Structure-Function Studies of GDNF Family Ligand-RET signalling

REVIEW OF THE LITERATURE . 1 1. Neurotrophic factors inside and outside of the nervous system . 1 1.1 Classifi cation of neurotrophic factors . 2 1.1.1 Neurotrophins . 2 1.1.2 Neurokines . 3 1.1.3 MANF family.3 1.1. 4 GDNF and its family ligands . 3 2. Neurotrophic factor receptors . 6 2.1 Receptors of neurotrophins . 7 2.2 Receptors of neurokines . 8 2.3 Receptors of GDNF family ligands . 9 2.3.1 GFRα receptors . 9 2.3.2 RET receptor tyrosine kinase . 13 3. The GFL-GFRα-RET complex . 14 3.1 The structure of GFLs . 16 3.2 The structure of GFRαs . 17 3.3 The structure of RET . 19 4. RET-dependent GFL-signalling . 20 4.1 Activity of the RET kinase domain . 20 4.2 Downstream signalling . 21 4.3 Signalling by different ligands . 23 4.4 Subcellular localisation of RET . 23 4.4.1 GFL signalling and lipid rafts . 24 4.5 Cross-talk with other pathways . 25 5. The GFL/GFRα/RET signalling pathway and human diseases . 26 5.1 Gain-of-function mutations in RET . 26 5.2 Loss-of-function mutations in RET . 29 5.3 Parkinson’s disease . 31 6. Therapeutic use of GFLs . 32 AIMS OF THE STUDY . 34 MATERIALS AND METHODS . 35 1. RET phosphorylation assays (used in I-IV) . 35 1.1 With soluble GFRα1 receptors.35 1.2 With GPI-anchored GFRα1 receptors . 35 2. Neurite outgrowth assays (I-II) . 35 2.1 With soluble GFRα1 receptors.35 2.2 With GPI-anchored GFRα1 receptors . 36 3. Co-immunoprecipitation of SHC/RET and GRB2/RET (III) . 36 4. AKT phosphorylation assay in the presence of Brefeldin A (III) . 36 5. AKT, ERK and STAT3 phosphorylation assays with ER-retained RET (III) . 37 6. Phospho-RET ELISA assays (IV) . 37 7. Other methods . 37 RESULTS AND DISCUSSION . 38 1. Functional characteristics of the mouse GFRα4-GPI receptor (I) . 38 1.1 Biochemical and functional characterisation of the mouse GFRα4 receptor . 38 1.2 Recruitment of RET to lipid rafts by the GFRα4 receptor . 39 1.3 The biological activity of the GFRα4 receptor . 40 2. The role of domain 1 in the function of GFRα1 (II) . 41 2.1 Characterisation of soluble and GPI-anchored GFRα1 receptors.41 2.2 Differences in GDNF binding and biological activity between truncated and full-length GFRα1 . 41 2.3 The effect of GFRα1 concentration on RET phosphorylation . 42 2.4 Domain 1 stabilises the GFRα1-GDNF interaction, affects RET phosphorylation and contributes to the biological activity in vitro . 43 3. Activity of RETMEN 2B in the endoplasmic reticulum (III) . 44 3.1 Technical limitations and approaches . 44 3.2 Localisation and phosphorylation of RET precursor . 44 3.3 Interactions between RETMEN 2B precursor and adapter proteins SHC and GRB2 in the ER . 45 3.4 Downstream signalling mediated by RETMEN 2B precursor in the ER . 46 3.5 The precursor of RETMEN 2B is biologically active . 46 4. Structure of the GDNF-GFRα1 complex (IV) . 48 4.1 GDNF-GFRα1-SOS complex . 48 4.2 Interactions between GDNF, GFRα1, SOS and RET . 48 4.3 Comparison of GDNF-GFRα1 to ARTN-GFRα3 . 49 4.4 Role of heparin in the GDNF-GFRα1-RET complex . 50 CONCLUSIONS . 51 ACKNOWLEDGEMENTS . 52 REFERENCES . 54 ORIGINAL PUBLICATIONS I-IV LIST OF ORIGINAL PUBLICATIONS Jianmin Yang, Maria Lindahl, Päivi Lindholm, I. Heidi Virtanen, Eleanor Coffey, Pia Runeberg-Roos, Mart Saarma (2004). PSPN/GFRα4 has a signifi cantly weaker capacity than GDNF/GFRα1 to recruit RET to rafts, but promotes neuronal survival and neurite outgrowth. FEBS Lett. 569:267-71. Heidi Virtanen II. , Jianmin Yang, Maxim M. Bespalov, Jukka O. Hiltunen, VeliMatti Leppänen, Nisse Kalkkinen, Adrian Goldman, Mart Saarma, Pia RunebergRoos (2005). The fi rst cysteine-rich domain of the receptor GFRα1 stabilizes the binding of GDNF. Biochem J. 387:817-24. Pia Runeberg-Roos, III. Heidi Virtanen, Mart Saarma (2007). RET(MEN 2B) is active in the endoplasmic reticulum before reaching the cell surface. Oncogene 26:7909-15. Vimal Parkash, Veli-Matti Leppänen, IV. Heidi Virtanen, Jaana M. Jurvansuu, Maxim M. Bespalov, Yulia A. Sidorova, Pia Runeberg-Roos, Mart Saarma, Adrian Goldman (2008). The structure of the glial cell line-derived neurotrophic factor-coreceptor complex: insights into RET signaling and heparin binding. J Biol Chem. 283:35164-72.