Download The Glial Cell–Derived Neurotrophic Factor Signaling Pathway

Part F.
The Glial Cell–Derived Neurotrophic
Factor Signaling Pathway
35
Signaling Pathways of Glial Cell–Derived Neurotrophic Factor
LOUIS F. REICHARDT
G
lial cell–derived neurotrophic factor (GDNF), a distant relative
of transforming growth factor- (TGF-), was purified as a factor that promotes the survival and differentiation of embryonic mesencephalic dopaminergic neurons (Lin et al., 1993). A closely related
protein, neurturin, was purified as a factor that promotes the survival
of sympathetic neurons (Kotzbauer et al., 1996). Subsequently, two
additional members of the GDNF family of ligands—artemin and
persephin—were identified in EST databases (Baloh et al., 1998; Milbrandt et al., 1998; Masure et al., 1999). Each of these proteins has
been shown to function as a survival and differentiation factor for subpopulations of cultured neurons (reviewed in Airaksinen et al., 1999;
Baloh et al., 2000a; Bennett, 2001; Butte, 2001; Manie et al., 2001;
Takahashi, 2001).
The search for GDNF receptors was initially frustrating and led to
the characterization of a glycosylphosphatidylinositol (GPI)-anchored
binding protein now named GFR1 that seemed unlikely to function
as a single subunit receptor for this neurotrophic factor (Treanor et
al., 1996). But when mice lacking GDNF were examined (Moore et
al., 1996; Pichel et al., 1996), they proved to have an almost identical phenotype to mice that lacked the orphan receptor tyrosine kinase
c-ret (Schuchardt et al., 1994). In each instance, severe deficits were
seen in development of the metanephric kidney and the enteric nervous system, suggesting that they act through the same signaling path-
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way. Indeed, it was rapidly demonstrated that GFR1 is the major ligand-binding subunit in a signaling complex that includes c-ret (e.g.
Durbec et al., 1996a; Jing et al., 1996). More recent work has demonstrated the existence of four members of the GFR family (GFR1–4)
that, after ligand engagement, activate the c-ret tyrosine kinase
(Airaksinen et al., 1999; Klein et al., 1997; Enokido et al., 1998; Lindahl et al., 2001) with GDNF, neurturin, artemin, and persephin functioning as reasonably specific ligands for GFR1, GFR2, GFR3,
and GFR4, respectively. In prior work, inactivating mutations of cret had been shown to be responsible for many cases of Hirschsprung’s
disease, which is caused by a failure of enteric neuron precursors to
populate normally the intestine (Edery et al., 1994). In addition, mutations or gene fusions that activate the tyrosine kinase activity of cret had been shown to be oncogenic, resulting in multiple endocrine
neoplasia and medullary thyroid carcinoma (Goodfellow, 1994). Consequently, the signaling pathways activated by GDNF have been of
keen interest to both developmental biologists and oncologists.
LIGAND–RECEPTOR INTERACTIONS
The members of the GDNF family are secreted disulfide-bonded homodimers that are distant relatives of TGF- (Lin et al., 1993;
Kotzbauer et al., 1996; Baloh et al., 1998; Milbrandt et al., 1998; Ma-
Signaling Pathways of Glial Cell–Derived Neurotrophic Factor
Fig1
sure et al., 1999; Rosenblad et al., 2000). The three-dimensional structure of GDNF has been determined at high resolution (Eigenbrot and
Gerber, 1997). This structural information has made it possible to identify the surface residues in GDNF that are responsible for mediating
interactions with GFR1. The structural information has guided experiments in which domains are swapped between GDNF family members. Individual amino acids within domains involved in receptor interactions have been mutated to confirm their functions (Baloh et al.,
2000b; Eketjall et al., 1999).
The GFR family (GFR1–4) consists of four glycosylphosphatidylinositol (GPI)-anchored membrane proteins that bind preferentially to GDNF, neurturin, artemin, and persephin, respectively
(Airaksinen et al., 1999; Scott and Ibanez, 2001). Although each member of this family has a single preferred ligand, several have been
shown to bind to additional members of the GFR family with lower
affinity. While association of a GFR receptor with c-ret is promoted
by ligand engagement (Treanor et al., 1996), in the absence of ligand
some of the GFR receptors also interact with c-ret with lower affinities (Sanicola et al., 1997; Eketjall et al., 1999). Association between
a GFR subunit and c-ret has been shown to broaden the ligand-binding ability of the complex (Sanicola et al., 1997). For example, some
mutants of GDNF are defective in binding to GFR1 and are able to
bind and activate signaling in cells that express both GFR1 and cret (Eketjall et al., 1999).
When first isolated, clones of GFR1 were shown to encode a 461
amino acid long protein that includes a cleavable signal sequence. The
presence of a C-terminal hydrophobic sequence without any C-terminal hydrophilic region, preceded by a group of three small amino acids,
defined a cleavage and recognition site for possible addition of a glycosylphosphatidylinositol (GPI)-linked membrane anchor (Jing et al.,
1996; Treanor et al., 1996). GFR1 was shown to be associated with
the membrane from which it was released by phosphatidylinositolspecific phospholipase C. The presence of GFR1 was essential for
autophosphorylation of c-ret by GDNF. Interestingly, both soluble and
membrane-attached GFR1 were effective in mediating GDNF-promoted c-ret autophosphorylation, suggesting that GFR receptors can
act in trans. Each of the other members of the GFR family has subsequently been shown to have similar properties (Buj-Bello et al.,
1997; Thompson et al., 1998). In addition, transcription of the mouse
GFR4 gene generates interesting developmentally regulated and
tissue-specific splice variants, several of which are predicted to result
in expression of secreted soluble proteins and one of which is predicted to generate a trans-membrane isoform (Lindahl et al., 2000).
Splice variants predicted to result in expression of both GPI-attached
and secreted receptors have also been reported in rat and human tissues (Masure et al., 2000; Lindahl et al., 2001). The functional importance of these variants has not yet been investigated.
In initial characterizations, the extracellular domains of GFR subunits were observed to be cysteine-rich. In subsequent analyses, three
domains in the extracellular portions of the GFR receptors have been
defined (Lindahl et al., 2000; Scott and Ibanez, 2001). The central domain, which is the most conserved, has been shown to determine ligand-binding specificity for GDNF and for neurturin. Because there are
different crucial ligand-binding determinants for these two trophic factors, it was possible to construct a GFR1–GFR2 chimeric receptor
that binds both ligands with similar efficiency (Scott and Ibanez,
2001). Although GFR1–3 in mammals and GFR1–4 in chick share
these three domains, the mouse, rat, and human GFR4 receptors lack
the most N-terminal cysteine-rich domain (Lindahl et al., 2000, 2001;
Masure et al., 2000; Zhou et al., 2001). None of the splice variants of
GFR4 alter the central domain.
RET was originally identified as an oncogene activated by fusion
of a tyrosine kinase domain to other proteins (Takahashi et al., 1985).
When first sequenced, the protooncogene c-ret appeared to be a receptor tyrosine kinase that contained a cleavable signal sequence, an
approximately 600 amino acid extracellular domain, a transmembrane
domain, and a cytoplasmic domain with a tyrosine kinase plus several tyrosines that could potentially serve after phosphorylation as recruitment sites for signaling proteins (Takahashi et al., 1988, 1989)
(Fig. 35–1). C-ret was unusual because it lacked the immunoglobu-
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lin, leucine-rich repeats and the fibronectin-type III repeats that are
present in the extracellular domains of other members of the receptor
tyrosine kinase family (Schneider, 1992; Iwamoto et al., 1993). In addition, differential splicing 3 of exon 19 generates short (9 a.a.),
medium (43 a.a.), and long (51 a.a.) isoforms of the cytoplasmic domain, all of which contain a functional tyrosine kinase but differ in
their docking sequences for adapter proteins (Tahira et al., 1990;
Lorenzo et al., 1995, 1997). Of these, only the short and long isoforms
are expressed at significant levels in vivo.
Initial analyses indicated that the approximately 600 amino acid
long extracellular domain lacked homology to known proteins. Subsequent analysis, though, indicated the presence of conserved repeats
homologous in position and sequence to the Ca2-binding domains of
cadherins (Schneider, 1992; Hill et al., 2001). Recent molecular modeling of the extracellular domain of c-ret has confirmed the presence
of four cadherin-like domains, followed by a cysteine-rich, membraneproximal domain that is similar to the membrane-proximal domain of
the cadherin-related protein Flamingo (Anders et al., 2001). Similar
to cadherins, c-ret binds Ca2, and the presence of Ca2 is essential
for crosslinking of 125I-GDNF to c-ret, but not to GFR1, on cells
that express both receptor subunits (Anders et al., 2001). This suggested that Ca2 stabilizes the structure of the c-ret extracellular domain. The presence of Ca2 is also essential for surface expression of
c-ret, suggesting that Ca2 is essential for stabilization of c-ret inside
the endoplasmic reticulum (van Weering et al., 1998). Since GFR
and GDNF homologs have not been identified in the Drosophila
genome, c-ret may have evolved from a cell adhesion molecule.
Although GDNF family ligands provide the most direct pathway
for activation through GFR subunits of c-ret, an alternative pathway
has recently been described in neurons that coexpress trkA, a receptor tyrosine kinase that is activated by nerve growth factor (NGF)
(Tsui-Pierchala et al., 2002). Mature sympathetic neurons have lost
their dependence on NGF for survival, but they continue to require it
for process outgrowth and differentiation. NGF-mediated activation
of TrkA results in delayed phosphorylation of c-ret in mature sympathetic neurons both in vitro and in vivo (Tsui-Pierchala et al., 2002).
Only the long isoform Ret 51, not the short isoform Ret 9, is phosphorylated in response to NGF. Optimal responses to NGF in vitro by
these neurons depends on c-ret, but not on the GFR subunit function. Thus, in some neurons NGF transactivates the long isoform of
c-ret, which, in turn, facilitates the normal maturation of these cells.
The mechanism by which this occurs is not known, but the slow kinetics of c-ret phosphorylation suggest that trkA does not directly
phosphorylate c-ret.
SIGNALING ACTIVATED BY c-ret
Because of its important roles in normal development and in oncogenesis, signaling pathways regulated by c-ret and oncogenic ret variants have been intensely studied and are frequently reviewed (van
Weering and Bos, 1998; Mason, 2000; Saarma, 2000; Manie et al.,
2001; Takahashi, 2001). Ligand engagement of a GFR receptor in
cells that also express c-ret results in dimerization and activation of
the c-ret tyrosine kinase activity. Immediate substrates of this kinase
include six tyrosines in the c-ret cytoplasmic domain (Liu et al., 1996)
(Fig. 35–1). Of these, four tyrosines (Y-905, Y-1015, Y-1062, Y-1096)
have been shown to provide docking sites for cytoplasmic signaling
proteins (Hayashi et al., 2000; Coulpier et al., 2002). Phosphorylated
Y905 has been shown to provide a docking site for Grb-7 and Grb-10
(Pandey et al., 1995, 1996). It is not certain what roles the Grb-7/10/14
family of adaptor proteins play in c-ret-mediated signaling, but these
proteins have been shown to regulate cell migration and survival (Han
et al., 2001). Recent work, for example, indicates that Grb-10 functions as a coactivator of Akt (Jahn et al., 2002). Y-1096 is present in
the long, but not in the short or middle, isoforms of c-ret and has been
shown to provide a docking site for the adaptor Grb-2 (Liu et al., 1996).
Recruitment of Grb-2 provides a potential mechanism for activation of
the PI-3 kinase–Akt and Ras–Map kinase pathways.
Phosphorylated Y-1015 mediates docking of PLC-1, which is activated by phosphorylation and then acts to hydrolyze phosphatidy-
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DEFINED PATHWAYS
Figure 35–1. Interactions of GDNF with GFR1 and c-ret. Depicted on the
left is GDNF binding to GFR1 that is preferentially localized in cholesterol
and sphingolipid-rich lipid rafts. GDNF binding to GFR1 results in activation of src-family tyrosine kinases that are also present in lipid rafts. Downstream signaling events (not shown) include activation of MAP kinase, CREB,
and phospholipase-C. Shown also in the left panel is c-ret attached to two proteins—ACAP and ENIGMA—that associate with this receptor irrespective of
its activation state. ACAP provides a docking site for protein kinase A that has
been shown to phosphorylate S-696, thereby regulating signaling through Y687. Depicted on the right is the recruitment by the GDNF–GFR1 complex
of c-ret to the lipid raft. Recruitment of c-ret results in activation of its tyrosine kinase and phosphorylation of several tyrosines in its cytoplasmic domain.
Phosphorylation of Y-905 results in recruitment of two adapters Grb-7 and
Grb-10 that activate poorly characterized downstream signaling pathways.
Phosphorylation of Y-1015 permits recruitment and activation of phospholipase-C1. Activation of PLC results in increased cytoplasmic Ca2 and activation of Ca2- and diacylglycerol-regulated protein kinases. Phosphorylation
of Y-1062 results in recruitment of several different adaptors, including IRS1,
FRS2, Shc, Dok-4, and Dok-5. These adaptors have been shown to activate
several intracellular signaling pathways, including cascades controlled by Ras,
Erk-1/2, Erk-5, PI-3 kinase, and rac (see text for details). Phosphorylation of
Y-1096 permits recruitment of Grb-2 and results in activation of ras through
SOS. Localization of c-ret in lipid rafts has been shown to promote the recruitment of FRS2. Localization of c-ret to rafts is essential for normal activation of several of the signaling pathways summarized above and described
in more detail in the text.
linositides to generate inositol tris-phosphate and diacylglycerol. Inositol tris-phosphate induces release of Ca2 stores, increasing levels
of cytoplasmic Ca2. This results in activation of the various enzymes
that are regulated by cytoplasmic Ca2, including Ca2-calmodulinregulated protein kinases and phosphatases and Ca2-regulated isoforms of protein kinase C. Formation of diacylglycerol (DAG) stimulates the activities of DAG-regulated isoforms of protein kinase C.
One of these DAG-regulated isoforms, PKC, is required for activation of the mitogen activated protein kinase (ERK) cascade and ERKdependent differentiation in many neuronal cells (Corbit et al., 1999,
2000). It appears to act between Raf and MEK in the ERK signaling
cascade. Activation of PLC-1 through Y-1015 is essential to observe
the full oncogenic and proliferative activities of ret (Borrello et al.,
1996).
By far the most important mediator of signaling by c-ret appears to
be Y-1062 (Fig. 35–1). Interestingly, while Y-1062 is present in the
long, medium, and short isoforms of c-ret, splicing introduces different sequences immediately 3 of the codon for Y-1062, so the amino
acid sequence immediately C-terminal of Y-1062 differs between the
RET 9, RET 43, and RET 51 isoforms, thus providing the potential
for differential recognition of signaling proteins at this site (Tahira et
al., 1990; Myers et al., 1995; Lorenzo et al., 1997). The presence of
Y-1062 is required for the oncogenic and proliferative activities of cret isoforms (Melillo et al., 2001b). The signaling pathways activated
by phosphorylation of this site are extensive and include the Ras, PI3 kinase, Jun kinase, p38MAP kinase, Erk-1/2 and Erk-5 kinases, and
Rac (Hayashi et al., 2001; Fukuda et al., 2002). Depending on cell
type, phosphorylation of this site promotes cell survival, proliferation,
differentiation, motility, or oncogenesis. When phosphorylated, this
site has been shown to recruit several different adaptor proteins, including IRS1, FRS2, Shc, Dok-4 and Dok-5 (Arighi et al., 1997;
Hayashi et al., 2000; Grimm et al., 2001; Melillo et al., 2001a, 2001b).
Shc recruitment provides pathways through Grb-2 and the Ras exchange factor SOS (son of sevenless) for activation of Ras. Activation of Ras by SOS has many downstream consequences, including
stimulation of PI-3 kinase, activation of the c-raf/ERK pathway, and
stimulation of the p38MAP kinase pathway (Xing et al., 1998).
Alternatively, Shc recruitment of Grb-2 has also been shown to promote association of the adapter Gab-1, which, in turn, binds and facilitates activation through phosphorylation of PI-3 kinase and the
downstream signaling pathways. Recruited IRS1 has been shown to
associate directly with, thereby facilitating, activation of PI-3 kinase
Signaling Pathways of Glial Cell–Derived Neurotrophic Factor
(Melillo et al., 2001a). FRS2 recruitment and phosphorylation provides multiple docking sites for the Grb-2–SOS complex, thereby promoting activation of RAS, the ERKs, and PI-3 kinase (Melillo et al.,
2001b). FRS2 also provides a site for recruitment of Crk that, in turn,
recruits the exchange factor C3G, thereby activating the small G protein Rap1 (York et al., 2000). This provides a mechanism for activation of B-raf and the ERKs that does not depend on Ras. Because
Rap1 is localized to endosomes, efficient activation of this pathway
appears to require endocytosis of ligand–receptor complexes (York et
al., 2000; Howe et al., 2001). In PC-12 cells, FRS2-mediated signaling has been shown to be essential for the prolonged activation of the
ERK cascade that is required to promote differentiation of these cells
(Grewal et al., 1999; York et al., 2000). FRS2 also provides a docking site for the tyrosine phosphatase Shp2 (Hadari et al., 1998). Shp2
has been shown to enhance activation of the Ras-Raf-MEK-ERK pathway by a mechanism that is not clear. Gab-1 also has been shown to
nucleate formation of a complex that includes Shp2 (Shi et al., 2000).
P62dok is an adaptor protein that has been shown to associate with
several receptor tyrosine kinases. P62dok also associates with RasGAP
and has been shown to inhibit ERK kinase activation. There are at
least five dok homologs that associate with c-ret in a two-hybrid assay (Grimm et al., 2001). Two of these, dok-4 and dok-5, are novel.
Association of these adaptors with c-ret depends on phosphorylation
of Y-1062. In contrast to p62dok, these two adaptors do not interact
with RasGAP, and overexpression enhances rather than inhibits ERK
activation. Ligand engagement of c-ret in PC-12 cells results in neurite outgrowth that requires Y-1062. Fusion of sequences from either
dok-4 or dok-5, but not dok-2, to a mutated c-ret lacking Y-1062 also
permits GDNF-dependent neurite outgrowth by PC-12 cells (Grimm
et al., 2001). The same fusions also are able to mediate GDNF-dependent survival of these cells in a serum-free medium. Thus these
two adaptors are likely to be important in mediating neuronal responses to GDNF and its family members.
The differentiation-promoting responses observed in PC-12 cells
and neurons differ fundamentally from the proliferative responses observed in other cells. Clearly, responses to c-ret activation are celltype specific, depending on the repertories of the intracellular signaling proteins that are present in different cell types. Responses of
different cell types to neurotrophins also depend on cell type, with
some cells showing proliferative and others differentiative responses
(Huang and Reichardt, 2001). Among other targets of probable importance for promoting differentiation, activation of c-ret has been
shown to up-regulate the cyclin-dependent kinase inhibitor p27kip1
(Baldassarre et al., 2002).
In addition to classical adaptors, the Y-1062 site is also essential
for recruiting a PDZ protein named ENIGMA (Durick et al., 1996,
1998). ENIGMA is an interesting protein that consists of an N-terminal PDZ domain plus three Lim domains, one of which associates
specifically with c-ret in an interaction that depends on Y-1062. This
interaction does not depend on phosphorylation of this tyrosine, however. While the function of ENIGMA in c-ret signaling is uncertain,
it is essential for the mitogenic activity of a cAMP-dependent kinase
RI–c-ret fusion protein named Ret/Ptc2 that causes papillary thyroid
carcinoma (Durick et al., 1998). ENIGMA is believed to promote mitogenesis and oncogenesis by localizing Ret/Ptc2 to the membrane
through its PDZ domain. Since Y-1062 is essential for recruitment of
adaptors, such as Shc, that are essential for the mitogenic and oncogenic activities of Ret/Ptc2, dimerization is essential not only to activate the tyrosine kinase activity but also to permit simultaneous localization of Ret/Ptc2 to the membrane through ENIGMA and
recruitment of these adaptors. Because Y-1062 recruits so many different adaptors, which, in turn, nucleate formation of different signaling complexes that affect cell function, it will be a major challenge
for future scientists to understand the factors that bias c-ret-mediated
signaling responses into specific pathways, such as proliferation versus differentiation.
The protein kinase A–anchoring protein ACAP-79 also associated
with c-ret in a ligand-independent manner (Fukuda et al., 2002).
ACAP-79 provides a high-affinity docking site for the type II regula-
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tory subunit of cAMP-dependent protein kinase. In response to cAMP,
S-696 in the cytoplasmic domain of c-ret is phosphorylated, and phosphorylation at this site promotes a guanine nucleotide exchange factor activity for Rac-1 and promotes lamellipodia formation in the presence of GDNF. Mutation of S-696 or Y-1062 decreases Rac-1 guanine
nucleotide exchange factor activity, but mutation of Y-687 rescues the
phenotype of the S-696 mutation. These data suggest that guanine nucleotide exchange factor activity is promoted by signals depend on
phosphorylation of Y-1062, but is inhibited by a signal that emanates
from phosphorylated Y-687. cAMP-dependent phosphorylation of S696 prevents signaling from phosphorylated Y-687. These data illuminate a novel mechanism through which cAMP regulates a tyrosine
kinase–mediated signaling pathway.
SIGNALING THROUGH GFR SUBUNITS
GFR subunits function in large part as binding partners for GDNF
family ligands. Initially, GFR subunits were believed to only function as binding subunits of the c-ret receptor complex. However, recent work has demonstrated that these subunits have broader and more
direct roles in mediating signaling by GDNF (Saarma, 2001). Among
other functions, the presence of a GFR subunit appears to inhibit
basal activation of the c-ret kinase in the absence of physiological ligands (Trupp et al., 1998). Since inhibition has only been observed in
COS cells in which c-ret was overexpressed alone or in combination
with a GFR subunit, the physiological significance of this observation is uncertain.
Similar to other GPI-anchored membrane proteins, GFR subunits
are preferentially localized to cholesterol and sphingolipid-rich lipid
rafts (Poteryaev et al., 1999; Trupp et al., 1999), where src-family kinases are also concentrated (Lee et al., 2001). GDNF application to
cells expressing GFR1, but not c-ret, has been shown to activate src
kinase (Trupp et al., 1999). In turn, GFR1-mediated src kinase promotes phosphorylation of MAP kinase, CREB, and phospholipaseC1 (Trupp et al., 1998; Poteryaev et al., 1999). Activation of the
pathways through GFR1 alone is much less prolonged than activation through c-ret (Trupp et al., 1998), but since many cells express
GFR subunits that do not coexpress c-ret, this signaling pathway
probably has physiological consequences. A recent report suggests that
ligand engagement of GFR2 does not activate the signaling pathways that are activated by ligand engagement of GFR1 in the absence of c-ret (Pezeshki et al., 2001), so it is not certain that c-ret-independent signaling is promoted by all four of the GFR subunits.
One of the most important functions of ligand-bound GFR subunits now appears to be recruitment of c-ret to lipid rafts, where it acquires new signaling properties. Disruption of the specialized lipid raft
domains by cholesterol depletion has been shown to severely attenuate c-ret-dependent GDNF signaling (Tansey et al., 2000). GDNF
binding to the GFR1 subunit results in relocalization of c-ret from
the bulk membrane to the detergent-resistant lipid raft domain (Tansey
et al., 2000). In this specialized domain, c-ret interacts with signaling
proteins that it does not associate with outside of the rafts. For example, c-ret outside of rafts is found in association with Shc, but not
FRS2, while the c-ret in rafts is preferentially associated with FRS2,
but not Shc (Paratcha et al., 2001). In addition, raft-associated c-ret
interacts with and activates c-src, but not other src-related tyrosine kinases. Despite the theoretical existence of other pathways to PI-3 kinase through Y-1062, c-src is essential in neurons for activation of the
PI-3 kinase–Akt pathway and GDNF-dependent survival and neurite
outgrowth (Tansey et al., 2000; Encinas et al., 2001). Intriguingly,
both soluble and membrane-tethered GFR subunits are able to recruit c-ret to lipid rafts and potentiate downstream signaling by GDNF
(Paratcha et al., 2001). The mechanism by which ligand-bound GFR
in trans recruits c-ret to rafts is not certain, but it must differ from the
mechanism through which ligand-bound, membrane-tethered GFR
subunits recruit c-ret in cis, because trans recruitment requires an active c-ret kinase, while cis-recruitment does not. Since GFR subunits
have been shown to be released from cells that do not express c-ret
(Trupp et al., 1997; Yu et al., 1998; Worley et al., 2000; Paratcha et
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DEFINED PATHWAYS
al., 2001), the ability of a ligand-bound GFR subunit to promote activation and localization of c-ret to lipid rafts is very likely to be biologically important.
FUNCTIONS OF GDNF FAMILY-MEDIATED
SIGNALING PATHWAYS
GDNF is strongly expressed in the developing gut (Moore et al., 1996).
In GDNF, GFR1, and c-ret mutants, enteric neurons fail to populate
the intestine, and only a few of these neurons are present in the stomach and esophagus (Schuchardt et al., 1994; Durbec et al., 1996b;
Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996; Cacalano
et al., 1998; Enomoto et al., 1998). The phenotypes of these mice are
similar to, but more severe than, those of humans with Hirschsprung’s
disease in which deficits in enteric neuron populations in the lower
intestine result in intestinal obstruction (Edery et al., 1997; Gabriel et
al., 2002). Indeed, Hirschsprung’s disease is often associated with heterozygous mutations that result in deficits in signaling through c-ret
or components of the c-ret signaling pathway (Edery et al., 1994;
Romeo et al., 1994; Amiel et al., 1996; Attie et al., 1996; Kusafuka
and Puri, 1998; Parisi and Kapur, 2000; Gabriel et al., 2002). Mice
heterozygous for GDNF have been shown to have frequent obstructions of the lower intestine that result in very high levels of postnatal
lethality (Shen et al., 2002), and some humans with Hirschsprung’s
disease also have mutations in GDNF (Eketjall and Ibanez, 2002).
The GDNF to c-ret signaling pathway appears to act at many steps
in differentiation of the enteric nervous system (Gershon, 1998; Pachnis et al., 1998). A pool of neural crest cells from the hindbrain gives
rise to most of the enteric nervous system. In the absence of GDNF
signaling, there are deficits in the initial appearance of these cells in
the gut anlage, reflecting defects in migration, as well as increases in
apoptosis of this pool (Durbec et al., 1996b; Taraviras et al., 1999).
In culture and in intestinal explants, GDNF and neurturin have been
shown to promote survival, proliferation, and differentiation of enteric
neuron precursors (Hearn et al., 1998; Heuckeroth et al., 1998; Taraviras et al., 1999). Localized extrinsic sources of GDNF also attract
these cells in what is most likely a chemotropic response (Young et
al., 2001). Activation of the PI-3/Akt pathway has been shown to be
essential for the proliferative response of quail enteric precursors
(Focke et al., 2001). In transplantation experiments it has been possible to show that c-ret-expressing neural precursors can invade the gut
and populate it efficiently from a c-ret mutant embryo, demonstrating
that the GDNF to c-ret pathway is required in the neural crest–derived
progenitor cells and not in the nonneural cells of the developing gut
(Natarajan et al., 1999). Intriguingly, with development, enteric precursors shift from a proliferative to a trophic and differentiative response to GDNF (Worley et al., 2000). At later stages of development, c-ret expression is restricted to a population of enteric precursors
that is committed to differentiate into neurons (Lo and Anderson,
1995).
Interestingly, the short and long isoforms of c-ret differ in their abilities to support development of the enteric nervous system (de Graaff
et al., 2001). By targeted exon deletion, mice have been generated that
only express the short or long isoform. Animals that express RET-51,
but not RET-9, lack enteric neurons in the colon, while animals that
only express RET-9 appear completely normal. During development,
enteric neural precursors expressing only RET-51 never populate the
distal gut. This is not simply because they migrate more slowly than
normal precursors. The same deficit is observed in organ cultures of
the embryonic gut in which the overall growth and elongation of the
gut is quite limited. Consequently, the observations suggest that there
are differences in the signaling required to permit invasion of the proximal and distal segments of the gut.
Absence of signaling by GDNF family members also results in
deficits in additional neuronal populations, including sensory, motor,
and autonomic populations (Moore et al., 1996; Sanchez et al., 1996;
Heuckeroth et al., 1999; Nishino et al., 1999; Rossi et al., 1999, 2000;
Enomoto et al., 2000, 2001; Laurikainen et al., 2000; Hashino et al.,
2001). Analyses of targeted mutants have demonstrated survival requirements for c-ret, two of the GDNF family members, and three of
the GFR subunits. GDNF family members can be transported retrogradely from targets (Leitner et al., 1999) and can function as targetderived trophic factors, similar to nerve growth factor (Oppenheim et
al., 1995; Hashino et al., 2001).
In addition, c-ret signaling has been shown to be required for migration of neural precursors of sympathetic neurons (Enomoto et al.,
2001). As a result, the superior cervical ganglion is misplaced in both
the GFR3 and c-ret mutants (Nishino et al., 1999; Enomoto et al.,
2001). Deficits in neuronal number and delays in neuronal differentiation are seen throughout the sympathetic chain in the c-ret mutant
(Enomoto et al., 2001). In addition, sympathetic axons are misrouted,
almost certainly because the axons fail to respond to artemin. Artemin
is expressed in the vasculature that provides an initial guidance cue
for these axons and has been shown to be chemotropic for these axons in vitro. In both wild-type and mutant embryos, almost all cell
death in sympathetic ganglia occurs after down-regulation of c-ret expression. In the mutant, there is a similar elevation in cell death in
both c-ret-expressing and non-expressing neurons. As a result, the elevated cell death observed in the mutant has been attributed to failure of neurons to contact sources of other trophic factors. It does not
appear to be caused by direct dependence on artemin or other members of the GDNF family.
Both GDNF and neurturin are required for normal development of
several parasympathetic ganglia, with GDNF required early and neurturin later in development (Enomoto et al., 2000; Rossi et al., 2000;
Hashino et al., 2001). The sequential requirement for GDNF and neurturin correlates with changes in their expression in target tissues and
with changes in expression of the GFR1 and GFR2 receptors in the
neurons that innervate these tissues (Rossi et al., 2000; Hashino et al.,
2001).
In addition to these striking effects on development of sympathetic
and parasympathetic neurons, GDNF mutants have been shown to
have deficits in both neural crest–derived and placode-derived sensory ganglia (Moore et al., 1996; Sanchez et al., 1996). It is not certain whether these deficits reflect a requirement for trophic factor support by either neural precursors or mature neurons, or both.
GDNF also regulates the survival of motor and autonomic neurons
in the spinal cord. In the GDNF mutant, deficits were seen in some,
but not all, cranial motor nuclei and in motor neurons in the spinal
cord (Moore et al., 1996). Because there are distinct profiles of GFR
subunit expression in various motor neuron populations, it has seemed
attractive to imagine that different muscles express different GDNF
family members that provide trophic support for specific populations
of motor neurons (Mikaels et al., 2000). Absence of GDNF or GFR1
has been shown to result in selective loss of defined subpopulations
of motor neurons that express GFR1 and c-ret (Garces et al., 2000;
Oppenheim et al., 2000). Interestingly, no deficits were observed in
GFR2 mutants (Garces et al., 2000). In zebrafish, GFR1 is expressed in a specific primary motor neuron, CaP, while GDNF is expressed specifically in the muscle that it innervates (Shepherd et al.,
2001). Ectopic expression of GDNF results in perturbations in growth
by the CaP axon. Depletion of GDNF with morpholino antisense oligos did not, though, result in death or abnormal axon growth by the
CaP neuron. GDNF also has been shown to prevent the death of preganglionic neurons in the intermediolateral column of the spinal cord
after the removal of their target, the adrenal gland (Schober et al.,
1999). Since GDNF is expressed in adrenal chromaffin cells, it is almost certainly functioning as a target-derived trophic factor.
GDNF-dependent survival of chick motor neurons depends on signaling through the PI-3 kinase–Akt pathway, similar to survival of
other neurons promoted by neurotrophins, such as nerve growth factor (NGF) or brain derived growth factor (BDNF) (Soler et al., 1999;
Huang and Reichardt, 2001). There are differences in the essential survival pathways activated by GDNF and neurotrophins, however. In
contrast to BDNF, GDNF has been shown to elevate levels of X-linked
inhibitor of apoptosis (XIAP) and neuronal inhibitor of apoptosis
(NAIP) proteins in motor neurons after sciatic nerve axotomy (Perrelet et al., 2002). Inhibition of NAIP or XIAP activity prevents
GDNF-, but not BDNF-mediated neuroprotective effects.
The GDNF family of ligands has numerous additional effects that
Signaling Pathways of Glial Cell–Derived Neurotrophic Factor
are of functional importance in the mature nervous system. They have
been shown to protect neuronal subpopulations from excitotoxicity
(Pérez-Navarro et al., 1999; Bonde et al., 2000; Gratacos et al., 2001;
Marco et al., 2002). As described, they are also neuroprotective after
axotomy (Perrelet et al., 2002). In cell culture they have been shown
to promote synapse formation (Wang et al., 2002). In vivo, overexpression of GDNF in muscle increases the number of fibers innervated
by single motor neurons and promotes constant remodeling of neuromuscular junctions (Keller-Peck et al., 2001).
Similar to neurotrophins, GDNF and its relatives have been shown
to regulate expression of a variety of ion channels and receptors (Bradbury et al., 1998; Fjell et al., 1999; Brene et al., 2000; Cummins et
al., 2000; Boettger et al., 2002). In some instances, GDNF appears to
regulate the same channels as neurotrophins, but there are also numerous examples of differential regulation. Of potential clinical importance, GDNF has been shown to be analgesic in a model of neuropathic pain and is believed to act by regulating expression of Na
channels in nociceptive sensory neurons (Boucher et al., 2000). In addition to chronic effects, which are mediated largely through gene expression, GDNF has been shown to acutely regulate the activity of
channels in subpopulations of neurons. For example, GDNF has been
shown to potentiate the excitability of cultured midbrain dopaminergic neurons through reversible inhibition of transient A-type K channels (Yang et al., 2001). GDNF activates the MAP kinase cascade in
these neurons, and MAP kinase has been shown to directly phosphorylate these channels. Inhibition of the MAP kinase cascade prevents
the effect of GDNF, so it is likely that phosphorylation by MAP kinase directly regulates the activity of these channels. Similar to BDNF,
GDNF has been shown to enhance Ca2 entry and potentiate release
of acetylcholine in Xenopus spinal cord–skeletal muscle co-cultures
(Wang et al., 2001). Long-term exposure to GDNF increases expression of a Ca2-binding protein, frequenin. Interestingly, inhibition of
frequenin expression with anti-sense oligos or of its function with a
specific antibody prevents the effects of GDNF on Ca2 entry. Overexpression of frequenin results in a similar phenotype to that observed
following GDNF application, and the response to each occludes the
response to the other. Both GDNF and frequenin have been shown to
increase the opening probability of N-type, but not L-type, Ca2 channels. Thus GDNF acts through frequenin to regulate Ca2 channel
function by a mechanism that is not yet understood.
Signaling mediated by GDNF family members also is important for
development outside of the nervous system. In particular, GDNF signaling through GFR1 and c-ret is essential for normal development
of the metanephric kidney where GDNF is expressed at high levels in
the metanephric mesenchyme while GFR1 and c-ret are expressed
in the ureteric bud (Pachnis et al., 1993; Hellmich et al., 1996; Kreidberg, 1996; Moore et al., 1996). In the absence of any of these three
proteins, the ureteric bud fails to invade and branch within the
metanephric mesenchyme, so mature kidneys are absent from the vast
majority of animals (Schuchardt et al., 1994, 1996; Moore et al., 1996;
Pichel et al., 1996; Sanchez et al., 1996; Cacalano et al., 1998;
Enomoto et al., 1998; Tomac et al., 2000). Even mice with only one
copy of the GDNF gene have frequent unilateral kidney agenesis, and
the average size of their kidneys is reduced by 25% with fewer
nephrons and a reduced ureteric duct volume (Moore et al., 1996;
Cullen-McEwen et al., 2001).
Intriguingly, despite the similarities in inductive interactions in formation of the pro, meso, and metanephric kidneys, in mice
GDNF/GFR1/c-ret signaling is essential only for development of the
metanephros. Consistent with this, no deficit in pronephric kidney development is observed following GDNF depletion in zebrafish using
anti-sense morpholino-oligonucleotides (Shepherd et al., 2001). In
contrast, GDNF signaling through GFR1 has been shown to be essential for normal migration of pronephric duct cell precursors in axolotl (Drawbridge et al., 2000). The observations in axolotl suggest
that GDNF may contribute to pronephric development in other species,
but that other factors may compensate when it is absent or present at
abnormally low concentrations.
In rodents, GDNF signaling appears to regulate initial formation of
the ureteric bud because application of GDNF is capable of inducing
415
bud formation from other regions of the wolffian duct (Sainio et al.,
1997). GDNF signaling through GFR1 and c-ret, however, is important throughout kidney development, not just during initial invasion of the ureteric bud. In organ culture, interference with GDNF signaling inhibits branching of the ureteric bud, while application of
exogenous GDNF strongly promotes ureteric bud growth and branching (Vega et al., 1996; Pepicelli et al., 1997; Ehrenfels et al., 1999).
Interestingly, the short and long isoforms of c-ret also differ in their
abilities to support kidney development (de Graaff et al., 2001). In the
presence of only RET-51 the ureteric bud initiates invasion of the
metanephric mesenchyme, but later steps in kidney development do
not occur normally. In contrast, expression of RET-9 alone supports
all steps in kidney development. In the c-ret mutant background, forced
expression of ligand-dependent or constitutively active RET-9, but not
RET-51, rescues kidney development. Thus there must be differences
in the essential c-ret-mediated signaling pathways at various stages of
kidney development.
GDNF acts in part by stimulating and directing cell motility. GDNF
has been shown to be chemotactic, attracting ureteric buds to localized sources of GDNF (Sainio et al., 1997). GDNF also stimulates the
motility of kidney-derived epithelial cells (Tang et al., 1998). Some
groups have observed that GDNF increases cell proliferation in the
tips of the branches of the ureteric bud (Pepicelli et al., 1997). Activation of the PI-3 kinase pathway is required for the responses of both
kidney-derived epithelial cells and ureteric buds to GDNF (Tang et
al., 2002).
Many of the growth factors and transcription factors that regulate
kidney development act, at least partly, through regulation of GDNF
or c-ret expression. Once the primary ureteric bud invades the
metanephric mesenchyme, expression of c-ret is focused in the tips of
the branches of the ureteric bud, while GDNF expression is induced
in the mesenchyme surrounding these tips. Ectopic expression of cret throughout the ureteric bud–derived epithelia, for example, alters
the normal pattern of kidney morphogenesis, possibly because inappropriately localized c-ret competes with bud-tip-localized c-ret for
GDNF (Srinivas et al., 1999). Expression of GDNF or c-ret (or both)
is strongly reduced in several mutants that prevent normal kidney development, such as Emx2 (Miyamoto et al., 1997), Eya1 (Xu et al.,
1999), Pax2 (Brophy et al., 2001), double mutants of Hoxa11 and
Hoxd11 (Patterson et al., 2001), and a double mutant of two retinoic
acid receptors Rara and Rarb2 (Batourina et al., 2001). In the latter
instance, c-ret expression in the ureteric bud has been shown to depend indirectly on vitamin A–mediated signaling through receptors
localized in the metanephric stromal cells (Lelievre-Pegorier et al.,
1998; Moreau et al., 1998; Mendelsohn et al., 1999; Batourina et al.,
2001). Forced Hoxb7 promoter-driven expression of c-ret in the kidney anlage rescues kidney development in the absence of normal vitaminA–mediated signaling. Therefore, the reduction in c-ret expression clearly explains the deficits observed in vitamin A–deficient
animals and in animals that lack the two retinoic acid receptors. In
contrast, the domain of expression of GDNF in the intermediate mesenchyme is expanded anteriorly in double Foxc1 and Foxc2 mutants,
resulting in frequent presence of duplex kidneys and double ureters
(Kume et al., 2000). Expression of several genes believed to be important in kidney development, including Wnt-11, Wnt-4, and Ld (limb
deformity), are down-regulated when GDNF to c-ret signaling in the
kidney is inhibited through use of a Ret–Ig fusion protein (Pepicelli
et al., 1997; Ehrenfels et al., 1999). By combined use of expression
analysis and genetics, signaling pathways upstream and downstream
of GDNF signaling are gradually being assembled.
Signaling mediated by GDNF family members is also essential for
normal differentiation of other tissues. For example, GDNF and its receptors are expressed in the anlage of teeth, and GDNF has recently
been shown to be essential for their normal development (de Vicente
et al., 2002). In its absence, ameloblasts and odontoblasts fail to develop fully, resulting in an absence of the enamel matrix and predentin
layers. Expression of GDNF, neurturin, and their receptors in skin
changes in correlation with different phases of hair follicle growth and
regression (Botchkareva et al., 2000). Hair follicle regression is delayed by exogenous GDNF or neurturin and is accelerated in
416
DEFINED PATHWAYS
GFR1/ and GFR2/ mice (Botchkareva et al., 2000), indicating that these factors control the phase shift from growth to regression. As a final example of special interest, GDNF is expressed in Sertoli’s cells in the testes, where it is induced by follicle-stimulating
hormone (Meng et al., 2000; Tadokoro et al., 2002). GDNF promotes
proliferation of undifferentiated spermatogonia and decreases their
sensitivity to differentiation signals. As a result, the testes of mice
with one copy of a GDNF null allele are depleted in stem cell reserves,
while the testes of males in which GDNF is overexpressed accumulate undifferentiated spermatogonia (Meng et al., 2000). In older mice,
this results in development of malignant tumors expressing germ cell
markers (Meng et al., 2001).
GDNF SIGNALING PATHWAYS AND HUMAN DISEASE
The human genetics of the GDNF signaling pathway has provided interesting insights of general interest into the anatomy of receptor tyrosine kinase signaling. This has been possible because abnormalities
in this signaling pathway are associated with Hirschsprung’s disease,
which is caused by deficiencies in signaling through this pathway, and
several forms of cancer, which are associated with excessive activation of this pathway (reviewed in Hansford and Mulligan, 2000; Jhiang, 2000; Manie et al., 2001; Takahashi, 2001; Gabriel et al., 2002;
McCabe, 2002; Passarge, 2002). Hirschsprung’s disease is a human
disorder caused by failure of enteric neurons to populate the caudal
intestine. Long-segment and short-segment Hirschsprung’s disease are
distinguished by the length of intestine not populated by enteric neurons (Gabriel et al., 2002). Four classes of mutations in c-ret have
been identified that are associated with Hirschsprung’s disease. Each
reduces or compromises signaling through this receptor tyrosine kinase. One class of mutations alters important amino acid residues in
the extracellular domain of c-ret that reduce c-ret maturation and transport to the surface membrane (Iwashita et al., 1996; Ito et al., 1997;
Takahashi et al., 1999; Manie et al., 2001). Molecular modeling of the
cadherin domains indicates that each of the amino acids mutated in
this class of Hirschsprung’s disease is important for stabilizing the
structure of a domain, so these mutations almost certainly reduce maturation and surface expression through effects on protein folding (Anders et al., 2001). Thus there is comparatively little c-ret available to
be activated by GDNF family members.
Not surprisingly, the severity of Hirschsprung’s disease correlates
inversely with the amount of mutant ret transport to the surface with
the transport of mutants, resulting in long-segment disease more severely impaired than the transport of mutants and giving rise to shortsegment disease (Iwashita et al., 1996). A second class of mutants is
located in the kinase domain of c-ret; they reduce or abolish c-ret’s
tyrosine kinase activity (reviewed in Takahashi, 2001). These mutations occur in conserved amino acids shared with other tyrosine kinases. Finally, two classes of mutations affect signal transduction initiated by c-ret. Several have been shown to inhibit the activation of
PLC1. Others affect amino acid residues close to the crucial Y-1062
site and have been shown to inhibit Shc, FRS2, and IRS1 binding to
this site and to impair activation of the PI-3 kinase–Akt and Ras–Erk
pathways (Geneste et al., 1999; Ishiguro et al., 1999; Melillo et al.,
2001a, 2001b). It seems likely that they also affect binding by some
of the other adaptor proteins known to recognize this site, such as
Dok-4 and Dok-5. Thus, analysis of a genetically inherited human disease has resulted in dissection of the essential domains of a receptor
tyrosine kinase.
Finally, recent work indicates that overexpression of c-ret induces
apoptosis in some cells, which is suppressed by the presence of GDNF
(Bordeaux et al., 2000). Overexpression of several of the mutants associated with Hirschsprung’s disease also promotes apoptosis, but the
apoptosis induced by these mutants cannot be suppressed by GDNF.
C-ret-promoted apoptosis appears to involve cleavage of the cytoplasmic domain by a caspase-dependent mechanism. The molecular
details of this signaling pathway are not yet understood.
Mutations in other components of the GDNF signaling pathway
may also contribute to appearance of a Hirschsprung’s disease phenotype (Gabriel et al., 2002). Several mutations in GDNF and neur-
turin have been identified in patients with Hirschsprung’s disease, and
some of the GDNF mutants have been shown to result in expression
of a GDNF with reduced binding affinity for GFR1 (Doray et al.,
1998; Eketjall and Ibanez, 2002). While none of these alone seems
likely to cause Hirschsprung’s disease, they may contribute to pathogenesis in collaboration with other genetic alterations. It seems possible that contributions to the Hirschsprung’s disease phenotype could
also be made by alleles of genes encoding proteins in the c-ret signaling pathway.
Ret was first identified as an oncogene present in a human lymphoma that was the product of recombination between two unlinked
DNA segments, generating a fusion protein including a tyrosine kinase (Takahashi et al., 1985; Takahashi and Cooper, 1987). Since this
initial discovery, mutations or fusions of c-ret have been associated
with several different types of cancer, including papillary thyroid carcinomas (PTC), multiple endocrine neoplasia type 2 (MEN2),
pheochromocytoma, and parathyroid hyperplasia (Jhiang, 2000).
Analysis of oncogenic forms of c-ret has identified several classes of
mechanisms that render the activity of its tyrosine kinase partially or
completely independent of the ligand. In the first, the cytoplasmic domain of c-ret is fused to the product of another gene, resulting in formation of a constitutively dimerized fusion protein (Takahashi et al.,
1985). Eight different genes have been identified that are rearranged
to form fusions with ret in PTCs (Jhiang, 2000). Approximately 5%
to 30% of spontaneous papillary thyroid carcinomas are associated
with gene fusions of this type. As discussed previously, proliferative
and oncogenic responses by one of these fusion proteins have been
shown to depend on interaction of the soluble fusion protein with the
LIM and PDZ-domain-containing protein ENIGMA that relocalizes
ret from the cytoplasm to the membrane (Durick et al., 1996, 1998).
It seems likely that other fusion proteins of this type also require
ENIGMA to promote oncogenesis efficiently.
A second type of mutation generates an unpaired cysteine in the extracellular domain of c-ret that results in the constitutive dimerization
and activation of membrane-associated ret (Santoro et al., 1995). These
are dominant oncogenic mutations that result in MEN2 class A disease. In at least some cases, full activation of the mutant receptor requires the presence of a GDNF ligand, indicating that dimerization
alone does not create an optimal conformation for activation of the ret
tyrosine kinase activity (Mograbi et al., 2001).
In the third class of mutation, an alteration in the c-ret kinase domain (M918T) increases the basal kinase activity of c-ret and alters
its substrate preferences without promoting constitutive dimerization
and results in MEN2 class B disease (Santoro et al., 1995; Liu et al.,
1996). Ret (M918T) can be further activated by GDNF family ligands, so the pattern of expression of these ligands may contribute to
the neoplastic phenotype. Not unexpectedly, many of the tyrosines
phosphorylated in c-ret in response to GDNF application are also phosphorylated in these oncogenic ret proteins, including notably Y-1062
(Liu et al., 1996). Phosphorylation of Y-1096 is not observed in the
MEN2B mutant (M918T), but there is increased phosphorylation of
Y-1062, and a new phosphorylation site is created (Liu et al., 1996;
Salvatore et al., 2001). Thus, differences in tyrosine kinase activity
and substrate specificity are both thought to contribute to differences
in the cancers that are associated with different ret mutations. Many
studies have shown that adapters such as Shc, Grb-2, FRS2, IRS1, and
PLC-1 are recruited by oncogenic mutants of Ret protein, thereby
activating the Ras-Map kinase, PI-3 kinase–Akt, PLC, and other signaling pathways within affected cells (Hansford and Mulligan, 2000;
Jhiang, 2000; Takahashi, 2001).
CONCLUSION
GDNF and its relatives have proven to have unusually interesting signaling pathways that are important in normal development of the nervous system and other organs. Because of its involvement in both development and oncogenesis, studies have provided a model of how
human genetics can contribute to an understanding of receptor function, cell biology, and disease. Characterization of mouse mutants
lacking these proteins or their receptors have illuminated the diversity
Signaling Pathways of Glial Cell–Derived Neurotrophic Factor
of mechanisms through which trophic factors regulate cell behavior
and development, both inside and outside of the nervous system. These
proteins have also been shown to regulate the electrical properties of
neurons through control of transmitter receptors and ion channels. Because they protect neurons against a variety of toxic insults, they are
strong candidates to be developed as therapeutic agents to treat Parkinson’s and other diseases. Uncontrolled activity of the common receptor c-ret contributes to development of several different cancers. The
signaling cascades characterized as part of the effort to understand the
oncogenic activity of ret have proven to be of general interest and, in
the future, may provide targets for therapeutic treatments of many cancers in addition to those involving c-ret and the GDNF signaling pathway.
ACKNOWLEDGMENTS
I wish to thank Drs. Ursula Funfschilling, Beatriz Rico, and Sam Pleasure for
critical review of the manuscript. I also thank Ms. Sonia Brown for design of
Figure 35–1. Work in my laboratory has been supported by grants from the
National Institutes of Health and Howard Hughes Medical Institute. I am an
investigator in the Howard Hughes Medical Institute.
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