Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.     

The role of glial cell line‐derived neurotrophic factor family member artemin in neurological disorders and cancers

Artemin (ARTN) is a member of the glial cell line‐derived neurotrophic factor (GDNF) family ligands (GFLs), which encompasses family members, GDNF, neurturin (NRTN) and persephin (PSPN). ARTN is also referred to as Enovin or Neublastin, and bears structural characteristics of the TGF‐β superfamily. ARTN contains a dibasic cleavage site (RXXR) that is predicted to be cleaved by furin to yield a carboxy‐terminal 113 amino acid mature form. ARTN binds preferentially to receptor GFRα3, coupled to a receptor tyrosine kinase RET, forming a signalling complex for the regulation of intracellular pathways that affect diverse outcomes of nervous system development and homoeostasis. Standard signalling cascades activated by GFLs via RET include the phosphorylation of mitogen‐activated protein kinase or MAPK (p‐ERK, p‐p38 and p‐JNK), PI3K‐AKT and Src. Neural cell adhesion molecule (NCAM) is an alternative signalling receptor for ARTN in the presence of GFRα1, leading to activation of Fyn and FAK. Further, ARTN also interacts with heparan sulphate proteoglycan syndecan‐3 and mediates non‐RET signalling via activation of Src kinases. This review discusses the role of ARTN in spinal cord injury, neuropathic pain and other neurological disorders. Additionally, ARTN plays a role in non‐neuron tissues, such as the formation of Peyer's patch‐like structures in the lymphoid tissue of the gut. The emerging role of ARTN in cancers and therapeutic resistance to cancers is also explored. Further research is necessary to determine the function of ARTN in a tissue‐specific manner, including its signalling mechanisms, in order to improve the therapeutic potential of ARTN in human diseases.     

Beyond the cell surface: New mechanisms of receptor function

The text book view of cell surface receptors depicts them at the top of a vertical chain of command that starts with ligand binding and proceeds in a lineal fashion towards the cell nucleus. Although pedagogically useful, this view is incomplete and recent findings suggest that the extracellular domain of cell surface receptors can be a transmitter as much as a receiver in intercellular communication. GFRα1 is a GPI-anchored receptor for GDNF (glial cell line-derived neurotrophic factor), a neuronal growth factor with widespread functions in the developing and adult nervous system. GFRα1 partners with transmembrane proteins, such as the receptor tyrosine kinase RET or the cell adhesion molecule NCAM, for intracellular transmission of the GDNF signal. In addition to this canonical role, GFRα1 can also engage in horizontal interactions and thereby modify the function of other cell surface components. GFRα1 can also function as a ligand-induced adhesion cell molecule, mediating homophilic cell-cell interactions in response to GDNF. Finally, GFRα1 can also be released from the cell surface and act at a distance as a soluble factor together with its ligand. This plethora of unconventional mechanisms is likely to be a feature common to several other receptors and considerably expands our view of cell surface receptor function.     

Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

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.     

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.     

Platelet-derived growth factors and their receptors: Structural and functional perspectives

The four types of platelet-derived growth factors (PDGFs) and the two types of PDGF receptors (PDGFRs, which belong to class III receptor tyrosine kinases) have important functions in the development of connective tissue cells. Recent structural studies have revealed novel mechanisms of PDGFs in propeptide loading and receptor recognition/activation. The detailed structural understanding of PDGF–PDGFR signaling has provided a template that can aid therapeutic intervention to counteract the aberrant signaling of this normally silent pathway, especially in proliferative diseases such as cancer. This review summarizes the advances in the PDGF system with a focus on relating the structural and functional understandings, and discusses the basic aspects of PDGFs and PDGFRs, the mechanisms of activation, and the insights into the therapeutic antagonism of PDGFRs. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation

The Eph receptors and their ephrin ligands play crucial roles in a large number of cell–cell interaction events, including those associated with axon pathfinding, neuronal cell migration and vasculogenesis. They are also involved in the patterning of most tissues and overall cell positioning in the development of the vertebrate body plan. The Eph/ephrin signaling system manifests several unique features that differentiate it from other receptor tyrosine kinases, including initiation of bi-directional signaling cascades and the existence of ligand and receptor subclasses displaying promiscuous intra-subclass interactions, but very rare inter-subclass interactions. In this review we briefly discuss these features and focus on recent studies of the unique and expansive high-affinity Eph/ephrin assemblies that form at the sites of cell–cell contact and are required for Eph signaling initiation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

RET modulates cell adhesion via its cleavage by caspase in sympathetic neurons

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.     

The structure of the glial cell line-derived neurotrophic factor-coreceptor complex: insights into RET signaling and heparin binding

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR alpha 1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.     

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aβ-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ～50% of the nodose neurons. GFRα2 was expressed in ～50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.     

二聚化:受体酪氨酸激酶活化的重要机制

受体酪氨酸激酶家族是一类具有内源性蛋白酪氨酸激酶活性的生长因子受体。它们具有相似的分子结构 ,其配体介导的受体活化主要是通过二聚化的机制来实现的。配体介导同源或异源的受体二聚化 ,不同的配体以不同的机制介导受体的二聚化。本文介绍了受体酪氨酸激酶家族不同亚类受体在其配体介导下二聚化的机制 ,并着重介绍了表皮生长因子受体家族各成员间的异二聚化及其引起的胞内信号转导途径的多样化     

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr⁶⁸⁷ in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr⁶⁸⁷ and association with components of the Tyr¹⁰⁶² signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser⁶⁹⁶, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr⁶⁸⁷ as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

Tyrosine phosphorylation of protein kinase D2 mediates ligand-inducible elimination of the Type 1 interferon receptor

Type 1 interferons (including IFNα/β) activate their cell surface receptor to induce the intracellular signal transduction pathways that play an important role in host defenses against infectious agents and tumors. The extent of cellular responses to IFNα is limited by several important mechanisms including the ligand-stimulated and specific serine phosphorylation-dependent degradation of the IFNAR1 chain of Type 1 IFN receptor. Previous studies revealed that acceleration of IFNAR1 degradation upon IFN stimulation requires activities of tyrosine kinase TYK2 and serine/threonine protein kinase D2 (PKD2), whose recruitment to IFNAR1 is also induced by the ligand. Here we report that activation of PKD2 by IFNα (but not its recruitment to the receptor) depends on TYK2 catalytic activity. PKD2 undergoes IFNα-inducible tyrosine phosphorylation on specific phospho-acceptor site (Tyr-438) within the plekstrin homology domain. Activated TYK2 is capable of facilitating this phosphorylation in vitro. Tyrosine phosphorylation of PKD2 is required for IFNα-stimulated activation of this kinase as well as for efficient serine phosphorylation and degradation of IFNAR1 and ensuing restriction of the extent of cellular responses to IFNα.     

Receptor tyrosine kinase activation: From the ligand perspective

Receptor tyrosine kinases (RTKs) are single-span transmembrane receptors in which relatively conserved intracellular kinase domains are coupled to divergent extracellular modules. The extracellular domains initiate receptor signaling upon binding to either soluble or membrane-embedded ligands. The diversity of extracellular domain structures allows for coupling of many unique signaling inputs to intracellular tyrosine phosphorylation. The combinatorial power of this receptor system is further increased by the fact that multiple ligands can typically interact with the same receptor. Such ligands often act as biased agonists and initiate distinct signaling responses via activation of the same receptor. Mechanisms behind such biased agonism are largely unknown for RTKs, especially at the level of receptor–ligand complex structure. Using recent progress in understanding the structures of active RTK signaling units, we discuss selected mechanisms by which ligands couple receptor activation to distinct signaling outputs.     

Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.     

Structural basis for the specific recognition of IL-18 by its alpha receptor

Interleukin 18 (IL-18), a member of the IL-1 family of cytokines, is an important regulator of innate and acquired immune responses. It signals through its ligand-binding primary receptor IL-18Rα and accessory receptor IL-18Rβ. Here we report the crystal structure of IL-18 with the ectodomain of IL-18Rα, which reveals the structural basis for their specific recognition. It confirms that surface charge complementarity determines the ligand-binding specificity of primary receptors in the IL-1 receptor family. We suggest that IL-18 signaling complex adopts an architecture similar to other agonistic cytokines and propose a general ligand-receptor assembly and activation model for the IL-1 family.     

Signaling mechanisms through gp130: A model of the cytokine system

The interleukin-6 cytokine family plays roles in a wide variety of tissues and organs, including the immune hematopoietic and nervous systems. Gp130 is a signal-transducing subunit shared by the receptors for the IL-6 family of cytokines. The binding of a ligand to its receptor induces the dimerization of gp 130, leading to the activation of JAK tyrosine kinase and tyrosine phosphorylation of gpl30. These events lead to the activation of multiple signal-transduction pathways, such as the STAT, Ras-MAPK and PI-3 kinase pathways whose activation is controlled by distinct regions of gp130. We propose a model showing that the outcome of the signal transduction depends on the balance or interplay among the contradictory signal transduction pathways that are simultaneously generated through a cytokine receptor in a given target cell.