Defining Minimal Binding Regions in Regulator of Presynaptic Morphology 1 (RPM-1) Using Caenorhabditis elegans Neurons Reveals Differential Signaling Complexes

Scott T Baker,Brock Grill

doi:10.1074/jbc.m116.748004

Scott T Baker, Brock Grill

Open Access

https://doi.org/10.1074/jbc.m116.748004

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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.

Highlights

The Pam/Highwire/RPM-1 (PHR)2 proteins are conserved intracellular signaling proteins that include human Pam/ MYCBP2, Drosophila Highwire, and Caenorhabditis elegans regulator of presynaptic morphology 1 (RPM-1) [1, 2]
Multiple Domains in RPM-1 Are Sufficient for Binding to FSN-1—Our previous work showed that FSN-1 binding domain 1 (FBD1) of RPM-1 is sufficient for binding to FSN-1, and we identified point mutations in FBD1 that reduce binding in 293 cells [24]
This raised two points: there were likely to be other sites in RPM-1, besides FBD1, that were sufficient for binding to FSN-1, and identification of these sites was likely to require in vivo biochemistry from transgenic C. elegans

Summary

Introduction

The Pam/Highwire/RPM-1 (PHR) proteins are conserved intracellular signaling proteins that include human Pam/ MYCBP2, Drosophila Highwire, and Caenorhabditis elegans regulator of presynaptic morphology 1 (RPM-1) [1, 2]. The RBD covers a relatively large region of RPM-1 and is very close to FBD1 in the primary RPM-1 protein sequence At present, it remains uncertain whether residues in RPM-1 required for binding to RAE-1 impact binding to FSN-1 and vice versa. We use transgenic C. elegans to expand our structurefunction analysis of RPM-1 and FSN-1 in vivo This approach revealed two new domains in RPM-1, FBD2 and FBD3, which are sufficient for binding to FSN-1. Because these interactions were not observed previously using 293 cells [24], our results show that binding of FSN-1 to portions of RPM-1 requires in vivo expression in worm neurons. Transgenic overexpression experiments with fragments of RPM-1 and rescue experiments with RPM-1 deletion constructs indicate that the FBD2 and FBD3 are required for RPM-1 function in vivo

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Conclusion