Houghton, Francesca Mary;
(2023)
Molecular characterisation of GFRalpha-1 assemblies in synaptic adhesion.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Glial-cell line derived neurotrophic factor (GDNF) is a promising treatment for Parkinson’s disease by supporting dopaminergic neuronal survival through RET receptor tyrosine kinase activation. RET-independent functions for GDNF-GFR⍺1 have been described including ligand-dependent cell adhesion at synapses and signalling through an alternative GDNF receptor NCAM. The aim of this thesis was to explore RET-independent roles of GDNF-GFR⍺1 by using structural and biophysical methods to define the underlying molecular mechanisms. Structural characterisation of zebrafish GDNF-GFR⍺1 assemblies revealed a barrelshaped decameric adhesion assembly in which two GFR⍺1 pentamers bridge five GDNF dimers. GDNF-dependent GFR⍺1 trans-adhesion was reconstituted in liposomes, allowing the pentameric interface to be validated. The interface overlaps with the known RET binding site and binding of RET extracellular module to GDNFGFR⍺1 is shown to disrupt the adhesion assembly. An equivalent assembly for mammalian GDNF-GFR⍺1 could not be detected in vitro but the same pentameric interface was validated using cellular assays as being essential for trans-synaptic adhesion. Structural and biophysical characterisation of the interaction between GDNF, GFR⍺1 and NCAM revealed NCAM V-shaped dimers mediate a high affinity ternary complex with GDNF-GFR⍺1 dependent on GFR⍺1-D1 domain. Efforts to reconstitute the interaction between NCAM and GDNF-GFR⍺1 using purified components was not successful. Finally, heparan sulfate is shown to bind to GFR⍺1 at the D1-D2-D3 junction and promotes a conformational compaction in the GFR⍺1-D1 domain. Sulfated glycosaminoglycans (GAGs) disrupt high affinity NCAM interactions in vitro and the GDNF-mediated synaptic adhesion. A model is proposed where GFR⍺1 conformation drives GDNF-dependent trans-adhesion and GFR⍺1-NCAM interaction, or in the presence of RET and GAGs, promotes trophic support. This model challenges the prevailing view that GFR⍺1 receptor acts simply as a passive co-receptor, giving a new appreciation of GFR⍺1 function.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Molecular characterisation of GFRalpha-1 assemblies in synaptic adhesion |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | CC BY-NC: Copyright © The Author 2023. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10179281 |
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