Neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily. We have reported that rat NLRR-3 gene expression is regulated mainly through the Ras-mitogen-activated protein kinase (MAPK) signaling pathway. NLRR-3 was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration (0.01 ng/ml) of epidermal growth factor (EGF), but the amplification of MAPK phosphorylation by NLRR-3 was no longer observed when the carboxyl terminal 30 amino acid stretch that contains clathrin-mediated endocytosis motifs was deleted. A green fluorescent protein-tagged NLRR-3 localized at the plasma membrane was efficiently internalized in COS-7 cells, but internalization of a carboxyl terminal-deleted version (NLRR*C) was less efficient. The presence of clathrin-adaptor protein complexes containing NLRR-3 in brain lysate was confirmed by immunoprecipitation and glutathione S-transferase pull-down experiments, and affinity column chromatography revealed that the carboxyl terminal region of NLRR-3 interacts with ß-adaptin. We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles.