Cell biology of GDNF and its receptors. Pia Runeberg-Roos Institute of Biotechnology

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1 Cell biology of GDNF and its receptors Pia Runeberg-Roos Institute of Biotechnology

2 1. Introduction to GDNF and its receptors 2. Basic cell biology clinical use A few examples: How is GDNF secreted? Is folding & glycosylation important? Where is the signaling complex formed? How/where is the receptor inactivated? How can the same mutation in RET be both activating and inactivating?

3 What is a neurotrophic factor? A protein that promotes survival of some population of neurons. The same protein may also promote neuronal proliferation, differentiation, migration - or density of innervation. The same protein may also affect other cell types than neurons. Cranial Peripheral Autonomic A. Central nervous system B. Peripheral nervous system B1. Somatic nervous system B2. Autonomic nervous system sympathetic nervous system parasympathetic nervous system

4 How do neurotrophic factors work? Classical neurotrophic factors: They are extracellular proteins They need receptors on the cell surface The receptors transmit the signal from the outside to the inside of the cell The ligand / receptor interaction must be specific in order to achieve specific biological reactions

5 How do receptors work? Receptors may consist of one protein or several proteins. Part of the receptor must penetrate the membrane Ibáñez 1998 Binding of the ligand leads to a conformational change or assembly of the receptor complex Many receptors are kinases or phosphatases Neurotrophins: NGF, BDNF, NT-3 and NT-4 GDNF family ligands: GDNF, NRTN, ARTN, PSPN Neurokines: CNTF, IL6, LIF, cardiotrophin 1

6 The extracellular GDNF/receptor complex triggers intracellular responses Four GDNF family ligands: GDNF, Glial cell line-derived Neurotrophic Factor NRTN, Neurturin ARTN, Artemin PSPN, Persephin Four ligand binding co-receptors: GFRα1, Gdnf Family Receptor α1 GFRα2, Gdnf Family Receptor α2 GFRα3, Gdnf Family Receptor α3 GFRα4, Gdnf Family Receptor α4 Airaksinen & Saarma 2002 One transmembrane receptor: RET, REarranged during Transfection

7 Dimeric GDNF induces dimerisation of the receptors Dimeric GDNF binds and dimerises GFRα1. Tetrameric GDNF/GFRα1 dimerises RET. RET connects the extracellular side to the intracellular side. The intracellular domain of RET is an enzyme with tyrosine kinase activity. Dimerisation of RET triggers autophosphorylation. Structure of dimeric GDNF, Eigenbrot et al., 1997

8 GDNF-signalling is driven by the dimeric nature of the ligand GDNF soluble homodimeric protein GFRa1 GPI-anchored to the membrane 3 homologous subdomains, D1-3 GDNF binds to D2 cellmembrane RET 4 cadherin-like domains a cystein-rich domain a transmembrane domain a juxtamembrane domain a kinase domain a C-terminal tail GPI = Glycosylphosphatidylinositol

9 How is the GDNF-signal perceived? The phosphorylated tyrosine residues on RET create new interaction sites for intracellular signalling proteins. These interactions lead to new intracellular signalling cascades. Fukuda et al 2005

10 GDNF is a Glial cell line-derived Neurotrophic Factor GDNF was originally found when searching for a neurotrophic factor which would prevent the continued degeneration of midbrain dopaminergic neurons a hallmark of Parkinson s disease. Although GDNF was identified, cloned and characterized based on its ability to promote dopamine uptake in dissociated rat embryo midbrain cultures (Lin et al., 1993), this protein soon turned out to be very broadly expressed and crucially important for the development of especially the enteric nervous system and kidney. GDNF is by now known as a multifunctional protein with the capacity to induce survival, proliferation, migration as well as differentiation.

11 GDNF and its receptors from a clinical point of view Developmental functions Development of the enteric nervous system (RET) Development of the kidney (RET) Spermatogenesis (GDNF) Diseases Hirschsprung disease (RET) Congenital anomalies of kidney or urinary tract (RET) Familial medullary thyroid cancer (RET) Multiple endocrine neoplasia, type 2A (RET) Multiple endocrine neoplasia, type 2B (RET) Papillary thyroid carcinoma (RET) Potential clinical use Parkinson s disease (GDNF, NRTN) Neuropatic pain (ARTN, GDNF) Amyotrophic lateral sclerosis (GDNF) Spinal cord injury (GDNF) Depression (GDNF) Cerebral ischemia (GDNF) Addiction (GDNF) Male fertility (GDNF)

12 Is there more to learn? We know the structure of the ligand We have an idea on how the receptor complex forms We know how signalling is mediated We know the biological responses We have a lot of expression data We know for which cell types signalling is crucial The molecule is in clinical trials

13 CNS Striatum Hippocampus Motor nucleus n. Cortex cerebri Locus coeruleus Substantia innominata Cerebellum Olfactory tubercle Locus coeruleus Corpus pineale Clark s column Dorsal horn of spinal cord Pons Thalamic nucleus Cingulate cortex Peripheral tissues Mesenchyme in many areas Teeth Tongue epithelium Tongue papillae Muscle tissue Retina Nasal cavity Skin Vibrissae Inner ear Ear canal Eye muscles Glomus caroticum Kidney GI-tract PubMed: RET 4556 papers?? PubMed: GDNF 3173 papers PubMed: GFRa1 263 papers PubMed: neurturin 316 papers PubMed: artemin 139 papers PubMed: GFRa2 104 papers PubMed: persephin 86 papers PubMed: GFRa3 53 papers PubMed: GFRa4 13 papers

14 How is GDNF secreted? Is folding & glycosylation important? Where is the signaling complex formed? How/where is the receptor inactivated? What is the role of heparin-binding? Is GDNF recycled to the cell surface? How can the same mutation in RET be both activating and inactivating? What form of GDNF should be cloned for cell-based or virus-based treatment? Does it make a difference if one produces GDNF in mammalian cells or E.coli? Is the site of application important? Can the inactivation be modified? How well is GDNF spreading in the tissue? Can GDNF be made more stable? Mechanisms of folding during secretion?

15 How is GDNF secreted? (1) Background: PRE sequence for translocation to the ER PRO sequence for folding/ targeting/ inactivation Splice variants of GDNF: (α) and (β) variants differ only in their PRO sequence Pre(α)pro GDNF - with a longer prosequence (58 residues) Pre(β)pro GDNF - with a shorter prosequence (31 residues) Function of the PRO sequence in GDNF? Lonka-Nevalaita et al 2010

16 How is GDNF secreted? (2) Hartmann et al 2001 Background/ secretion of BDNF: Nonneuronal cells constitutive secretion Neurons & neuroendocrine cells active secretion in response to a specific signal constitutive secretion Example: A mutated proregion (Val/Met) of BDNF changed hippocampal activity and memory function impaired intracellular distribution and secretion no effect on constitutive secretion impaired folding affects trafficking to secretory vesicles? Secretion of GDNF: Pre(α)pro GDNF constitutive secretion Pre(β)pro GDNF activity dependent secretion SgII = Secretogranin II marker of regulated secretory vesicles Lonka-Nevalaita et al 2010

17 How is GDNF/NRTN secreted? (3) On the secretion of NRTN: Choice of PRO sequence effects secretion Choice of cell type affects secretion Fjord-Larsen et al 2005

18 How is GDNF secreted? (4) Conclusions: Secretion is affected by the prosequence as well as the cell type. For cell- and virus-based treatments it is very important to characterize and verify the secretion of GDNF.

19 Treatment by adding GDNF as a protein (1) pump Production of GDNF in E.coli Advantages: High yield Production of GDNF in mammalian cells: Disadvantages: Low yield (gene therapy) (encapsulated cells)

20 Treatment by adding GDNF as a protein (2) Posttranslational modifications of GDNF in mammalian cells C-bridges: 3 intermolecular & 1 intramolecular bridges glycosylation: two potential N-linked glycosylation sites proteolytic processing: signal sequence, prosequence, mature protein crystal structure: only the underlined part is solved Primary sequence of human GDNF: MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATI KRLKR SPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIF RYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI

21 Treatment by adding GDNF as a protein (3) Production of GDNF in E.coli Advantages: High yield Disadvantages: Quality? - inclusion bodies - folding? - no glycosylation Production of GDNF in mammalian cells: Advantages: High quality - control in the ER Disadvantages: Low yield High/low expression vectors: Overloading of the mammalian secretory system can cause artefacts: - secretion of aggregates - secretion of intracellular proteins - secretion of uncleaved proteins - secretion of unglycosylated proteins High yield production of GDNF in E.coli Horger et al., 1998: GDNF was expressed in E. coli and purified using a modification of a method described in earlier work (Henderson et al., 1994). The insoluble fraction after lysis of E. coli cells was suspended in 6 M guanidine hydrochloride buffer, ph 8, and treated with sodium sulfite (0.1 M) and sodium tetrathionate (10 mm) to sulfonate the cysteine residues (4 C for 16 hr). After dialysis against 4 M urea buffer and centrifugation, monomeric GDNF was partially purified by anion exchange chromatography and then refolded in a buffer containing 4 M urea, 15% glycerol, 0.1 M phosphate, and 4 mm cysteine, ph 8, at 4 C. The GDNF dimer was then purified by anion exchange, cation exchange, and hydrophobic interaction chromatography. The final pool was dialyzed exhaustively against 1 mm HCl, aliquoted, and lyophilized.

22 Treatment by adding GDNF as a protein (4) Stability of GDNF In media 1 h 2. In media 24 h 3. On cell type 1, 24 h 4. On cell type 2, 24 h Conclusions: The source of GDNF can be of crucial importance for the results. GDNF from E.coli In media 1 h 2. In media 24 h 3. On cell type 1, 24 h 4. On cell type 2, 24 h GDNF from mammalian cells

23 Where is the signaling complex formed? (1) GDNF - a soluble protein - its extracellular spreading is restricted by its affinity to heparan sulfated proteoglycans GFRa1 - a GPI-anchored protein - is transported to specific domains of the cell membrane: lipid rafts - is transported to specific areas in polar cells = neurons, endothelial cells (?) - can be solubilised from the membrane, but has a restricted spreading in the tissue due to binding to extracellular heparan sulfated proteoglycans RET - a transmembrane protein

24 Where is the signaling complex formed? (2) Conclusions: Striatum Medial Forebrain Bundle Substantia Nigra For treating patients one needs to choose the site of application carefully. Currently too little information is available. Kirik et al. (2004) Nature Neuroscience

25 30 min 15 min no ligand WT WT DNM2-K44A DNM2-K44A DNM2-WT DNM2-WT Where is the receptor active? (1) Rab5-GFP RET merge GDNF α-ret α-pret α-perk1/ 2 α-erk1/ 2 α-pakt α-akt RET is internalised by clathrin coated pits. Internalisation is needed for full activation. RET activates AKT at the cell surface RET activates ERK after internalisation Rab5-GFP = marker for clathrin coated pits, DNM2-K44A = transfected with Dynamin dominant negative variant, DNM2-WT = transfected with Dynamin wildtype, WT = not transfected cells, Richarson & Mulligan, Oncogene 2006

26 How/where is the receptor inactivated? (1) Human RET Two well characterised splice variants, differing in the C-terminal tail Two different proteins RET9 ( amino acids) RET51 ( amino acids) Note! The lower band = ER-located uncompletely glycosylated (RET9 or RET51) The upper band = Cell surface located, fully glycosylated (RET9 or RET51) RET9 and RET51 form independent complexes RET51 has a faster turnover than RET9 RET51 is more ubiquitylated than RET9 Ubiquitination - a process by which proteins are marked for proteasome degradation, CHX = cycloheximide, an inhibitor of protein synthesis, Scott et al 2005, J Biol Chem

27 How/where is the receptor inactivated? (2) Neurons in compartmentalised cultures CB = cell bodies = soma DA = distal axons = neurite Epox = epoxomycin, a proteasome inhibitor Tsui & Pierchala 2010, J. Neurosci. The level of Ret9 expression dictates whether the retrograde survival of sympathetic neurons can be supported by GDNF GDNF does not support the long-term retrograde survival of sympathetic neurons. The axonal degradation of activated Ret limits the retrograde signaling capacity of GDNF

28 How/where is the receptor inactivated? (3) Conclusions: Alternative splicing can regulate the half-life and function of a growth factor receptor. The local degradation of Ret in axons dictates whether GDNF family ligands act as retrograde survival factors. The site of application is important. DRG = dorsal root ganglion sensory neurons SCG = sympathetic neurons of the superior cervical ganglion Tsui & Pierchala 2010, J. Neurosci.

29 How can the same mutation in RET be both activating and inactivating? (1) Mutation = C609Y HSCR, Hirschsprung Disease FMTC, Familial Medullary Thyroid Cancer Dvorakova et al, 2005, J Pediatric Sugery

30 How can the same mutation in RET be both activating and inactivating? (2) Romea et al, 1998, J Internal Medicine

31 How can the same mutation in RET be both activating and inactivating? (3) CLD, cadherin-like domains CRD, cystein-rich domain TMD, transmembrane domain JMD, juxtamembrane domain TKD, tyrosine kinase domain TAIL, C-terminal tail HSCR, Hirschsprung Disease FMTC, Familial Medullary Thyroid Cancer MEN 2A, Multiple endocrine neoplasia, type 2A MEN 2B, Multiple endocrine neoplasia, type 2B P-TYR, phosphotyrosine

32 How can the same mutation in RET be both activating and inactivating? (4)

33 How can the same mutation in RET be both activating and inactivating? (5) Kuznetsov & Nigam, N Engl J Med 1998

34 How can the same mutation in RET be both activating and inactivating? (5) Kuznetsov & Nigam, N Engl J Med 1998

35 Too little is known about how GDNF, GFRa1 and RET are circulated inside, out of and back into the cells GDNF binds to heparan sulfated proteoglycans - why?

36 Too little is known about how GDNF, GFRa1 and RET are circulated inside, out of and back into the cells Can GDNF and/or its receptors be recycled to the cell surface? Davis & Dickey, 2008, Annu. Rev. Physiol.

37 How is GDNF secreted? Is folding & glycosylation important? Where is the signaling complex formed? How/where is the receptor inactivated? What is the role of heparin-binding? Is GDNF recycled to the cell surface? How can the same mutation in RET be both activating and inactivating? What form of GDNF should be cloned for cell-based or virus-based treatment? Does it make a difference if one produces GDNF in mammalian cells or E.coli? Is the site of application important? Can the inactivation be modified? How well is GDNF spreading in the tissue? Can GDNF be made more stable? Mechanisms of folding during secretion?