorylation even in cells treated with heparinase III
(Fig. 8a-c).
|
![]() |
Discussion |
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---|
Our enzyme data shows that GDNF signalling requires heparan but not
dermatan or chondroitin sulphates, and addition of exogenous GAG chains shows
that exogenous dermatan and chondroitin sulphates are much less effective than
is heparan sulphate at inhibiting GDNF signalling. The specificity of GDNF for
heparan sulphate is a common but not universal feature of growth factors.
FGFs, for example, use only heparan sulphates but HGF can use heparan or
dermatan sulphates (Ashikari et al.,
1995; Lyon et al.,
2002
). Of these classes of glycosaminoglycan, heparan sulphates
show the greatest range of structural variations in vivo, differing in amounts
and positions of deacetylation, N-sulphation, O-sulphation and uronic acid
epimerisation. Where specificity has been examined, it is clear that
heparan-sulphate binding growth factors show specificity for a very limited
range of possible heparan sulphate sequences. Furthermore, these differ subtly
between different proteins. FGF-1, for example, requires heparan sulphate
domains composed of a cluster of
IdoA(2SO4)-GlcNSO3(6SO4)-IdoA(2SO4)-trisaccharide
motifs, FGF-2 binds with highest affinity to a
IdoA(2SO4)-GlcNSO3-IdoA(2SO4) trisaccharide,
while HGF binds clusters of
IdoA(2SO4)-GlcNSO3(6SO4), or three repeats of
IdoA-GalNAc(4SO4) when it binds to dermatan sulphate instead
(Ashikari et al., 1995
;
Lyon et al., 1998
;
Kreuger et al., 2001
). As yet
we know nothing about the binding specificity of GDNF for different types of
heparan sulphate, except for the fact that some sulphated residues must be
involved (or chlorate ions, which competitively block sulphation, would not
have had an effect in our experiments); presumably, however, GDNF too has a
specificity for particular heparan sequences.
If GDNF does indeed require specific types of heparan sulphate, the
involvement of these GAGs might add another layer of regulation to the GDNF
signalling system in vivo. Tissues in which GDNF is active, such as developing
kidney and brain, are rich in heparan sulphate proteoglycans such as syndecans
and glypicans, and each member of these families has its own distinct
spatiotemporal distribution (reviewed by
Bovolenta and Fernaud-Espinosa,
2000; Bandtlow and Zimmerman, 2000;
Davies et al., 2001
).
Crucially, the specific composition of heparan sulphate chains borne by these
molecules is also developmentally controlled, changing in both length and
6-O-sulphation during development of the mouse brain
(Brickman et al., 1998
).
Indeed, expression of different heparan sulphate sulphotransferase enzymes,
important in heparan sulphate synthesis, is itself regulated during brain
development (Guimond et al.,
2001
). This raises the possibility that changes in expression of
heparan sulphates in development may exert a powerful modulatory effect on
cells' sensitivity to GDNF, or on their relative sensitivity to different
growth factors, even when they bear the high-affinity receptor tyrosine
kinases at a constant level. Certainly at the crude level of whole tissues, it
is known that different sources of heparan sulphates can have quite different
affinities for a specific growth factor; pig liver heparan sulphate has a
strong binding affinity for HGF while pig aorta heparan sulphate has none
(Ashikari et al., 1995
).
Since interactions between growth factors and GAGs were first identified,
there have been a number of models for how these interactions aid signalling.
In the best-studied example, the FGF1 signalling system, interactions with
heparan sulphate perform several tasks simultaneously. To begin with, two FGF1
monomers are linked together into a biologically active complex by means of
their interaction with a shared heparan sulphate chain; crystallography this
FGF12HS1 heterotrimer reveals that the FGF1 monomers
share no protein-protein interface, and are kept together solely by the
heparan sulphate (DiGabriele et al.,
1998). The same chain of heparan sulphate also binds to one
molecule of the FGF receptor tyrosine kinase, though the other FGFR monomer,
which is recruited to make a `dimer' of receptor tyrosine kinases, is
apparently recruited through protein interactions alone; the whole complex
therefore has the structure FGF12FGFR2HS (Pellegrini et
al., 2001). A heparan decasaccharide is sufficient to promote assembly of this
complex in vitro.
How similar might be the role of heparan sulphate in signalling by GDNF?
Though we do not yet have direct evidence for binding of heparan sulphate by
GDNF, there are several good reasons to assume that such binding takes place.
First, GDNF binds well to heparin, a close structural relative of heparan
sulphate; this fact was used for GDNF's initial isolation
(Lin et al., 1993). Second,
the binding of GDNF to cells that have GFR
1 but no Ret shows binding
with two distinct affinities, a high affinity binding
Kd=1.5x10-12 and a low affinity one with
Kd=3.30x10-10
(Jing et al., 1996
). It is
likely that the low affinity interaction reflects binding to heparan sulphate,
especially since the dissociation constants are so similar to that of HGF for
its receptor tyrosine kinase and for heparan sulphate
[Kd=4.6x10-12 and
Kd=2.8x10-10, respectively
(Arakaki et al., 1992
)]. We now
report a very similar high abundance, low-affinity GDNF receptor, and show
that its abundance is significantly decreased when cells are preincubated in
sodium chlorate, an inhibitor of heparan sulphate synthesis. Third, GDNF has a
good consensus heparan sulphate binding sequence [by the criteria of Hileman
et al. (Hileman et al., 1998
)]
GKGRRG at amino acids 28-33 and a weaker consensus SRSRRL at
87-93. Neither of these sites is part of the GDNF-GFR
/Ret interface
(Baloh et al., 1999
), so might
be available for binding to heparan sulphate. The first of these sites is
known from homologue-scanning mutagenesis studies to be uneccesary for GDNF
function, but removal of the second reduces the activity of GDNF by 40-60%,
depending on the assay system used (Baloh
et al., 1999
). This second site is near the end of an alpha helix
that spirals up away from the GDNF-GFR
/Ret interface, and may therefore
be easily accessible. Even assuming that GDNF does bind heparan sulphate,
however, the interaction cannot be responsible for GDNF dimerisation, for that
is achieved by a covalent linkage via a disulphide bridge
(Hui et al., 1999
). In that
respect, the GDNF and FGF systems are certainly different.
Might heparan sulphate cross-link GDNF to its receptors, and help to
stabilise the interaction? GDNF is thought to assemble into a complex first
with GFR1, and only then to associate with Ret; indeed, there is good
evidence that the GDNF/GFR
1 complex can initiate signalling via the
Src-like-kinase pathway in its own right even in the absence of Ret
(Poteryaev et al., 1999
) and
presence or absence of Ret on cells that possess GFR
1 has no effect on
the observed affinities of GDNF for those cells
(Jing et al., 1996
).
GFR
1 has a possible heparan sulphate binding consensus [by the criteria
of Hileman et al. (Hileman et al.,
1998
)] NRRKCHKA at amino acids 188-196 at the N terminal
boundary of helix 5, a much weaker consensus MKKEKN at amino acids
93-98 at the N-terminal boundary of helix 3, and a few other clusters of
R&K elsewhere. Loss of the N-terminal domain of GFR
1, including the
MKKEKN consensus, greatly reduces the ability of GFR
1 to bind GDNF, but
it is not clear whether the MKKEKN site is the important part of this domain
(Scott and Ibanez, 2001
).
Neither of these sites is itself the GDNF binding site, as identified by
mutagenesis studies (Scott and Ibanez,
2001
); it is therefore possible, in principle, that GFR
1
binds heparan sulphate and might therefore be cross-linked to GDNF by their
binding the same oligosaccharide, as well as by direct protein-protein
interactions. Determining whether this is so awaits detailed structural
studies.
How else might heparan sulphate assist GDNF signalling? Since heparan sulphates are borne by proteoglycans of the cell surface and the pericellular matrix, one function of GDNF binding with low affinity to heparan sulphate might simply be to concentrate the growth factor in the vicinity of its high affinity receptors. Our finding that exogenous heparan sulphate inhibits GDNF signalling supports this model, for it suggests that that heparan sulphates are most useful to the system when they are located at the plasma membrane rather than diffusing freely. The model of concentration at the plasma membrane is also supported by our observation that very high concentrations of GDNF can include Ret phosphorylation even in cells depleted of GAGs, for if the concentration of GDNF in bulk medium is high enough, there will be no need for heparan-mediated concentration of GDNF near the plasma membrane. These observations do not, however, exclude the possibility that heparan sulphate also acts by the complex-stabilising model discussed above. They do, however, suggest that it cannot act only by stabilising complexes, for in that case soluble heparan sulphate would be expected to perform as well as proteoglycan-linked material on the surface of the cell. An important caveat to this last remark must be borne in mind, though; the exogenous heparan sulphate added by us might differ significantly (in precise patterns of sulphation, epimerisation etc) from the heparan sulphate with which GDNF normally interacts, and in that case our exogenous heparan might be able to compete with natural heparan sulphate but be unable to promote signalling for reasons of internal structure rather than its location.
In summary, we have shown that cell surface-associated heparan sulphate plays an important role in signalling by GDNF. It will be interesting to determine, in future work, the specific types of heparan involved, how they are involved, and whether expression of these types of heparan sulphate is modulated in a biologically meaningful way during development, disease and regeneration.
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Acknowledgments |
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