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Surface expression of TTYH2 is attenuated by direct interaction with ?-COP.

ABSTRACT: TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, ?-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTYH2-C) as a bait. Using in vitro and in vivo binding assays, we confirmed the protein-protein interactions between TTYH2 and ?-COP. We also found that the surface expression and activity of TTYH2 were decreased by co-expression with ?-COP in the heterologous expression system. In addition, ?-COP associated with TTYH2 in a native condition at a human colon cancer cell line, LoVo cells. The over-expression of ?-COP in the LoVo cells led to a dramatic decrease in the surface expression and activity of endogenous TTYH2. Collectively, these data suggested that ?-COP plays a critical role in the trafficking of the TTYH2 channel to the plasma membrane. [BMB Reports 2019; 52(7): 445-450].

SUBMITTER: Ryu J

PROVIDER: S-EPMC6675250 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Surface expression of TTYH2 is attenuated by direct interaction with β-COP.

Ryu Jiwon J Kim Dong-Gyu DG Lee Young-Sun YS Bae Yeonju Y Kim Ajung A Park Nammi N Hwang Eun Mi EM Park Jae-Yong JY

BMB reports 20190701 7

TTYH2 is a calcium-activated, inwardly rectifying anion channel that has been shown to be related to renal cancer and colon cancer. Based on the topological prediction, TTYH2 protein has five transmembrane domains with the extracellular N-terminus and the cytoplasmic C-terminus. In the present study, we identified a vesicle transport protein, β-COP, as a novel specific binding partner of TTYH2 by yeast two-hybrid screening using a human brain cDNA library with the C-terminal region of TTYH2 (TTY ...[more]

PMID: 30670146

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Project description:Bestrophin-1 (Best1) is a calcium-activated anion channel (CAAC) that is expressed broadly in mammalian tissues including the brain. We have previously reported that Best1 is expressed in hippocampal astrocytes at the distal peri-synaptic regions, called microdomains, right next to synaptic junctions, and that it disappears from the microdomains in Alzheimer's disease mouse model. Although Best1 appears to be dynamically regulated, the mechanism of its regulation and modulation is poorly understood. It has been reported that a regulatory protein, 14-3-3 affects the surface expression of numerous membrane proteins in mammalian cells.The protein-protein interaction between Best1 and 14-3-3? was confirmed by yeast-two hybrid assay and BiFC method. The effect of 14-3-3? on Best1-mediated current was measured by whole-cell patch clamp technique.We identified 14-3-3? as novel binding partner of Best1 in astrocytes: among 7 isoforms of 14-3-3 protein, only 14-3-3? was found to bind specifically. We determined a binding domain on the C-terminus of Best1 which is critical for an interaction with 14-3-3?. We also revealed that interaction between Best1 and 14-3-3? was mediated by phosphorylation of S358 in the C-terminus of Best1. We confirmed that surface expression of Best1 and Best1-mediated whole-cell current were significantly decreased after a gene-silencingof 14-3-3? without a significant change in total Best1 expression in cultured astrocytes. Furthermore, we discovered that 14-3-3?-shRNA reduced Best1-mediated glutamate release from hippocampal astrocyte by recording a PAR1 receptor-induced NMDA receptor-mediated current from CA1 pyramidal neurons in hippocampal slices injected with adenovirus carrying 14-3-3?-shRNA. Finally, through a structural modeling, we found critical amino acid residues containing S358 of Best1 exhibiting binding affinities to 14-3-3?.14-3-3? promotes surface expression of Best1 channel in astrocytes through direct interaction.

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Surface expression of TTYH2 is attenuated by direct interaction with ?-COP.

Publications

Surface expression of TTYH2 is attenuated by direct interaction with β-COP.

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