Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling
Robinson, Ross A.; Griffiths, Samuel C.; van de Haar, Lieke L.; Malinauskas, Tomas; van Battum, Eljo Y.; Zelina, Pavol; Schwab, Rebekka A.; Karia, Dimple; Malinauskaite, Lina; Brignani, Sara; van den Munkhof, Marleen H.; Düdükcü, Özge; De Ruiter, Anna A.; Van den Heuvel, Dianne M.A.; Bishop, Benjamin; Elegheert, Jonathan; Aricescu, A. Radu; Pasterkamp, R. Jeroen; Siebold, Christian
(2021) Cell, volume 184, issue 8, pp. 2103 - 2120.e31
(Article)
Abstract
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here,
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we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
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Keywords: axon regeneration, cell migration, cell surface receptors, complex structure, morphogen signaling, Neogenin, Netrin, protein-protein interactions, repulsive guidance molecule, signal transduction, Bioengineering, Medicine (miscellaneous), Biomedical Engineering, General Biochemistry,Genetics and Molecular Biology
ISSN: 0092-8674
Publisher: Cell Press
Note: Funding Information: We thank the staff of beamlines I03 and B21 at the Diamond Light Source (DLS), UK, and M. Broekhoven, K. Harlos, N. Mitakidis, D. Staunton, and T. Walter for technical support. We thank the following people for their generous gifts of reagents: A. Berns, A. Chédotal, D. Davis, R. Giger, K.-L. Guan, M. Kiliman, C. River, T. Skutella, and G. Smit. Work was supported by Cancer Research UK ( C20724/A14414 and C20724/A26752 to C.S.), European Research Council ( 647278 to C.S.), Medical Research Council UK ( MR/L017776/1 to C.S. and A.R.A.; MR/L009609/1 and MC_UP_1201/15 to A.R.A.), the Netherlands Organisation for Scientific Research ( ALW-VICI ), The People Program (Marie Curie Actions) of the European Union’s Seventh Framework (FP7) under REA grant agreement number 289581 (NPlast), the UU Strategic Theme “Dynamics of Youth ”, ALS Stichting Nederland (TOTALS), and Stichting ParkinsonFonds (to R.J.P.). R.J.P. is funded through the Gravitation program of the Dutch Ministry of Education, Culture, and Science and the Netherlands Organisation for Scientific Research ; S.C.G. was funded by a Wellcome PhD studentship ( 099675/Z/12/Z ). Electron microscopy provision was provided through the DLS UK National Electron Bio-Imaging Centre (eBIC) (proposal EM20223 ) and the OPIC Electron Microscopy Facility (funded by Wellcome JIF ( 060208/Z/00/Z ) and equipment ( 093305/Z/10/Z ) grants). Computation was performed at the Oxford Biomedical Research Computing (BMRC) facility. Support from the Wellcome Core Award 090532/Z/09/Z is acknowledged. Funding Information: We thank the staff of beamlines I03 and B21 at the Diamond Light Source (DLS), UK, and M. Broekhoven, K. Harlos, N. Mitakidis, D. Staunton, and T. Walter for technical support. We thank the following people for their generous gifts of reagents: A. Berns, A. Chédotal, D. Davis, R. Giger, K.-L. Guan, M. Kiliman, C. River, T. Skutella, and G. Smit. Work was supported by Cancer Research UK (C20724/A14414 and C20724/A26752 to C.S.), European Research Council (647278 to C.S.), Medical Research Council UK (MR/L017776/1 to C.S. and A.R.A.; MR/L009609/1 and MC_UP_1201/15 to A.R.A.), the Netherlands Organisation for Scientific Research (ALW-VICI), The People Program (Marie Curie Actions) of the European Union's Seventh Framework (FP7) under REA grant agreement number 289581 (NPlast), the UU Strategic Theme “Dynamics of Youth”, ALS Stichting Nederland (TOTALS), and Stichting ParkinsonFonds (to R.J.P.). R.J.P. is funded through the Gravitation program of the Dutch Ministry of Education, Culture, and Science and the Netherlands Organisation for Scientific Research; S.C.G. was funded by a Wellcome PhD studentship (099675/Z/12/Z). Electron microscopy provision was provided through the DLS UK National Electron Bio-Imaging Centre (eBIC) (proposal EM20223) and the OPIC Electron Microscopy Facility (funded by Wellcome JIF (060208/Z/00/Z) and equipment (093305/Z/10/Z) grants). Computation was performed at the Oxford Biomedical Research Computing (BMRC) facility. Support from the Wellcome Core Award 090532/Z/09/Z is acknowledged. C.S. R.J.P. and A.R.A. designed the project. R.A.R. S.C.G. T.M. and B.B. produced recombinant proteins, R.A.R. S.C.G. T.M. and J.E. performed biophysical experiments; R.A.R. S.C.G. and C.S. determined X-ray structures; T.M. L.M. and D.K. determined the cryo-EM structure; R.A.S. carried out IF and PLA experiments; L.L.v.d.H. performed mouse breeding, scRNA-seq analysis, collapse, and migration experiments; E.Y.v.B. and M.H.v.d.M. conducted in utero electroporation experiments; P.Z. performed co-IP; S.B. performed collapse assays; L.L.v.d.H. Ö.D. and A.A.d.R. performed immunohistochemistry and in situ hybridization; D.M.H.v.d.H. generated transgenic mice. R.A.R. S.C.G. L.L.v.d.H. T.M. R.J.P. and C.S. wrote the paper, with all authors commenting. The authors declare no competing interests. Publisher Copyright: © 2021 The Authors
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