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.