Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/08—Antiepileptics; Anticonvulsants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to methods for screening for agonists and antagonists of Ret-independent intracellular signaling.
• Glial cell line - derived neurotrophic factor (GDNF) (Lin et al, 1993), neurturin
• NTN neurotrophic factor
• PSP persephin
• ART a recently discovered artemin
• GDNF is a survival factor for embryonic midbrain dopaminergic neurons (Beck et al, 1995; Lin et al, 1993; Tomac et al., 1995), spinal motor neurons (Henderson et al., 1994; Oppenheim et al., 1995; Yan et al, 1995) , locus coeruleus noradrenergic neurons (Arenas et al., 1995), and subpopulations of peripheral sensory, sympathetic, and parasympathetic neurons (Buj-Bello et al., 1995; Trupp et al., 1995; reviewed by Airaksinen et al., 1999 and Saarma & Sariola, 1999).
• the pattern of neurotrophic activity of GDNF is therefore promising for its potential use in the treatment of Parkinson disease, Alzheimer disease, motoneuron diseases and several other neurodegenerative diseases.
• the biological importance of the GDNF family is illustrated by the phenotype of GDNF null mice which display deficits in primary sensory, sympathetic and motor neurons. These mice also fail to develop kidneys and most of the enteric nervous system and they die at birth (Moore et al., 1996; Pichel et al, 1996; Sanchez et al, 1996).
• the intracellular mechanism of GDNF' s action is far from understood.
• GDNF has been thought to act through a multi-component receptor system including a glycosyl- phosphatidyl-inositol (GPI)-anchored GDNF family receptor ⁇ l (GFR ⁇ l) (Jing et al., 1996; Treanor et al., 1996) and a transmembrane receptor tyrosine kinase, Ret (Durbec et al., 1996; Trupp et al., 1996).
• GPI glycosyl- phosphatidyl-inositol
• GFR ⁇ l glycosyl- phosphatidyl-inositol
• Ret transmembrane receptor tyrosine kinase
• GFR ⁇ l lacking an intracellular domain, has originally been assessed as a binding site for GDNF, serving only in the presentation of the GFR ⁇ l /GDNF complex to Ret (Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1997).
• Ret and GPI-anchored GFR ⁇ l are necessary receptors for GDNF (Cacalano et al, 1998; Enomoto et al., 1998) since mice lacking Ret, GDNF or GFR ⁇ l all share a similar phenotype and die soon after birth.
• Ret and GFR l expression patterns although similar, exhibit differences in many tissues (Trupp et al., 1997; Enomoto et al, 1998, Golden et al., 1999, Kokaia et al, 1999), which may be a sign of the distinct signaling from GFR ⁇ receptors alone or in conjunction with Ret tyrosine kinase in trans (Yu et al., 1998).
• We recently showed both in vitro and in vivo Ylikoski et al., 1998), for example, that GDNF promotes survival of postnatal cochlear sensory neurons expressing GFR ⁇ l mRNA but lacking Ret mRNA. This difference in expression patterns may be a sign of distinct Ret-independent signaling triggered by activation of GRF ⁇ receptors.
• RN33B cells were described therein as expressing four putative receptors for GDNF, none of which was c-Ret. Two of the receptors were later determined to be GFR ⁇ l and GFR ⁇ 2 (reported as GDNFR ⁇ and GDNFR ⁇ , respectively, U.S. Patent Application Serial No. 08/861,990, incorporated herein by reference). The mechanism of the Ret-independent signaling, however, was not known or described.
• GPI-anchored membrane proteins have not been conclusively shown to exhibit independent intracellular signaling functions, evidence suggesting this possibility has been increasing (Simons and Ikonen, 1997; Friedrichson and Kurzchalia, 1998; Harder et al., 1998; Varma and Mayor, 1998; Viola et al., 1999). It has been shown, for example, that GPI-anchored proteins in the immune system can mediate intracellular signaling events, such as activation of the small G-proteins, Src-type tyrosine kinases and elevation of intracellular free calcium concentration ([Ca 2+ ],) (Green et al, 1997; Brown and London, 1998; Viola et al., 1999). GPI-anchored independent signaling has not previously been shown in cells of the nervous system, however.
• the aim of the invention is to further elucidate Ret independent intracellular signaling.
• DRG dorsal root ganglion
• the present invention provides methods for screening for compounds that are agonists or antagonists of GPI-anchored receptor mediated intracellular signaling, more specifically, GFR ⁇ l -dependent, Ret-independent intracellular signaling, and methods for preventing and treating neuronal diseases comprising the use of such compounds.
• the present invention relates to methods for identifying compounds which are agonists of intracellular signaling effected by GPI-anchored receptors in nervous system cells comprising incubating nervous system cells having such receptors with a test compound and determining whether intracellular signaling has been effected in the cells.
• the present invention relates to methods for identifying compounds which are antagonists of intracellular signaling effected by GPI-anchored receptors in nervous system cells comprising incubating nervous system cells having such receptors with a test compound in the presence of a sufficient amount of an agonist of such signaling, and determining whether such signaling is decreased in the cells, as compared to controls run in the absence of the compound.
• the present invention relates to a method for identifying compounds which are antagonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GFR ⁇ l receptors, but not Ret, with a compound to be tested in the presence of a sufficient amount of an agonist of such signaling, and determining whether such signaling is decreased in the cells, as compared to controls run in the absence of the compound.
• the present invention relates to a method for identifying compounds which are agonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GFR ⁇ l receptors, but not Ret, with a compound previously determined to bind GFR ⁇ l and determining whether the compound causes an increase in [Ca 2+ ] f .
• the present invention relates to a method for identifying compounds which are antagonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GFR ⁇ l receptors, but not Ret, with a compound to be tested in the presence of a sufficient amount of an agonist of GFR ⁇ l -dependent, Ret- independent intracellular signaling effective for increasing [Ca 2+ ], and determining whether cells incubated with the compound have decreased [Ca 2+ ] f levels as compared with controls not incubated with the compound.
• the present invention relates to a method for identifying compounds which are agonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GFR ⁇ l receptors, but not Ret, with a compound to be tested, preparing a cell lysate, immunoprecipitating the lysate with anti-GFR ⁇ l antibodies to form an immunoprecipitate, and performing assays to measure kinase phosphorylation on that immunoprecipitate.
• the present invention relates to a method for identifying compounds which are antagonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GFR ⁇ l receptors, but not Ret, with a compound to be tested in the presence of a sufficient amount of an agonist of GFR ⁇ l -dependent, Ret- independent intracellular signaling to effect said kinase phosphorylation, preparing a cell lysate, immunoprecipitating that lysate with anti-GFR ⁇ l antibodies to form an immunoprecipitate, and performing assays to measure kinase phosphorylation on that immunoprecipitate, then comparing the results to controls run in the absence of the compound to be tested.
• the invention relates to a method for identifying agonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GPI-anchored GFR ⁇ l receptors, but not Ret, with a compound to be tested and determining whether Src-kinase is activated.
• the invention relates to a method for identifying antagonists of GFR ⁇ l -dependent, Ret-independent intracellular signaling by incubating cells which express GPI-anchored GFR ⁇ l receptors, but not Ret, with a compound to be tested and a sufficient amount of an agonist of GFR ⁇ l -dependent, Ret-independent intracellular signaling to activate Src kinase and determining whether incubation with the compound has resulted in less activation of Src kinase as compared to controls not incubated with the compound.
• the present invention relates to methods for effecting cellular responses in nervous system cells comprising administering an effective amount of either an agonist or antagonist of GPI-anchored intracellular signaling.
• the present invention relates to a method for identifying agonists of intracellular signaling effected by GFR ⁇ receptors comprising incubating lipid rafts prepared from cells having such receptors with a test compound, and determining whether Src-type kinase is activated
• the present invention relates to a method for identifying antagonists of intracellular signaling effected by GFR ⁇ receptors comprising incubating lipid rafts prepared from cells having such receptors with a test compound in the presence of a sufficient amount of an agonist of GFR ⁇ -dependent signaling to activate Src-type kinase, and determining whether Src-type kinase activation is reduced in the presence of the test compound, as compared with controls run without the test compound.
• the present invention relates to methods for treating neuronal diseases comprising the administration of an agent which is an agonist or antagonist of GPI- anchored intracellular signaling. In further aspects, the present invention relates to methods for treating neuronal diseases comprising the administration of agents which are agonists and/or antagonists of GPI-anchored intracellular signaling.
• Figures 1 A-G depict GDNF-evoked rapid and long-lasting [Ca 2+ ]j changes in wild type mouse DRG neurons. The vertical bars depict delta[Ca 2+ ], changes of 100 nM.
• A. Neurons were loaded with 10 ⁇ M Ca-Green 1AM. Increasing concentrations of 10-100 ng/ml GDNF, applied to the bath as indicated, evoked rapid and long-lasting elevations in [Ca 2+ ]j (n 15 separate experiments, at least 3-4 neurons recorded in each experiment).
• B 100 ng/ml GDNF, heat-inactivated at 98 °C for 15 min did not evoke changes in [Ca 2+ ], (8 recorded cells).
• thapsigargin (5 ⁇ M) (Tha) was applied at the end of the experiments to prove the functionality of the internal calcium stores.
• C. RT-PCR shows that wild type DRG neurons express Ret (284 bp fragment), GFR ⁇ l (746 bp fragment), and GFR ⁇ 2 (429 bp fragment) mRNA.
• Neurofilament light chain (NF-L, 644 bp fragment) was used as a positive control for neuronal mRNAs.
• H 2 O lane depicts a negative control without added mRNA. The size of molecular weight markers is shown on the right.
• GDNF 100 ng/ml-evoked long-lasting Ca 2+ elevation in Ret " ' " DRG neurons (the two traces are representative of 41 recordings. The vertical bars depict d[Ca 2+ ]j changes of 100 nM.
• C. Pre-treatment with PI-PLC (1 U/ml; for 1 hr at 37 °C) abolished the effect of GDNF because of removal of the GPI-anchored proteins from the membrane.
• thapsigargin 5 ⁇ M
• FIGS 3 A-E depict GDNF-evoked calcium entry in Ret " ' " DRG neurons.
• A. In part of the DRG neurons, application of nominally Ca 2+ free extracellular solution (no added EGTA) resulted in a delayed transient [Ca 2+ ], elevation possibly due to activation of capacitative calcium entry (15 recorded neurons). This [Ca 2+ ]j overshoot was not observed when calcium concentration in the nominally Ca 2+ free external solution was clamped to about 1 nM with 2 mM EGTA (14 recorded neurons; data not shown). A return to 2 mM external Ca 2+ (washout) resulted in a pronounced [Ca 2+ ]j overshoot indicating an increased membrane permeability for Ca 2+ .
• FIG. 4 A-D depict GDNF activation of GFR ⁇ l -associated Src kinases, MAP kinases and CREB in Ret " ' " DRG neurons.
• the immunoprecipitate was subjected to an in vitro kinase assay and revealed a major phosphorylated ⁇ 60 kD band.
• WB Western blotting
• D GDNF induced profound increase in CREB Ser-133 phosphorylation.
• the numbers below lanes indicate the fold induction of CREB phosphorylation relative to control.
• the lower panel shows the reprobing of the same filter with anti-CREB antibodies and demonstrate comparable amount of CREB protein in all lanes.
• FIGS 5 A-C depict GDNF-stimulated Src-type kinases associated with GFR ⁇ l in the Ret negative human SHEP neuroblastoma cell line.
• the cells were non-treated (0 min) or treated with 100 ng/ml GDNF for the time indicated.
• the optical density of the bands was determined using a phosphoimager and a TINA program and is presented as fold increase relative to control (GDNF non-treated cells).
• Lower panel the precipitates after the kinase assay were probed by Western blotting (WB) with pan-Src antibodies, which recognize Fyn, Yes and Src.
• WB Western blotting
• pan-Src antibodies which recognize Fyn, Yes and Src.
• C Left panel (GFR ⁇ l): the postnuclear lysate from SHEP cells non-treated (-) or pre-treated (+) with GDNF (100 ng/ml, lmin) was precipitated with GFR ⁇ l antibodies.
• FIGS 6 A-C depict GDNF-evoked phosphorylation of MAPK, CREB and ATF-1 in the Ret-negative SHEP neuroblastoma cell line.
• A. Addition of GDNF evoked transient and profound increase of p42/p44 MAPK phosphorylation (left panel). The numbers below lane indicate the fold induction of p42 phosphorylation relative to control. Lower panel shows the re-probing of the same filter with anti-GFR ⁇ l antibodies and demonstrates comparable amount of GFR ⁇ l protein in all lanes. The results shown are representative of four independent experiments.
• B
• Figures 7 A-C depict GDNF-activated Src type kinase and MAP kinase in NIH 3T3 fibroblasts stably transfected with GFR ⁇ l .
• A. The kinase assay experiments. The major ⁇ 60 kD proteins can be co-precipitated with GFR ⁇ l after GDNF (100 ng/ml) pre-treatment both in Neuro2A-20 neuroblastoma cells expressing Ret (1) and in the NIH3T3 fibroblast cells (2) (both cell lines were stably transfected with GFR ⁇ l).
• Neuro2A neuroblastoma cells expressing Ret but not endogenous GFR ⁇ l was treated with GDNF in the presence of a soluble GFR ⁇ l (GFR ⁇ l/Fc chimeric protein; 1 ⁇ g/ml) lacking a GPI anchor. Soluble GFR ⁇ l induced GDNF-dependent phosphorylation of p42/p44 MAPK. The numbers below lanes indicate fold induction of p42 MAPK phosphorylation relative to control (non-treated with GDNF).
• FIGS 8 A-C depict GDNF-increased PLC ⁇ tyrosine phosphorylation in SHEP neuroblastoma cells.
• A. SHEP cells were incubated with the indicated concentrations of GDNF for 1 min, and then lysed. Tyrosine-phosphorylated proteins were immunoprecipitated with 4G-10 anti-phosphotyrosine antibodies ( ⁇ -PY) and than probed for PLC ⁇ with anti-PLC ⁇ antibodies by Western blotting as described in the Materials and Methods. GDNF evoked a dose-dependent increase in PLC ⁇ tyrosine phosphorylation (n 3 experiments). The samples were normalized by protein amount.
• ⁇ -PY anti-phosphotyrosine antibodies
• FIGS 9 A-B depict a schematic representation of the proposed Ret-independent GDNF- evoked signaling pathway.
• A. The GDNF -triggered membrane signaling most probably occurs within lipid rafts, as GFR ⁇ l protein can be co-precipitated with Src type kinases in Triton X- 100 insoluble membrane fractions.
• B. GDNF-evoked activation of GFR ⁇ l induces Src type kinase (in particular, pp62 Yes kinase in SHEP cells) activation and subsequent phosphorylation of PLC ⁇ and MAP kinases.
• PLC ⁇ activation leads to IP 3 -dependent release of Ca 2+ from internal calcium stores.
• Src-dependent phosphorylation of MAPK lead to its translocation to the nucleus and CREB activation..
• GPI-linked proteins associated with lipid rafts are able to mediate intracellular signaling events in vitro and in vivo (Green et al., 1997; Simons and Ikonen, 1997; Mayor et al., 1998).
• the existence of microdomains of GPI-anchored proteins was recently shown in living cells (Varma & Mayor, 1998; Friedrichson & Kurzchalia, 1998).
• the GFR ⁇ proteins, as described above, are GPI- anchored.
• GDNF can evoke potent intracellular signaling through a Ret- independent, GPI-anchored GFR ⁇ l -mediated pathway.
• GPI-anchored GFR ⁇ l can solely evoke the induction of PLC ⁇ signaling pathway that is dependent on the activation of Src Family kinases and results in the long-lasting sustained [Ca 2+ ]i.
• GDNF activates Ret-independent Src kinase-mediated ERK1/ERK2 (MAPK)and CREB in Ret " ' " DRG neurons and in the different Ret-negative cell lines.
• MAPK Ret-independent Src kinase-mediated ERK1/ERK2
• Src kinase activity can be determined indirectly by looking at the tyrosine phosphorylation state or kinase activity of its substrates that include, but is not limited to, pl30Cas (Sakai et al., 1997) and FAK (Polte et al., 1997). Methods for determining MAPK and CREB activation are described herein.
• Activation of the GFR ⁇ l -dependent, Ret-independent signaling pathways may stimulate different cellular responses than those of the Ret-dependent pathway. For instance, neuronal survival, neurite extension, enhancement of neurotransmitter synthesis, or other cellular responses to GDNF may be preferentially enhanced by one pathway over the other. Agonists or antagonists of one of these signaling mechanisms may provide for more specific cellular responses than that of GDNF itself and thereby gain therapeutic advantage over GDNF. Methods for identifying agonists and antagonists of Ret-dependent intracellular signaling is described in U.S. Patent Application Serial No. 08/861 ,990, incorporated herein by reference. Methods to develop agonists of both signaling pathways, antagonists of both pathways, agonists or antagonists of just one pathway, or compounds that agonize one pathway but antagonize the other pathway are contemplated herein.
• GFR ⁇ l -dependent, Ret-independent signaling may promote the survival and function of specific neuronal populations. Auditory neurons, which receive the impulses from the sensory auditory hair cells and transmit them to the brain, respond to GDNF both in vitro and in vivo. GDNF has been shown to protect neurons of the inner ear. (Tay et al., 1998; Shoji et al., 1998; and Keithley et al, 1998.) GFR ⁇ l is expressed on auditory neurons in the absence of Ret. Therefore, a mechanism is described herein that suggests how the Ret-independent signaling works and how this population of neurons may be supported by compounds that specifically mimic GDNF dimerization of GFR ⁇ l at the receptor or at the subsequent intracellular signaling events.
• effect means an alteration or change.
• An effect can be positive, such as causing an increase in some material, or negative, e.g., antagonistic or inhibiting.
• agonist refers to a compound or composition that can stimulate or positively influence the intracellular signaling pathways described herein, or augment or synergize the activity of any other compound or composition thereon.
• antagonist refers to a compound or composition that can inhibit, suppress, block or negatively influence the intracellular signaling pathways described herein.
• the term "sufficient amount” as used herein refers to a quantity of an agent that will result in the referred to effect.
• bind refers to the interaction between ligands and their receptors, the binding being of a sufficient strength and for a sufficient time to allow the detection of said binding under the conditions of the assays disclosed herein.
• administration includes but is not limited to, oral, subbuccal, transdermal, parenteral, subcutaneous and topical. A common requirement for these routes of administration is efficient and easy delivery.
• the term "effective amount,” refers to the amount required to achieve an intended purpose for both prophylaxis or treatment without undesirable side effects, such as toxicity, irritation or allergic response. Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the art. Human doses can readily be extrapolated from animal studies (Katocs et al, Chapter 27 In: Remington 's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).
• the dosage required to provide an effective amount of a formulation will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al, Chapter 3 In: Goodman & Gilman 's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al, eds., McGraw-Hill, New York, NY, 1996).
• neural system cell refers to all cells present in or derived from the nervous system, including, but not limited to neuronal cells, such as neurons, and non-neuronal cells, such as glial cells.
• transformed cell refers to a cell that has been modified using procedures known in the art to express GFR ⁇ l and/or not to express Ret.
• neurodegenerative or retrogressive process within one or more cells of the nervous system, including even the death of nerves, axons, or tracts of the central nervous system.
• cellular response refers to, without limitation, any change in neuronal survival, neuronal plasticity, neurite extension, cell migration, or any enhancement or inhibition of neurotransmitter synthesis and/or release.
• lipid rafts refers to a structure of sphingolipids and cholesterol packed into moving platforms within the liquid bilayer of cell membranes, and includes the detergent insoluble, glycolipid-enriched fraction that remains after extraction with Triton X-100 or similar detergents.
• GPL GPI-anchored or GPI-linked as used herein in reference to a receptor refer to a receptor that is associated with GPL
• independent intracellular signaling in reference to a receptor as used herein refers to a receptor that evokes intracellular signaling without requiring and/or in the absence of co-receptors.
• GPI-anchored protein-coupled kinases and Src- type kinases in particular have been shown to evoke PLC ⁇ stimulation, [Ca 2+ ]j elevation and MAPK activation (Brown and London, 1998; Dikic et al., 1996; Finkbeiner and Greenberg, 1998; Khare et al., 1997; Lutrell et al, 1996, 1997; Thomas and Brugge, 1997).
• lipid rafts a structure of sphingolipids and cholesterol packed into moving platforms within the liquid bilayer (Sargiacomo et al., 1993; Simons and Ikonen, 1997; Brown and London, 1998; Luttrell et al, 1999; Viola et al., 1999). Whether or not such lipid rafts exist in DRG neurons is not known.
• Src-type kinases can be co-precipitated with GFR ⁇ l in DIGs from Ret " ' " DRG neurons as well as in the different Ret-negative but GFR ⁇ l- expressing cell lines.
• DIGs from SHEP neuroblastoma cells DIGs from SHEP neuroblastoma cells
• GDNF evoked a potent transient activation of Src kinase.
• GDNF evoked potent activation of p42/p44 MAPK and CREB in the different Ret-deficient cell lines. Again GDNF-evoked phosphorylation of MAPK was completely abolished with low doses of the selective Src kinase inhibitor, PP2.
• PP2 selective Src kinase inhibitor
• Src kinases and MAPK might significantly affect cell function since these kinases have been established to be crucially involved in mitogenesis, nerve-growth factor induced cell differentiation with neurite outgrowth, cell migration as well as in focal adhesion kinase (FAK) dependent cell motility (Khare et al., 1997; Thomas and Brugge, 1997).
• a potent MAPK activation such as observed in our experiments, might promote neuronal survival and neuronal plasticity (reviewed by Fukunaga K. & Miyamoto E., 1998 and Impey et al., 1999).
• soluble GFR ⁇ l capable of inducting MAPK phosphorylation in the presence of GDNF in Ret-expressed cells, was unable to evoke intracellular signaling in the Ret-negative parental NIH3T3 fibroblasts.
• an adapter protein exists, it would, unlike Ret, strictly require an association with the GPI anchor of GFR ⁇ l .
• Another possibility may be that enzymes activated in the rafts involved in anchor release might yield soluble phospho-oligosaccharides. These would then flip across the bilayer and may function as active second messengers in the cytosol.
• GDNF can also trigger lipid-lipid interaction and raft coalescence-dependent accumulation of intracellular phosphorylated proteins. Whether such signaling pathways are involved in the action of GDNF is presently unknown.
• GDNF evokes [Ca 2+ ], elevation and Ret- independent activation of Src-tyrosine kinase, PLC ⁇ , MAPK and CREB - coupled intracellular signaling pathways in Ret " ' " DRG neurons and in the Ret-negative cell lines.
• GDNF binds to the GPI-anchored GFR ⁇ l with subsequent activation of Src kinases associated with GFR ⁇ l followed by activation of MAPK, CREB, PLC ⁇ and sustained elevation of [Ca 2+ ],.
• the proposed signaling pathway is summarized in Fig. 9.
• mice Ret- deficient mice (Schuchardt et al., 1994) were identified from heterozygote matings by the absence of kidneys and PCR-based genotyping. GFR ⁇ 2-def ⁇ cient mice were obtained from homozygote matings (Rossi et al, 1999).
• fCa 2+ Ji measurement [Ca 2+ ]j was measured using a Bio-Rad MRC- 1024 confocal microscope equipped with an argon-krypton laser. The cells were loaded with 10 ⁇ M Calcium Green- 1AM (Molecular Probes), a membrane permeable Ca 2+ dye, by 30 min incubation in serum free cell-culture media at 37 °C and 5 % CO 2 .
• Transgenic GFR ⁇ 2 knock-out mice were produced in our laboratory. To isolate GFR ⁇ 2 genomic clones, we screened a mouse 129/Sv library (Stratagene) with a rat GRF ⁇ 2 cDNA fragment as a probe. A 6.7 kb Hindlll-XBAI fragment was used to construct the targeting vector. A 0.5 kb Notl-Xbal fragment of the GFR ⁇ 2 gene, containing part of the first coding exon with the translation initiation site, was replaced with a 2.0 kb cassette containing the neomycin-resistance gene (neo) driven by the PGK promoter and polyadenylation signal.
• neo neomycin-resistance gene driven by the PGK promoter and polyadenylation signal.
• Rl embryonic stem cells were electroporated with linearized plasmid and selected in G418 (250ug/ml). Resistant clones were screened by Southern blot analysis using a 5' outside probe that recognizes a 7.8 kb wild-type and 5.5 kb mutant band after a BamHI digest. Positive clones were further hybridized with neo and 3' outside probes to exclude random integration of the vector. Two injected clones gave germline transmission, when the chimeras were crossed toC57BL/6JOlaHsd. Single cell RT-PCR A negative pressure was applied to the micropipette, and the whole cell was harvested under visual control.
• the content of the pipette was expelled into a test tube containing TrizolTM reagent lysis buffer (Gibco BRL) and 1 ⁇ g of carrier tRNA.
• the total RNA was isolated and the RT-PCR reaction was performed using a TitanTM One Tube RT- PCR kit (Boehringer Mannheim) according to the manufacturer's instructions.
• One half of the material from each neuron was used to amplify GFR ⁇ l mRNA.
• the presence of total RNA in a sample was ensured by the amplification of a 216 bp fragment of cyclophilin mRNA using the second half of the material.
• the primers from a QuantumRNATM kit (Ambion, TX) were used.
• the primers used for analysis of GFR ⁇ l expression were as follows: 5'-GCGGCACCATGTTCCTAGCC-3' (SEQ ID NO: 1) and 5'-CAGACTCAGGCAGTTGGGCC-3' (SEQ ID NO: 2).
• the primers were designed to cross at least one intron and to exclude amplification from genomic DNA. Amplification was carried out for 45 cycles at 95°C for 45 s; 62°C for 45 s; and 72°C for 60 s.
• the resulting fragments were identified by Southern blotting with a 32 P-labelled cDNA insert of the mouse GFR ⁇ l clone and a cDNA fragment of cyclophilin. Radioactive signals were detected with a Fuji Bioimage analyzer BAS 2000. No fragments were obtained from the media of cultured neurons.
• RNA from cultured DRG neurons was divided into three equal parts and the RT-PCR was carried out as above.
• the primers for GFR ⁇ 2 were: 5'-TATTGGAGCATCCATCTGGG-3' (SEQ ID NO: 3) and 5'- AGCAGTTGGGCTTCTCCTTG-3' (SEQ ID NO: 4), and for Ret they were: 5'- ATGAAAGGGTACTGACCATGG-3' (SEQ ID NO: 5) and 5'- AGGACCACACATCACTTTGAG-3 ' (SEQ ID NO: 6).
• the PCR was carried out for 40 cycles under the conditions indicated above.
• the cells were washed twice with cold PBS/vanadate and lysed in TX-100 lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, ImM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors (Boehringer) on ice for 1 hour.
• the postnuclear lysates were pre-cleared by incubation with 50 ⁇ l of 50% Protein G- Sepharose for goat polyclonal antibodies or Protein A-Sepharose CL4B (Pharmacia) for rabbit polyclonal antibodies for 1 hour at +4°C.
• the supernatants were incubated with 0.4-0.5 ⁇ g of anti-GFR ⁇ l or anti-Yes polyclonal antibodies (Santa Cruz Biotechnology) overnight at +4°C followed by incubation with Protein G or A-Sepharose for 2 hours.
• the immunocomplexes were washed twice in TX- 100 lysis buffer without EDTA and twice in kinase buffer (25 mM Hepes, pH 7.4, 5 mM MgCl 2 , 5 mM MnCl 2 , 1 mM sodium orthovanadate).
• Immunocomplexes were incubated in a kinase buffer supplemented with 5-10 ⁇ Ci of [ ⁇ - 32 P]ATP for 20 minutes at 37°C. The samples were washed out from incorporated label and were subjected to 10% SDS-PAGE. Proteins were transferred to a Hybond ECL membrane (Amersham) using semi-dry blot apparatus (Schleicher & Schuell, Dassel, FRG). Labeled proteins were visualized with a Fuji Bioimage analyzer BAS 2000 or by autoradiography. Quantification of the optical density of the blots was performed using the TINA program.
• the semiconfluent cell monolayers were starved for 3 hours in serum-free medium, and then GDNF was applied for the indicated time.
• GDNF was applied for the indicated time.
• the DRG neurons were dissected from El 8 mice and maintained in NGF-containing medium for 2 hours. After this time the neurons were deprived of NGF by placing them in NGF-free medium in the presence of anti-NGF antibodies. After 2 hr without NGF, the neurons were stimulated with GDNF.
• a Src-family kinase inhibitor PP2 was added at the indicated concentrations to the cell monolayers 5 min before GDNF application.
• 1 ⁇ g/ml of soluble GFR ⁇ l lacking a GPI anchor was added 5 min prior to the GDNF application and was kept in the solution during the GDNF treatment.
• the cells were briefly washed with PBS/sodium vanadate and lysed in the buffer containing TBS, 2 mM EDTA, 1% NP- 40, 1 % Triton X- 100, 1 mM PMSF, 1 mM Na 3 VO 4 and a CompleteTM protease inhibitors cocktail (Boehringer Mannheim).
• Total cell protein for each extract was measured by MicroBSA (Pierce) and an equivalent amount of protein was resolved electrophoretically on 10% polyacrylamide gels. The proteins were transferred to a Hybond ECL (Amersham) membrane and the blot was probed with either MAPK (ERK1/2) or JNK Anti- ActiveTM pAbs (Promega) according to the manufacturer's instructions.
• the blots were then reprobed with GFR ⁇ l mAbs (Transduction Laboratories).
• GFR ⁇ l mAbs Transduction Laboratories
• Western blots were probed with the antibodies that specifically recognise the Ser- 133 phosphorylated form of CREB and then re-probed with the anti-CREB antibodies (New England Biolabs).
• GDNF (10 ng/ml) induced a rapid transient increase in [Ca 2+ ]j, whereas the concentrations over 10 ng/ml (10-100 ng/ml) evoked both a transient and slow long-lasting elevation in [Ca 2+ ]
• (Fig. 1A) (n 15 experiments; at least 3-4 neurons were recorded in each experiment).
• Fig. 1A heat- inactivated GDNF. Heat-inactivated GDNF did not evoke any changes of basal [Ca 2+ ]i (8 recorded cells) (Fig. IB).
• thapsigargin an inhibitor of SERCA pump at intracellular calcium stores. All cells responded to the application of thapsigargin with a profound elevation in [Ca 2+ ]j (two representative recordings shown on Fig. IB). Further control experiments were performed using cleavage of GPI-anchored proteins from the membrane with phosphatidylinositol-specific phospholipase C (PI-PLC) (7 recordings). None of the pre-treated neurons responded to the application of GDNF (100 ng/ml) (Fig. ID).
• Ret tyrosine kinase can activate cytoplasmic PLC ⁇ through the PLC ⁇ docking site (Borrello et al., 1996), and this could lead to IP 3 production and subsequent Ca 2+ release.
• DRG neurons isolated from Ret' " mice (Schuchardt et al., 1994).
• GFR ⁇ l mRNA 11 representative lanes shown on Fig. 2A
• GDNF triggers Ca 2+ release from the internal stores in Ret negative DRG neurons Activation of PLC ⁇ apparently leads to IP 3 production and subsequent Ca 2+ release from IP 3 sensitive stores.
• GDNF did not evoke an increase in [Ca 2+ ]i in comparison to the saline injected control neurons (Fig.
• GDNF activates a GFRal-coupled kinase, MAP kinases and cAMP response element binding protein (CREB) in Ret' ' DRG neurons
• BDNF neurotrophins
• GDNF stimulates GFRal-coupled p62Yes type kinase in SHEP neuroblastoma cells
• SHEP human neuroblastoma cells line for further exploration of GDNF-dependent non-Ret signaling.
• SHEP cells lack Ret mRNA but express GFR ⁇ l mRNA (Fig.5A) and an ample amount of GFR ⁇ l protein (data not shown).
• GDNF 100 ng/ml
• Fig. 5B Src- type kinase activity
• GDNF activates ERK1/ERK2, CREB and CREB-r elated protein ATF-1 in SHEP cells.
• GDNF induces activation ofERKl/ERK2 in NIH 3T3 fibroblasts via Src type kinases.
• NIH 3T3 fibroblasts stably transfected with GFR ⁇ l do not express Ret and were therefore used as a non-neuronal cell line for investigating the GDNF-evoked Ret-independent signaling.
• GDNF transiently activates PLC ⁇ but not JNK in SHEP cells.
• GDNF neurotrophic factor signaling: four masters, one servant? Molecular and Cellular Neuroscience, 13, 313-325. Arenas, E., Trupp, M., Akerud, P. and Ibanez, C.F. (1995) GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo. Neuron, 15, 1465-1473.
• GDNF is an age-specific survival factor for sensory and autonomic neurons. Neuron, 15, 821-828.
• GFR ⁇ l is an essential receptor component for GDNF in the developing nervous system and kidney. Neuron, 21, 53-62.
• JNKS c-Jun NH 2 -terminal protein kinases
• Davletov, B.A. Meunier, F.A., Ashton, A.C., Matsushita, H., Hirst, W.D., Lelianova, V.G., Wilkin, G.P., Dolly, J.O. and Ushkaryov, Y.A. (1998) Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca 2+ . EMBO Journal 17, 3909-3920.
• GFR alphal -deficient mice have deficits in the enteric nervous system and kidneys. Neuron, 21, 317-324. Finkbeiner, S., Tavazoie, S.F., Maloratsky, A., Jacobs, K.M., Harris, K.M. and Greenberg, M.E. (1997) CREB: A major mediator of neuronal neurotrophin responses. Neuron, ⁇ 9, 1031-1047.
• GD ⁇ F a potent survival factor for motoneurons present in peripheral nerve and muscle. Science, 266, 1062-1064.
• GDNF-induced activation of the Ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF.
• GDNFR-alpha a novel receptor for GDNF.
• GFR ⁇ -2 and GFR ⁇ -3 are two new receptors for ligands of the GDNF family. Journal of Biological Chemistry, 272, 33111-33117.
• GDNF a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 260, 1130-1132.
• Luttrell L.M., Hawes, B.E., van Biesen, T., Luttrell, D.K., Lansing, T.J.,
• Luttrell L.M., Ferguson, S.S.G., Daaka, Y., Miller, W.E., Maudsley, S.,Della Rocca, G.J., Lin, F.-T., Kawakatsu, H., Owada, K., Luttrell, D.K., Caron, M.G. and Lefkowitz, R.J. (1999) ⁇ -arrestin-dependent formation of ⁇ 2 adrenergic receptor-Src protein kinase complexes. Science, 283, 655-661.
• Guinea pig auditory neurons are protected by glial cell line-derived growth factor from degeneration after noise trauma. Hearing Research, 124, 17-26.

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