TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome-associated protein NA14.
TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjögren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.
Aki
Toshihiko
T
Center for Gene Research, Yamaguchi University, Yamaguchi, Japan.
Funakoshi
Takeshi
T
Nishida-Kitayama
Junko
J
Mizukami
Yoichi
Y
D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D047108:Embryonic Development / Q000502:physiology*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids; D021381:Protein Transport / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
D001324:Autoantigens; C110204:NA14 nuclear autoantigen; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled; C000606723:TPRA1 protein, human