Zebrafish GDNF and its co-receptor GFRα1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro.

Tuulia Saarenpää,Jaana Jurvansuu,Heidi Kaljunen,Adrian Goldman,Arun Kumar Mahato,Yulia Sidorova,Liying Yu,Mart Saarma,Igor Tascón,Konstantin Kogan,Jukka Kallijärvi

doi:10.1371/journal.pone.0176166

Tuulia Saarenpää, Jaana Jurvansuu + Show 9 more

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https://doi.org/10.1371/journal.pone.0176166

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Journal: PloS one	Publication Date: May 3, 2017
Citations: 14	License type: CC BY 4.0

Affiliation: University of Helsinki, University of Leeds

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1) and receptor tyrosine kinase “RET”, several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 μM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.

Highlights

Glial cell-line derived neurotrophic factor (GDNF), a secreted growth factor of the GDNF family ligands (GFLs) [1], is an important biomedical research target, especially since it has been shown to promote the survival of the midbrain dopaminergic neurons that degenerate in Parkinson’s disease [2]
We found that an N-terminal Flag and 8-His tag on the recombinant zGDNF significantly inhibited signaling through human RET (hRET) (S2A Fig)
Our results show that insect cells can express correctly folded and fully functional components of the zebrafish RET signaling system

Summary

Introduction

Glial cell-line derived neurotrophic factor (GDNF), a secreted growth factor of the GDNF family ligands (GFLs) [1], is an important biomedical research target, especially since it has been shown to promote the survival of the midbrain dopaminergic neurons that degenerate in Parkinson’s disease [2]. GDNF and the other GFLs, neurturin, persephin, and artemin, each selectively pair with one of the four glycophosphatidylinositol- (GPI-) anchored co-receptors “GDNF family receptor α’s” (GFRαs) [3] to form a tripartite complex that signals through receptor tyrosine kinase RET [4, 5]. Upon formation of the RET-GFRα1-GDNF complex with a stoichiometry of 2:2:2 [11] the tyrosine kinases of the RET dimer autophosphorylate and these phosphorylated tyrosine residues serve as a binding platform for a number of adaptor proteins activating intracellular signaling pathways [3]. The activation of the various RET intracellular signaling pathways is important for promoting neuronal survival, neurite formation and branching, synapse formation, spinal motoneuron survival and kidney development and spermatogenesis [12, 13]

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