Identification of tomoregulin-1 as a novel addicsin-associated factor

Addicsin is a novel factor encoding a 23-kDa hydrophobic protein that is highly upregulated in the amygdala nuclei of morphine-administered mice. It is a murine homolog of human JWA and rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a negative modulator of the neural glutamate transporter excitatory amino acid carrier 1 (EAAC1). Recent findings demonstrated that addicsin participates in various physiological processes by forming hetero- or homomultimeric complexes. However, the binding targets and molecular functions of addicsin remain largely unknown. To identify potential factors that associate with mouse addicsin, we performed a yeast two-hybrid screen using a 17-day-old mouse whole embryo cDNA library. We identified tomoregulin-1 (TR1) as a novel addicsin-associated factor. TR1, a type I transmembrane protein containing two follistatin-like modules and an epidermal growth factor-like domain, participates in nodal and bone morphogenetic protein signaling. Immunoprecipitation assays demonstrated that TR1 bound to addicsin, and that amino acids 145-188 of addicsin and amino acids 228-266 of TR1 were important for the formation of the addicsin-TR1 heterocomplex. The double-fluorescent immunohistochemical analysis revealed that addicsin and TR1 were coexpressed in neurons in the mature mouse brain regions tested. Moreover, TR1 showed a punctuate pattern throughout the cell, with preferential expression on the cell surface when expressed alone. However, TR1 predominantly redistributed to the endoplasmic reticulum (ER) when coexpressed with addicsin. Furthermore, coexpression of an addicsin mutant that lacked TR1 binding ability had little effect on the distribution of TR1. Biotinylation assays showed that coexpression of addicsin with TR1 suppressed the cell surface expression of TR1. Wound-healing assays demonstrated that the interaction of addicsin with TR1 had a significant effect on cell migration. These findings demonstrate that addicsin in the ER controls intracellular TR1 trafficking from the ER to plasma membrane and regulates cell migration through its interaction with TR1.