Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.     

Regulation of c-Ret,GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6-hydroxydopamine (6-OHDA) transiently increased c-Ret and GFRalpha1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c-Ret and GFRalpha1 was downregulated. GFRalpha2 expression was differentially regulated, as it decreased only 6 days after 6-OHDA injection. Triple-labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6-OHDA lesion.     

Target-derived GFRalpha1 as an attractive guidance signal for developing sensory and sympathetic axons via activation of Cdk5

Immobilized and diffusible molecular cues regulate axon guidance during development. GFRalpha1, a GPI-anchored receptor for GDNF, is expressed as both membrane bound and secreted forms by accessory nerve cells and peripheral targets of developing sensory and sympathetic neurons during the period of target innervation. A relative deficit of GFRalpha1 in developing axons allows exogenous GFRalpha1 to capture GDNF and present it for recognition by axonal c-Ret receptors. Exogenous GFRalpha1 potentiates neurite outgrowth and acts as a long-range directional cue by creating positional information for c-Ret-expressing axons in the presence of a uniform concentration of GDNF. Soluble GFRalpha1 prolongs GDNF-mediated activation of cyclin-dependent kinase 5 (Cdk5), an event required for GFRalpha1-induced neurite outgrowth and axon guidance. Together with GDNF, target-derived GFRalpha1 can function in a non-cell-autonomous fashion as a chemoattractant cue with outgrowth promoting activity for peripheral neurons.     

Determinants of ligand binding specificity in the glial cell line-derived neurotrophic factor family receptor alpha S

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 alpha (GFRalpha). All four GDNF family ligands utilize c-Ret as a common signaling receptor, whereas specificity is conferred by differential binding to four distinct GFRalpha homologues. To understand how the different GFRalphas 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 alpha helices and two beta strands. Distinct hydrophobic and positively charged residues in this central region were required for binding of GFRalpha1 to GDNF. Interaction of GFRalpha1 and GFRalpha2 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 GFRalpha1, 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.     

Insights into GFRalpha1 regulation of neural cell adhesion molecule (NCAM) function from structure-function analysis of the NCAM/GFRalpha1 receptor complex

The neural cell adhesion molecule NCAM binds glial cell line-derived neurotrophic factor (GDNF) through specific determinants located in its third immunoglobulin (Ig) domain. However, high affinity GDNF binding and downstream signaling depend upon NCAM co-expression with the GDNF co-receptor GFRalpha1. GFRalpha1 promotes high affinity GDNF binding to NCAM and down-regulates NCAM-mediated homophilic cell adhesion, but the mechanisms underlying these effects are unknown. NCAM and GFRalpha1 interact at the plasma membrane, but the molecular determinants involved have not been characterized nor is it clear whether their interaction is required for GFRalpha1 regulation of NCAM function. We have investigated the structure-function relationships underlying GFRalpha1 binding to NCAM in intact cells. The fourth Ig domain of NCAM was both necessary and sufficient for the interaction of NCAM with GFRalpha1. Moreover, although the N-terminal domain of GFRalpha1 had previously been shown to be dispensable for GDNF binding, we found that it was both necessary and sufficient for the efficient interaction of this receptor with NCAM. GFRalpha1 lacking its N-terminal domain was still able to potentiate GDNF binding to NCAM and assemble into a tripartite receptor complex but showed a reduced capacity to attenuate NCAM-mediated cell adhesion. On its own, the GFRalpha1 N-terminal domain was sufficient to decrease NCAM-mediated cell adhesion. These results indicate that direct receptor-receptor interactions are not required for high affinity GDNF binding to NCAM but play an important role in the regulation of NCAM-mediated cell adhesion by GFRalpha1.     

GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.     

Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFRalpha1 RET-specific agonists

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFRalpha1-GFRalpha4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRalpha1.RET or GFRalpha2.RET) identified sites along the second finger of GDNF critical for activating the GFRalpha1.RET and GFRalpha2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRalpha1. RET, but additional determinants are required for interaction with the other GFRalphas. This difference in the molecular basis of GFL-GFRalpha specificity allowed the production of GFRalpha1. RET-specific agonists and provides a foundation for understanding of GFL-GFRalpha.RET signaling at the molecular level.     

Direct interactions among Ret, GDNF and GFRalpha1 molecules reveal new insights into the assembly of a functional three-protein complex

The glial-cell-line-derived neurotrophic factor (GDNF) ligand activates the Ret receptor through the assembly of a multiprotein complex, including the GDNF family receptor alpha1 (GFRalpha1) molecule. Given the neuroprotective role of GDNF, there is an obvious need to precisely identify the structural regions engaged in direct interactions between the three molecules. Here, we combined a functional approach for Ret activity (in PC12 cells) to cross-linking experiments followed by MS-MALDI to study the interactions among the purified extracellular region of the human Ret, GDNF and GFRalpha1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFRalpha1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFRalpha1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested.     

Expression of GFRalpha1 receptor splicing variants with different biochemical properties is modulated during kidney development

The glial cell line-derived neurotrophic factor (GDNF) family coreceptor alpha1 (GFRalpha1) is a critical component of the RET receptor kinase signal-transducing complex. The activity of this multicomponent receptor is stimulated by the glial cell line-derived neurotrophic factor (GDNF) and is involved in neuronal cells survival and kidney development. GFRalpha1 pre-mRNA is alternatively spliced and produces two isoforms: GFRalpha1a, which includes the exon 5; and GFRalpha1b, which excludes it. Here we show that the Gfralpha1a isoform is predominantly expressed in neuronal tissues and in PC12 cells differentiated toward a neuronal phenotype. GFRalpha1 splicing is also regulated during kidney development, GFRalpha1a is the minor isoform before birth and then rapidly becomes the major form after birth. We established cell lines expressing either GFRalpha1 isoforms and demonstrated that the GFRalpha1b isoform binds GDNF more efficiently than GFRalpha1a. Consistently, GFRalpha1b promotes a stronger RET phosphorylation than GFRalpha1a. These results indicate that specific inclusion of the GFRalpha1 exon 5 in neuronal tissues or during kidney development may alter the binding properties of GDNF to GFRalpha1, and thus could constitute an additional regulatory mechanism of the RET signaling pathway.     

Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 involved in its biological function

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.     

Glial cell-line derived neurotrophic factor and neurturin regulate the expressions of distinct miRNA precursors through the activation of GFRalpha2

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are structurally related neurotrophic factors that have both been shown to prevent the degeneration of dopaminergic neurons in vitro and in vivo. NTN and GDNF are thought to bind with different affinities to the GDNF family receptor alpha-2 (GFRalpha2), and can activate the same multi-component receptor system consisting of GFRalpha2, receptor tyrosine kinase Ret (RET) and NCAM. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that regulate gene expression through translational repression or RNA degradation. miRNAs have diverse functions, including regulating differentiation, proliferation and apoptosis in several organisms. It is currently unknown whether GDNF and NTN regulate the expression of miRNAs through activation of the same multi-component receptor system. Using quantitative real-time PCR, we measured the expression of some miRNA precursors in human BE(2)-C cells that express GFRalpha2 but not GFRalpha1. GDNF and NTN differentially regulate the expression of distinct miRNA precursors through the activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2). This study showed that the expression of distinct miRNA precursors is differentially regulated by specific ligands through the activation of GFRalpha2.     

GFRalpha1 expression in cells lacking RET is dispensable for organogenesis and nerve regeneration

The GDNF family ligands signal through a receptor complex composed of a ligand binding subunit, GFRalpha, and a signaling subunit, the RET tyrosine kinase. GFRalphas are expressed not only in RET-expressing cells, but also in cells lacking RET. A body of evidence suggests that RET-independent GFRalphas are important for (1) modulation of RET signaling in a non-cell-autonomous fashion (trans-signaling) and (2) regulation of NCAM function. To address the physiological significance of these roles, we generated mice specifically lacking RET-independent GFRalpha1. These mice exhibited no deficits in regions where trans-signaling has been implicated in vitro, including enteric neurons, motor neurons, kidney, and regenerating nerves. Furthermore, no abnormalities were found in the olfactory bulb, which requires proper NCAM function for its formation and is putatively a site of GDNF-GFRalpha-NCAM signaling. Thus RET-independent GFRalpha1 is dispensable for organogenesis and nerve regeneration in vivo, indicating that trans-signaling and GFRalpha-dependent NCAM signaling play a minor role physiologically.     

The structure of GFRalpha1 domain 3 reveals new insights into GDNF binding and RET activation

Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.     

Crosstalk between Jagged1 and GDNF/Ret/GFRalpha1 signalling regulates ureteric budding and branching

Glial-Cell-Line-Derived Neurotrophic Factor (GDNF) is the major mesenchyme-derived regulator of ureteric budding and branching during nephrogenesis. The ligand activates on the ureteric bud epithelium a receptor complex composed of Ret and GFRalpha1. The upstream regulators of the GDNF receptors are poorly known. A Notch ligand, Jagged1 (Jag1), co-localises with GDNF and its receptors during early kidney morphogenesis. In this study we utilized both in vitro and in vivo models to study the possible regulatory relationship of Ret and Notch pathways. Urogenital blocks were exposed to exogenous GDNF, which promotes supernumerary ureteric budding from the Wolffian duct. GDNF-induced ectopic buds expressed Jag1, which suggests that GDNF can, directly or indirectly, up-regulate Jag1 through Ret/GFRalpha1 signalling. We then studied the role of Jag1 in nephrogenesis by transgenic mice constitutively expressing human Jag1 in Wolffian duct and its derivatives under HoxB7 promoter. Jag1 transgenic mice showed a spectrum of renal defects ranging from aplasia to hypoplasia. Ret and GFRalpha1 are normally downregulated in the Wolffian duct, but they were persistently expressed in the entire transgenic duct. Simultaneously, GDNF expression remained unexpectedly low in the metanephric mesenchyme. In vitro, exogenous GDNF restored the budding and branching defects in transgenic urogenital blocks. Renal differentiation apparently failed because of perturbed stimulation of primary ureteric budding and subsequent branching. Thus, the data provide evidence for a novel crosstalk between Notch and Ret/GFRalpha1 signalling during early nephrogenesis.     

GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.     

Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin

To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.     

Mammalian GFRalpha -4, a divergent member of the GFRalpha family of coreceptors for glial cell line-derived neurotrophic factor family ligands, is a receptor for the neurotrophic factor persephin

Four members of the glial cell line-derived neurotrophic factor family have been identified (GDNF, neurturin, persephin, and enovin/artemin). They bind to a specific membrane-anchored GDNF family receptor as follows: GFRalpha-1 for GDNF, GFRalpha-2 for neurturin, GFRalpha-3 for enovin/artemin, and (chicken) GFRalpha-4 for persephin. Subsequent signaling occurs through activation of a common transmembrane tyrosine kinase, cRET. GFRalpha-4, the coreceptor for persephin, was previously identified in chicken only. We describe the cloning and characterization of a mammalian persephin receptor GFRalpha-4. The novel GFRalpha receptor is substantially different in sequence from all known GFRalphas, including chicken GFRalpha-4, and lacks the first cysteine-rich domain present in all previously characterized GFRalphas. At least two different GFRalpha-4 splice variants exist in rat tissues, differing at their respective COOH termini. GFRalpha-4 mRNA is expressed at low levels in different brain areas in the adult as well as in some peripheral tissues including testis and heart. Recombinant rat GFRalpha-4 variants were expressed in mammalian cells and shown to be at least partially secreted from the cells. Persephin binds specifically and with high affinity (K(D) = 6 nm) to the rat GFRalpha-4 receptor, but no cRET activation could be demonstrated. Although the newly characterized mammalian GFRalpha-4 receptor is structurally divergent from previously characterized GFRalpha family members, we suggest that it is a mammalian orthologue of the chicken persephin receptor. This discovery will allow a more detailed investigation of the biological targets of persephin action and its potential involvement in diseases of the nervous system.     

Roles for GFRalpha1 receptors in zebrafish enteric nervous system development

Components of the zebrafish GDNF receptor complex are expressed very early in the development of enteric nervous system precursors, and are already present as these cells begin to enter the gut and migrate caudally along its length. Both gfra1a and gfra1b as well as ret are expressed at this time, while gfra2 expression, the receptor component that binds the GDNF-related ligand neurturin, is not detected until the precursors have migrated along the gut. Gfra genes are also expressed in regions of the zebrafish brain and peripheral ganglia, expression domains conserved with other species. Enteric neurons are eliminated after injection with antisense morpholino oligonucleotides against ret or against both Gfra1 orthologs, but are not affected by antisense oligonucleotides against gfra2. Blocking GDNF signaling prevents migration of enteric neuron precursors, which remain positioned at the anterior end of the gut. Phenotypes induced by injection of antisense morpholinos against both Gfra orthologs can be rescued by introduction of mRNA for gfra1a or for gfra2, suggesting that GFRalpha1 and GFRalpha2 are functionally equivalent.     

The major determinant of the heparin binding of glial cell-line-derived neurotrophic factor is near the N-terminus and is dispensable for receptor binding

GDNF (glial cell-line-derived neurotrophic factor), and the closely related cytokines artemin and neurturin, bind strongly to heparin. Deletion of a basic amino-acid-rich sequence of 16 residues N-terminal to the first cysteine of the transforming growth factor beta domain of GDNF results in a marked reduction in heparin binding, whereas removal of a neighbouring sequence, and replacement of pairs of other basic residues with alanine had no effect. The heparin-binding sequence is quite distinct from the binding site for the high affinity GDNF polypeptide receptor, GFRalpha1 (GDNF family receptor alpha1), and heparin-bound GDNF is able to bind GFRalpha1 simultaneously. The heparin-binding sequence of GDNF is dispensable both for GFRalpha1 binding, and for activity for in vitro neurite outgrowth assay. Surprisingly, the observed inhibition of GDNF bioactivity with the wild-type protein in this assay was still found with the deletion mutant lacking the heparin-binding sequence. Heparin neither inhibits nor potentiates GDNF-GFRalpha1 interaction, and the extracellular domain of GFRalpha1 does not bind to heparin itself, precluding heparin cross-bridging of cytokine and receptor polypeptides. The role of heparin and heparan sulfate in GDNF signalling remains unclear, but the present study indicates that it does not occur in the first step of the pathway, namely GDNF-GFRalpha1 engagement.     

Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase

Proximal signaling events and protein-protein interactions initiated after activation of the c-Ret receptor tyrosine kinase by its ligand, glial cell line-derived neurotrophic factor (GDNF), were investigated in cells carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85(PI3K), the adaptor Gab2, and the protein-tyrosine phosphatase SHP-2. In agreement with Ras-independent activation of PI3K by GDNF in neuronal cells, survival of sympathetic neurons induced by GDNF was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85(PI3K) and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF.