The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Eugene M. Johnson - One of the best experts on this subject based on the ideXlab platform.
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c src is required for glial cell line derived neurotrophic factor gdnf family ligand mediated neuronal survival via a phosphatidylinositol 3 kinase pi 3k dependent pathway
The Journal of Neuroscience, 2001Co-Authors: Mario Encinas, Malú G. Tansey, Jeffrey Milbrandt, Joan X Comella, Brian A Tsuipierchala, Eugene M. JohnsonAbstract:The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, Persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.
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c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)dependent pathway
2001Co-Authors: Mario Encinas, Malú G. Tansey, Joan X Comella, Brian A. Tsui-pierchala, Jeffrey Milbr, Eugene M. JohnsonAbstract:The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, Persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)anchored coreceptors GFR�1–�4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects
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gfrα mediated localization of ret to lipid rafts is required for effective downstream signaling differentiation and neuronal survival
Neuron, 2000Co-Authors: Malú G. Tansey, Robert H. Baloh, Jeffrey Milbrandt, Eugene M. JohnsonAbstract:The GDNF family ligands (GFLs: GDNF, neurturin, Persephin, and artemin) signal through RET and a gly-cosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRalpha1-alpha4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRalpha1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRalpha1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.
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functional mapping of receptor specificity domains of glial cell line derived neurotrophic factor gdnf family ligands and production of gfrα1 ret specific agonists
Journal of Biological Chemistry, 2000Co-Authors: Robert H. Baloh, Malú G. Tansey, Eugene M. Johnson, Jeffrey MilbrandtAbstract:Abstract 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 α-component (GFRα1-GFRα4)) 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 GFRα1·RET or GFRα2·RET) identified sites along the second finger of GDNF critical for activating the GFRα1·RET and GFRα2·RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRα1·RET, but additional determinants are required for interaction with the other GFRαs. This difference in the molecular basis of GFL-GFRα specificity allowed the production of GFRα1·RET-specific agonists and provides a foundation for understanding of GFL-GFRα·RET signaling at the molecular level.
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Artemin, a Novel Member of the GDNF Ligand Family, Supports Peripheral and Central Neurons and Signals through the GFRα3–RET Receptor Complex
Neuron, 1998Co-Authors: Robert H. Baloh, Malú G. Tansey, Patricia A. Lampe, Timothy J. Fahrner, Hideki Enomoto, Kelli S. Simburger, Melanie L. Leitner, Toshiyuki Araki, Eugene M. Johnson, Jeffrey MilbrandtAbstract:Abstract The glial cell line–derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRα1–GFRα4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRα3–RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRα1–RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRα1–RET) and NTN (GFRα2–RET), Artemin has a preferred receptor (GFRα3–RET) but that alternative receptor interactions also occur.
Mart Saarma - One of the best experts on this subject based on the ideXlab platform.
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heparan sulfate proteoglycan syndecan 3 is a novel receptor for gdnf neurturin and artemin
Journal of Cell Biology, 2011Co-Authors: Maxim M Bespalov, Evgeny Kulesskiy, Sarka Tumova, Heikki Rauvala, Yulia Sidorova, Anni Ahonenbishopp, Ana Cathia Magalhaes, Mikhail Paveliev, Claudio Rivera, Mart SaarmaAbstract:Glial cell line–derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson’s disease. Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except Persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL–syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3–dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid–releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.
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ablation of Persephin receptor glial cell line derived neurotrophic factor family receptor α4 impairs thyroid calcitonin production in young mice
Endocrinology, 2006Co-Authors: Paivi H Lindfors, Mart Saarma, Maria Lindahl, Jari Rossi, Matti S AiraksinenAbstract:Glial cell line-derived neurotrophic factor family receptor (GFRalpha) 4, the binding receptor for Persephin, is coexpressed with the signaling Ret receptor tyrosine kinase predominantly in thyroid calcitonin-producing C cells. We show by in situ hybridization and immunohistochemistry that the functional, glycolipid-anchored form of GFRalpha4 is produced in mouse only in the C cells but not in parathyroid gland or in the brain. C cells expressed functional GFRalpha4 throughout postnatal development, whereas Ret expression in these cells decreased postnatally and was undetectable in adults. To understand the physiological role of GFRalpha4, we produced GFRalpha4-deficient [knockout (KO)] mice. No differences were observed between wild-type and GFRalpha4-KO littermate animals in growth, gross behavior, or viability. The number and morphology of the thyroid C cells were indistinguishable between the genotypes in both newborn and adult age. However, thyroid tissue calcitonin content was reduced by 60% in newborn and by 45% in 3-wk-old GFRalpha4-KO mice compared with wild-type controls. In contrast, thyroid calcitonin levels were similar in adult animals. Consistent with the reduced calcitonin levels, bone formation rate in juvenile GFRalpha4-KO mice was increased. In conclusion, this study indicates a novel role for endogenous GFRalpha4 signaling in regulating calcitonin production in thyroid C cells of young mice.
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human glial cell line derived neurotrophic factor receptor α4 is the receptor for Persephin and is predominantly expressed in normal and malignant thyroid medullary cells
Journal of Biological Chemistry, 2001Co-Authors: Maria Lindahl, Urmas Arumae, Matti S Airaksinen, Dmitry Poteryaev, Tonis Timmusk, Italia Bongarzone, Antonella Aiello, Marco A Pierotti, Mart SaarmaAbstract:Abstract Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) α subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRα1, neurturin via GFRα2, artemin via GFRα3, whereas the mammalian GFRα receptor for Persephin (PSPN) is unknown. Here we characterize the human GFRα4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRα4 lack the first Cys-rich domain characteristic of other GFRα receptors. Unlabeled PSPN displaces 125I-PSPN fromGFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRα4 and Ret, and is able to promote autophosphorylation of Ret inGFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not otherGFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRα4 may restrict the MEN2 syndrome to these cells.
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other neurotrophic factors glial cell line derived neurotrophic factor gdnf
Microscopy Research and Technique, 1999Co-Authors: Mart Saarma, Hannu SariolaAbstract:Glial cell line-derived neurotrophic factor (GDNF) was first discovered as a potent survival factor for midbrain dopaminergic neurons and was then shown to rescue these neurons in animal models of Parkinson's disease. GDNF is a more potent survival factor for dopaminergic neurons and the noradrenergic neurons of the locus coeruleus than other neurotrophic factors, and an almost 100 times more efficient survival factor for spinal motor neurons than the neurotrophins. The members of the GDNF family, GDNF, neurturin (NTN), Persephin (PSP), and artemin (ART), have seven conserved cysteine residues with similar spacing, making them distant members of the transforming growth factor-β (TGF-β) superfamily. Like the members of the neurotrophin family, the GDNF-like growth factors belong structurally to the cysteine knot proteins. Like neurotrophins, GDNF family proteins are responsible for the development and maintenance of various sets of sensory and sympathetic neurons but, in addition, GDNF and NTN are also responsible for the development and survival of the enteric neurons, and NTN for parasympathetic neurons. All neurotrophins bind to the p75 low-affinity receptor, but their ligand specificity is determined by trk receptor tyrosine kinases. GDNF, NTN, PSP, and ART mediate their signals via a common receptor tyrosine kinase, Ret, but their ligand specificity is determined by a novel class of glycosyl-phosphatidylinositol (GPI)-anchored proteins called the GDNF family receptor α (GFRα). GDNF binds preferentially to GFRα1, NTN GFRα2, ART GRFα3, and PSP GFRα4 as a co-receptor to activate Ret. GFRα4 has until now been described only from chicken. Although the GDNF family members signal mainly via Ret receptor tyrosine kinase, there is recent evidence that they can also mediate their signals via GFRα receptors independently of Ret. The GDNF family of growth factors, unlike neurotrophins, has a well-defined function outside the nervous system. Recent transgenic and organ culture experiments have clearly demonstrated that GDNF is a mesenchyme-derived signaling molecule for the promotion of ureteric branching in kidney development. NTN, ART, and PSP are also expressed in the developing kidney, and NTN and PSP induce ureteric branching in vitro, but their true in vivo role in kidney morphogenesis is still unclear. Microsc. Res. Tech. 45:292–302, 1999. © 1999 Wiley-Liss, Inc.
Norio Kaneda - One of the best experts on this subject based on the ideXlab platform.
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establishment and characterization of a noradrenergic adrenal chromaffin cell line tsam5ne immortalized with the temperature sensitive sv40 t antigen
Cell Biology International, 2011Co-Authors: Susumu Kohno, Tomiyasu Murata, Kiyomi Hikita, Naoshi Koide, Norio KanedaAbstract:We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or Persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.
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neuronal differentiation elicited by glial cell line derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsam5d immortalized with temperature sensitive sv40 t antigen
Journal of Neuroscience Research, 2008Co-Authors: Tomiyasu Murata, Masaru Tsuboi, Kiyomi Hikita, Susumu Kohno, Naoshi Koide, Norio KanedaAbstract:To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not Persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation.
Jeffrey Milbrandt - One of the best experts on this subject based on the ideXlab platform.
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c src is required for glial cell line derived neurotrophic factor gdnf family ligand mediated neuronal survival via a phosphatidylinositol 3 kinase pi 3k dependent pathway
The Journal of Neuroscience, 2001Co-Authors: Mario Encinas, Malú G. Tansey, Jeffrey Milbrandt, Joan X Comella, Brian A Tsuipierchala, Eugene M. JohnsonAbstract:The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, Persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.
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gfrα mediated localization of ret to lipid rafts is required for effective downstream signaling differentiation and neuronal survival
Neuron, 2000Co-Authors: Malú G. Tansey, Robert H. Baloh, Jeffrey Milbrandt, Eugene M. JohnsonAbstract:The GDNF family ligands (GFLs: GDNF, neurturin, Persephin, and artemin) signal through RET and a gly-cosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRalpha1-alpha4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRalpha1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRalpha1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.
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functional mapping of receptor specificity domains of glial cell line derived neurotrophic factor gdnf family ligands and production of gfrα1 ret specific agonists
Journal of Biological Chemistry, 2000Co-Authors: Robert H. Baloh, Malú G. Tansey, Eugene M. Johnson, Jeffrey MilbrandtAbstract:Abstract 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 α-component (GFRα1-GFRα4)) 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 GFRα1·RET or GFRα2·RET) identified sites along the second finger of GDNF critical for activating the GFRα1·RET and GFRα2·RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRα1·RET, but additional determinants are required for interaction with the other GFRαs. This difference in the molecular basis of GFL-GFRα specificity allowed the production of GFRα1·RET-specific agonists and provides a foundation for understanding of GFL-GFRα·RET signaling at the molecular level.
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Artemin, a Novel Member of the GDNF Ligand Family, Supports Peripheral and Central Neurons and Signals through the GFRα3–RET Receptor Complex
Neuron, 1998Co-Authors: Robert H. Baloh, Malú G. Tansey, Patricia A. Lampe, Timothy J. Fahrner, Hideki Enomoto, Kelli S. Simburger, Melanie L. Leitner, Toshiyuki Araki, Eugene M. Johnson, Jeffrey MilbrandtAbstract:Abstract The glial cell line–derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRα1–GFRα4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRα3–RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRα1–RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRα1–RET) and NTN (GFRα2–RET), Artemin has a preferred receptor (GFRα3–RET) but that alternative receptor interactions also occur.
Malú G. Tansey - One of the best experts on this subject based on the ideXlab platform.
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c src is required for glial cell line derived neurotrophic factor gdnf family ligand mediated neuronal survival via a phosphatidylinositol 3 kinase pi 3k dependent pathway
The Journal of Neuroscience, 2001Co-Authors: Mario Encinas, Malú G. Tansey, Jeffrey Milbrandt, Joan X Comella, Brian A Tsuipierchala, Eugene M. JohnsonAbstract:The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, Persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.
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c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)dependent pathway
2001Co-Authors: Mario Encinas, Malú G. Tansey, Joan X Comella, Brian A. Tsui-pierchala, Jeffrey Milbr, Eugene M. JohnsonAbstract:The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, Persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)anchored coreceptors GFR�1–�4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects
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gfrα mediated localization of ret to lipid rafts is required for effective downstream signaling differentiation and neuronal survival
Neuron, 2000Co-Authors: Malú G. Tansey, Robert H. Baloh, Jeffrey Milbrandt, Eugene M. JohnsonAbstract:The GDNF family ligands (GFLs: GDNF, neurturin, Persephin, and artemin) signal through RET and a gly-cosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRalpha1-alpha4) that binds ligand with high affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRalpha1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRalpha1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.
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functional mapping of receptor specificity domains of glial cell line derived neurotrophic factor gdnf family ligands and production of gfrα1 ret specific agonists
Journal of Biological Chemistry, 2000Co-Authors: Robert H. Baloh, Malú G. Tansey, Eugene M. Johnson, Jeffrey MilbrandtAbstract:Abstract 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 α-component (GFRα1-GFRα4)) 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 GFRα1·RET or GFRα2·RET) identified sites along the second finger of GDNF critical for activating the GFRα1·RET and GFRα2·RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRα1·RET, but additional determinants are required for interaction with the other GFRαs. This difference in the molecular basis of GFL-GFRα specificity allowed the production of GFRα1·RET-specific agonists and provides a foundation for understanding of GFL-GFRα·RET signaling at the molecular level.
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Artemin, a Novel Member of the GDNF Ligand Family, Supports Peripheral and Central Neurons and Signals through the GFRα3–RET Receptor Complex
Neuron, 1998Co-Authors: Robert H. Baloh, Malú G. Tansey, Patricia A. Lampe, Timothy J. Fahrner, Hideki Enomoto, Kelli S. Simburger, Melanie L. Leitner, Toshiyuki Araki, Eugene M. Johnson, Jeffrey MilbrandtAbstract:Abstract The glial cell line–derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin [NTN], and Persephin [PSP]) signal through a multicomponent receptor system composed of a high-affinity binding component (GFRα1–GFRα4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFRα3–RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFRα1–RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRα1–RET) and NTN (GFRα2–RET), Artemin has a preferred receptor (GFRα3–RET) but that alternative receptor interactions also occur.