Yotiao, a Novel Protein of Neuromuscular Junction and Brain That Interacts with Specific Splice Variants of NMDA Receptor Subunit NR1

TLDR

The molecular machinery that immobilizes neurotransmitter receptors at postsynaptic sites is poorly understood, and NR1 clustering depends on the alternatively spliced C1 exon, suggesting a functional interaction with the cytoskeleton. Thus yotiao is an NR1‑binding protein potentially involved in cytoskeletal attachment of NMDA receptors. The yeast two‑hybrid screen identified yotiao, a novel coiled‑coil protein that interacts with NR1 in a C1 exon‑dependent manner. Yotiao mRNA is modestly expressed in brain and abundant in skeletal muscle and pancreas, the protein (~230 kDa) localizes to cerebral cortex, hippocampus, cerebellum, postsynaptic density, and neuromuscular junctions, where it colocalizes with NR1 and co‑immunoprecipitates with it.

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 approximately 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.