Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2

Naoka Yamamoto,Manabu Kashiwagi,Manami Ishihara,Takaaki Kojima,Andrés D Maturana,Shun’Ichi Kuroda,Tomoaki Niimi

doi:10.1074/jbc.ra118.005819

Naoka Yamamoto, Manabu Kashiwagi + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra118.005819

Copy DOI

Abstract

The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.

Highlights

The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs
To investigate whether NELL1 binds to proteins of the Robo family (Fig. 1A), we first performed binding assays in solution using FLAG-tagged NELL1 and the HA-tagged extracellular domain (ECD) of Robo1– 4
Because NELL2 binds to fibronectin type III (FNIII) domains of Robo3 [21], we performed solutionbinding assays using the FNIII domains of Robo1– 4 instead of the ECD

Summary

Results

To investigate whether NELL1 binds to proteins of the Robo family (Fig. 1A), we first performed binding assays in solution using FLAG-tagged NELL1 and the HA-tagged extracellular domain (ECD) of Robo . To determine which domain of Robo interacts with NELL1, deletion mutants of Fc-tagged Robo2-FNIII were prepared, and the binding activity was examined. A581T mutation showed no significant induction, double to quintuple mutants exhibited 5–13-fold induction of NELL1binding activity These results reinforce the above suggestion that several amino acids including Thr581 of mRobo contribute to the NELL1 binding. We performed the solid-phase binding assay under acidic conditions to investigate whether low pH affects the NELL1binding activity to the Robo2-ECD protein.

B FLAG-NELL1

Discussion

Experimental procedures