Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NR1

Abstract

The molecular machinery underlying neurotransmitter receptor immobilization at postsynaptic sites is poorly understood. The NMDA receptor subunit NR1 can form clusters in heterologous cells via a mechanism dependent on the alternatively spliced C1 exon cassette in its intracellular C-terminal tail, suggesting a functional interaction between NR1 and the cytoskeleton. The yeast two-hybrid screen was used here to identify yotiao, a novel coiled coil protein that interacts with NR1 in a C1 exon-dependent manner. Yotiao mRNA (11 kb) is present modestly in brain and abundantly in skeletal muscle and pancreas. On Western blots, yotiao appears as an ~230 kDa band that is present in cerebral cortex, hippocampus, and cerebellum. Biochemical studies reveal that yotiao fractionates with cytoskeleton-associated proteins and with the 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 colocalized 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 attachment of NMDA receptors. Consistent with a general involvement in postsynaptic structure, yotiao was also found to be specifically concentrated at the neuromuscular junction in skeletal muscle.