Defining Minimal Binding Regions in Regulator of Presynaptic Morphology 1 (RPM-1) Using Caenorhabditis elegans Neurons Reveals Differential Signaling Complexes*
- From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458
- ↵1 To whom correspondence should be addressed: Dept. of Neuroscience, The Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458. Tel.: 561-228-2110; Fax: 561-228-2111; E-mail: bgrill{at}scripps.edu.
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Edited by Henrik G. Dohlman
Abstract
The intracellular signaling protein regulator of presynaptic morphology 1 (RPM-1) is a conserved regulator of synapse formation and axon termination in Caenorhabditis elegans. RPM-1 functions in a ubiquitin ligase complex with the F-box protein FSN-1 and functions through the microtubule binding protein RAE-1. Using a structure-function approach and positive selection for transgenic C. elegans, we explored the biochemical relationship between RPM-1, FSN-1, and RAE-1. This led to the identification of two new domains in RPM-1 that are sufficient for binding to FSN-1, called FSN-1 binding domain 2 (FBD2) and FBD3. Furthermore, we map the RAE-1 binding domain to a much smaller region of RPM-1. Point mutations in RPM-1 that reduce binding to RAE-1 did not affect FSN-1 binding, indicating that RPM-1 utilizes different biochemical mechanisms to bind these molecules. Analysis of RPM-1 protein complexes in the neurons of C. elegans elucidated two further discoveries: FSN-1 binds to RAE-1, and this interaction is not mediated by RPM-1, and RPM-1 binding to FSN-1 and RAE-1 reduces FSN-1·RAE-1 complex formation. These results indicate that RPM-1 uses different mechanisms to recruit FSN-1 and RAE-1 into independent signaling complexes in neurons.
Footnotes
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↵* This work was supported by National Institutes of Health Grant 2R01 NS072129-06 (to B. G.) and National Science Foundation Grant IOS-1121095 (to B. G.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
- Received July 12, 2016.
- Revision received December 14, 2016.
- © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
