Abstract
Some ubiquitin-like (UBL) domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS) protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP) were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain.Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.
Highlights
Proteins can be modified by either a single ubiquitin moiety or polymeric ubiquitin chains to alter their stability, localization, binding partners, or physical conformation [1,2]
We refer to this protein as Transmembrane and ubiquitin-like domain-containing 1 (Tmub1)/Hepatocyte Odd Protein Shuttling (HOPS) because it represents well the domain characteristics which used in our identification
When we introduced the RNAi of Tmub1/ HOPS (134–152 bp) to the hippocampal neurons at day in vitro (DIV) 14 and incubated them for additional 2 d, the signals detected by the antiTmub1/HOPS antibody were significantly reduced throughout the entire neurons, a few signals of Tmub1/HOPS were still observed in the cell body (Figure 2E)
Summary
Proteins can be modified by either a single ubiquitin moiety or polymeric ubiquitin chains to alter their stability, localization, binding partners, or physical conformation [1,2]. Ubiquitination has been reported to regulate cell surface receptors [3], such as AMPARs [4], and c-aminobutyric acid A receptors (GABAARs) [5]. UBL proteins and UBL domain-containing proteins appear to regulate a wide variety of proteins of various processes [6,7]. Some UBL proteins and UBL domain-containing proteins have been reported to be involved in receptor regulation. One of the UBL domain-containing proteins, Plic-1/ubiquilin-1, regulates the cell surface number and subunit stability of GABAARs [9]. The GABAAR-associated protein (GABARAP/ubiquilin-2), which contains a UBL core domain in the C-terminus [10], traffics GABAARs to the plasma membrane in neurons [11]
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