YOTIAO, A NOVEL PROTEIN OF NEUROMUSCULAR-JUNCTION AND BRAIN THAT INTERACTS WITH SPECIFIC SPLICE VARIANTS OF NMDA RECEPTOR SUBUNIT NR1

The molecular machinery underlying neurotransmitter receptor immobiliz ation at postsynaptic sites is poorly understood. The NMDA receptor su bunit NR1 can form clusters in heterologous cells via a mechanism depe ndent on the alternatively spliced C1 exon cassette in its intracellul ar C-terminal tail, suggesting a functional interaction between NR1 an d the cytoskeleton. The yeast two-hybrid screen was used here to ident ify yotiao, a novel coiled coil protein that interacts with NR1 in a C 1 exon-dependent manner. Yotiao mRNA (11 kb) is present modestly in br ain and abundantly in skeletal muscle and pancreas. On Western blots, yotiao appears as an similar to 230 kDa band that is present in cerebr al cortex, hippocampus, and cerebellum. Biochemical studies reveal tha t yotiao fractionates with cytoskeleton-associated proteins and with t he postsynaptic density. With regard to immunohistochemistry, two anti -yotiao antibodies display a somatodendritic staining pattern similar to each other and to the staining pattern of NR1. Yotiao was colocaliz ed by double-label immunocytochemistry with NR1 in rat brain and could be coimmunoprecipitated with NR1 from heterologous cells. Thus yotiao is an NR1-binding protein potentially involved in cytoskeletal attach ment of NMDA receptors. Consistent with a general involvement in posts ynaptic structure, yotiao was also found to be specifically concentrat ed at the neuromuscular junction in skeletal muscle.