ORIGINAL ARTICLE

JCP07-0644.R1(21492)
Journal of

TPRA40/GPR175 Regulates
Early Mouse Embryogenesis
Through Functional Membrane
Transport by Sjögren’s
Syndrome-AssociatedProtein NA14

CellularPhysiology

TOSHIHIKO AKI, TAKESHI FUNAKOSHI,JUNKO NISHIDA-KITAYAMA, AND YOICHI MIZUKAMI*
Center for Gene Research, Yamaguchi University, Yamaguchi, Japan
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 timedependent manner and the translocation was inhibited by GFP-NA14DN, an N-terminal deletion mutant that cannot bind to microtubules
but binds to TPRA40. TPRA40DC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found thatthe effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DN. These observations
indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.
J. Cell. Physiol. 9999: 1–13, 2008. ß 2008 Wiley-Liss, Inc.

G protein-coupled receptors (GPCRs) comprise a large
superfamily of genes accounting for about 1% of the humangenome, and have attracted much attention because
approximately 50% of drugs target GPCRs (Drews, 1996).
GPCRs transmit signals from the extracellular environment to
the intracellular cytoplasm through activation by their cognate
ligands (Marchese et al., 1999; Civelli et al., 2001). Althoughlarge numbers of orphan GPCRs were discovered during thecourse of the genome project (Horn et al., 1998, 2003),
the cognate ligands and physiological functions of many of theseorphan receptors remain unknown. GPCRs regulate
intracellular signal transduction pathways, at least in part,through the generation of reactive oxygen species (ROS). For
example, the generation of ROS is required for the activation ofvascular smooth muscle cell proliferation by angiotensin II(Griendling et al., 1994) and thrombin (Patterson et al., 1999).
Angiotensin II and thrombin also activate Jak/STAT pathways
through the activation of Rac small GTPase and the subsequent
generation of ROS (Pelletier et al., 2003). During oxidative
stress caused by ischemia/reperfusion, we have demonstrated
that mitogen-activated protein kinase/extracellularsignal-regulated kinase (MAPK/ERK) translocates from thecytoplasm to the nucleus, and is activated in the nucleus in rat
heart (Mizukami and Yoshida, 1997). The nuclear translocation
and activation of ERK are dependent on the activations ofphosphoinositide-3 kinase (PI3K) and protein kinase C (PKC)z,
suggesting that the PI3K/PKCz/ERK pathway is activated duringoxidative stress (Mizukami and Yoshida, 1997; Mizukami et al.,1997, 2000). ERK maintains contractility and survival during
reperfusion in rat cardiomyocytes through the c-Myc-mediatedup-regulation of glycolytic a-enolase, as revealed by aproteomic approach (Mizukami et al., 2004). Since the PI3K/
ERK pathway is activated by GPCRs (Stephens et al., 1994;
ß 2 0 0 8 W I L E Y - L I S S , I N C .

Cantrell, 2001), it is possible that GPCRs might be responsible
for the activation of the PI3K/PKCz/ERK and the subsequentcellular responses during oxidative stress.
We have screened GPCRs whose expressions are regulated
during hypoxia and reoxygenation, and identified two orphan
GPCRs. The expression of TPRA40 (a 40 kDa transmembraneprotein regulated in adipocytes; corresponds to mouse
GPR175) declines rapidly during hypoxia and reoxygenation(Fujimoto et al., 2001), whereas GPR30 is up-regulated during
reoxygenation after hypoxia (Kimura et al., 2001). Although wehave revealed that GPR30 activates a p53-dependent apoptotic
pathway during oxidative stress caused by ischemia/reperfusion(Kimura et al., 2001), no physiological functions for TPRA40

Abbreviations: TPRA40, trans-membrane protein of 40 kDa
regulated in adipocytes; NA14, nuclear autoantigen of 14 kDa; SS,
Sjögren’s syndrome; MT, microtubule; GFP, green fluorescentprotein; GPCR, G protein-coupled receptor; shRNA, short hairpin
RNA; DAPI, 40 ,6-diamidino-2-phenylindole; SDS–PAGE, sodium
dodecyl sulfate–polyacrylamide gel electrophoresis; ROS, reactive
oxygen species; GST, glutathione S-transferase; RT, reverse
transcription; PCR, polymerase chain reaction.
Contract grant sponsor: Ministry of Education, Culture, Sports,
Science and Technology of Japan.
*Correspondence to: Yoichi Mizukami, Center for Gene Research,
Yamaguchi University, 1-1-1 Minami-Kogushi, Ube, Yamaguchi
755-8505, Japan. E-mail: mizukami@yamaguchi-u.ac.jp
Received 14 November 2007; Accepted 3 April 2008
Published online in Wiley InterScience(www.interscience.wiley.com.), 00 Month 2008.
DOI: 10.1002/jcp.21492

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have been revealed to date. TPRA40 is a seven transmembrane
domain protein whose expression is induced during thedifferentiation of 3T3-L1 cells from fibroblasts to adipocytes(Yang et al., 1999). We have demonstrated that TPRA40
contains a longer (84 amino acids) cytoplasmic region (Fujimoto
et al., 2001), compared with the relatively short (13 aminoacids) C-terminal putative cytoplasmic region of the initially
identified TPRA40 (Yang et al., 1999). The additional
71 amino acids include several consensus sequences forpost-translational modifications including PKC
phosphorylation, N-glycosylation, and myristoylation,
indicating the possible importance of the C-terminal region for
the functional expression of TPRA40 (Fujimoto et al., 2001).
TPRA40 shows low homology to other orphan receptors.
Although a combined phylogenetic/ligand analysis showed thateach GPCR can be assigned to one of four classes of GPCRs (A,B, C, and F/S), TPRA40 is classified into a fifth GPCR group thatcannot be assigned to any of the known classes (Vassilatis et al.,2003). It has been reported that the expression of TPRA40 isincreased in the epididymal fat of 24-month-old mice ascompared with young controls, and is also increased in obeseand diabetic mice as compared with age-matched controls(Yang et al., 1999), suggesting that TPRA40 expression isregulated by environmental conditions. Despite the lack ofstructural as well as physiological clues to uncover the functionof TPRA40, the regulation of TPRA40 expression duringoxidative stress, aging, and under pathophysiological conditions
suggests that TPRA40 should play important roles in cellularresponses related to diseases.
During an attempt to generate knockdown mice with areduced expression of TPRA40, we found that TPRA40
regulates the cell division of early mouse embryos. Using a yeast
two-hybrid system, nuclear autoantigen of 14 kDa (NA14), an
autoantigen of Sjögren’s syndrome, an inflammatory disease(Ramos-Morales et al., 1998), was identified as a TPRA40
binding protein. The functional plasma membrane expression of
TPRA40 is mediated by NA14, and TPRA40 involved in theregulation of cell division in early mouse embryos.
Experimental Procedures
Materials

Anti-FLAG mouse monoclonal antibody was purchased from
Sigma (St. Louis, MO). Anti- green fluorescent protein (GFP)
mouse monoclonal antibody was purchased from Santa Cruz(Santa Cruz, CA). Peroxidase-conjugated anti-mouseand -rabbit IgG antibodies were purchased from Promega(Madison, WI). Anti-actin antibody and anti-FLAG mousemonoclonal antibody-immobilized beads and glutathione
Sepharose 4B were purchased from Sigma andAmersham-Pharmacia (Buckinghamshire, UK), respectively.
Streptavidin magnet particles were from New England Biolabs(Beverly, MA). Peroxidase-conjugated streptavidin was from
Invitrogene (Carlsbad, CA). All other reagents werecommercially available.
Production of polyclonal antibody

A rabbit polyclonal antibody against TPRA40 was generatedusing a peptide corresponding to a region of rat TPRA40 (aminoacids 139–149) as an antigen. Sera were collected and theantibody was purified by ammonium sulfate precipitation.
Plasmids

To create TPRA40 short hairpin RNA (shRNA) expression
vectors, two pairs of synthesized oligo DNAs (type I; sense
strand 50 -TCCCAAATGCCAAGTGGACGAGGCTTTCAAGAGAAGCCTCGTC-CACTTGGCATTTTT-30 and
JOURNAL OF CELLULAR PHYSIOLOGY

antisense strand 50 -CAAAAAAAATGCCAAGTGGACGAGGCTTCTCTTGAAAGCCTCGTCCACTTGGCATTT-30 , type II; sense strand 50 -TCCCAAGATCCTGTGGGAAATCACCTTCAAGAGAGGTGATTTCCCACAGGATCTTTT-30 and antisense strand 50 -CAAAAAAAGATCCTGTGGGAAATCACCTCTCTTGAAGGTGATTTCCCACAGGATCTT-30 ) were annealed and ligated into
psiRNA-Hh1neo-CX-EGFP, which was constructed by ligating
the pCX-EGFP gene (Hasuwa et al., 2002) as a marker gene into
psiRNA-Hh1neo (Invivogene, San Diego, CA). The mutation
9 and 10 for TPRA40 shRNA-II were created with a point
mutation at the position 9 and 10 of the targeting sequence forthe shRNA-II, respectively, as indicated in boldfaces (Pusch
et al., 2003) (type II mutation 9; sense strand 50 -TCCCAAGATCCTATGGGAAATCACCTTCAAGAGAGGTGATTTCCCATAGGATCTTTT-30 and mutation 9; antisense strand
50 -CAAAAAAAG-ATCCTATGGGAAATCACCTCTCTTGAAGGTGATTTCCCATAGGATCTT-30 , type II
mutation 10; sense strand 50 -TCCCAAGATCCTGCGGGAAATCACCTTCAAGAGAGGTGATTTCCCGCAGGATCTTTT-30 and mutation 10; antisense strand 50 -CAAAAAAAGATCCTGCGGGAAATCACCTCTCTTGAAGGTGATTTCCCGCAGGATCTT-30 ). An N-terminal FLAG-tagged
TPRA40 (FLAG-TPRA40) expression vector was created byligating the full-length rat TPRA40 cDNA, which was cloned
previously (Fujimoto et al., 2001), into p3xFLAG-CMV10(Sigma). A C-terminal deletion mutant of FLAG-TPRA40(FLAG-TPRA40DC) was created by PCR using the sense
primer 50 -GCGAATTCAATGGCCAGCCTG-30 , antisense
primer 50 -CGGGATCCTCAGGGTTCAGA-30 , and FLAGTPRA40 as a template. Gateway Technology (Invitrogene) wasused to create the GFP- and GST-tagged NA14 expression
vectors. Human NA14 cDNA was amplified from human kidney
cDNA with sense primer 50 -GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGACCCAGCAGGGCGCG-30
and antisense primer 50 -GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACACACTTGCTGGGGCTGAG-30 .
The PCR product was cloned into pDONR207 to create theentry clone (pDONR207NA14). Then, N-terminal-tagged
FLAG-NA14, GFP-NA14, and glutathione S-transferase(GST)-NA14 expression vectors were created using a sitespecific recombination reaction according to the instruction
manual. A N-terminal GFP-tagged deletion mutant of NA14,
GFP-NA14DN, was created by ligating a N-terminal deletionfragment of human NA14 (amino acids 22–129) to pEGFP-C1(Clonetech, Palo Alto, CA). To create pT7-NA14, PCR was
carried out with sense primer 50 -TAATACGACTCACTATAGGATGACCCAGCAGGGCGC-30 , antisense primer
50 -TAATACGACTCACTATAGGACACTTGCTGGGGCGAG-30 , and pDONR207NA14 as a template. The PCR
product was ligated to pUC13 and used as a template for in vitrotranscription. The nucleotide sequences were confirmed bysequencing.
Injection to mouse embryos

The microinjection of vector DNA into mouse embryos
was performed using standard methods (Hogan et al., 1994). In
brief, fertilized one-cell embryos were collected from theoviducts of superovulated C57BL/6 female mice (8 weeks old).
The purified vector DNA (7 ng/ml) was injected into the pro
nuclei of the zygotes. The surviving embryos were cultured in a
microdrop of M16 medium in an atmosphere of 95% air/
5% CO2 at 378C, and were observed under a microscope for4 days after injection. The significant variation was unobservedin no injection control. A few empty vectors may be influenced
in the cell division. The development of embryos wasdetermined simultaneously in the comparative group (empty
vector vs. expression vector).

REGULATION OF EARLY EMBRYOGENESIS BY TPRA40

Yeast two-hybrid screening

The C-terminal fragment of rat TPRA40 (amino acids 286–369)
was subcloned into a bait vector pGBKT7 to fuse the GAL4
DNA binding domain. The yeast strain AH109 was firsttransformed with the bait vector and was subsequently
transformed with a prey vector of a human kidney cDNA library(pACT2) fused with the GAL4 transcriptional activation
domain. Approximately 2  106 transformants were screened
for histidine prototrophy in the presence of 5 mM
3-aminotriazole.
Cell culture and transfection

COS7 cells and HeLa cells were maintained in DMEM
supplemented with 10% FBS at 378C under a 5% CO2
atmosphere. All plasmids were transfected using
LipofectAMINE2000 (Invitrogen) according to the instructions
provided by manufacturer as described previously (Mizukami
et al., 2004).
FACS analysis

Cells harvested with PBE (5 mM EDTA in phosphate bufferedsaline with 0.5% bovine serum albumin) were fixed with
70% ethanol and incubated with phosphate-buffered saline
containing 5 mg/ml propidium iodide and 5 mg/ml RNase for
30 min at room temperature. At least, 10,000 cells werecounted per samples. FACS analyses were performed using a
BD Bioscience flow cytometer (FACSCalibur).
GST pull-down, immunoprecipitation,and immunoblotting

For GST pull-down experiments, COS7 cells grown toapproximately 80% confluence on 10 cm diameter dishes weretransfected with 10 mg GST-NA14 or FLAG-TPRA40 for 48 h.
Then, the cells were lysed in lysis buffer [1% Triton X-100,
20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate,
1 mM sodium orthovanadate, 1 mg/ml leupeptin, 1 mM
phenylmethanesulfonyl fluoride], and cellular extracts weremixed and incubated with glutathione Sepharose 4B. Then, thebeads were precipitated and washed three times with lysis
buffer, and the co-precipitated proteins were subjected toimmunoblotting. Immunoblotting was carried out as described
previously (Mizukami et al., 2004). For co-immunoprecipitation
experiments, COS7 cells were grown to approximately
80% confluence in 3.5 cm diameter dishes and transfected with
2 mg each of FLAG-TPRA40 vector and GFP-NA14 vector.
Forty-eight hours after transfection, the cells were washed with
PBS and lysed in lysis buffer. The cellular extracts were thenincubated with anti-FLAG antibody-immobilized beads
overnight at 48C. Then, the beads were collected bycentrifugation and washed three times with lysis buffer. The
precipitates were eluted from the beads by incubation in
SDS–PAGE sample buffer at 958C for 3 min, and were used for
immunoblotting. In vivo GST pull-down experiments were
performed as the co-immunoprecipitation experiments exceptthat the GST-NA14 vector and glutathione Sepharose 4B wereused instead of GFP-NA14 and anti-FLAG antibodyimmobilized beads, respectively.
Selection of transfected-cells by magnet particles

To select cells, the pMACS4.1 vector, which expresses a
truncated CD4 cell surface marker, was co-transfected withFLAG vectors as described previously (Mizukami et al., 2004).
Briefly, 24 h after transfection, the cells were selected with amagnet beads-bound antibody for CD4, followed by theselection by G418 (final concentration: 750 mg/ml). The
selected cells were observed under a bright field microscope on
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days 4–7 after transfection, and the numbers of cells werecounted.
Fluorescence microscopy

For immunofluorescence experiments, cells were grown toapproximately 50% confluence on coverslips and transfectedwith vectors as described previously (Kimura et al., 2001). Aftertransfection, the cells were fixed with 4% paraformaldehyde in
PBS for 10 min and made permeable by incubation in 0.5%
Triton X-100 in PBS for 5 min. Then, the cells were blockedwith 10% FBS in PBS for 30 min, and incubated with anti-FLAG
antibody overnight at 48C. After incubation with a
Cy3-conjugated anti-mouse IgG antibody and DAPI (final
concentration: 1 mg/ml) for 30 min, the cells were observed
under an inverted fluorescence microscope (IX70, Olympus,
JapanQ1). Images were processed using MetaMorph software(Universal Imaging CorpQ2.).
RNA interference

A small interference RNA (siRNA) mixture for NA14 wascreated using a Short Cut RNAi kit (New England Biolab)
according to the instructions. In brief, both the sense andantisense full-length RNAs of NA14 were created by in vitrotranscription using T7 RNA polymerase and pT7-NA14 plasmid
as a template. The sense and anti-sense RNAs were annealed tocreate double-stranded RNA, which was then digested to
21–23 base pair fragments by Short Cut RNase. Complete
digestion was checked by polyacrylamide gel electrophoresis
followed by ethidium bromide staining. The siRNA mixture wastransfected into cells at a concentration of 4 nM.
Cell surface labeling of FLAG-TPRA40

Cells were incubated with 4 mM sulfo-NHS-biotin (Pierce) in
PBS for 30 min at 48C, and inactivated by washing three times in
4 mM glycine in PBS. Cells were lysed by sonication in lysis buffer
and incubated with streptavidin magnet beads at 48C for 1 h.
Then, the beads were washed with lysis buffer three times andthe biotinylated proteins were eluted from the beads by
incubation in SDS–PAGE sample buffer at 958C for 3 min.
Reverse transcription (RT)-polymerase chain
reaction (PCR) method

Fertilized one-cell embryos were collected from the oviducts ofsuperovulated C57BL/6 female mice (8 weeks old), and were
cultured in a microdrop of M16 medium in an atmosphere of
95% air/5% CO2 at 378C for 2 days. The embryos were
resuspended in lysis buffer in Cell-to-Signal kit (Applied
Biosystems, Foster City, CA) after removal of zona pellucida.
First-strand cDNA was synthesized directly using chemicals
from the Cells-to-Signal kit and primed by pd (N)10 primers.
mRNA expressed in mouse brain was directly extracted byusing MagNA Pure LC system with MagNA Pure LC mRNA
Isolation Kit II (tissue), according to the manufacturer’s
instructions (Roche Diagonistics, Mannheim, Germany). RT
was performed with mRNA and random primer using
SUPERSCRIPT II (Invitrogene). The products of the RT reaction
were amplified by 40 cycles of PCR using KOD dash
(TOYOBO, Osaka, Japan) at 948C for 30 sec, 628C for 30 sec,
and 748C for 30 sec for NA14, and 948C for 30 sec, 628C for
2 sec, and 748C for 30 sec for TPRA40 and G3PDH. The
50 -ACCGGACCATCGCAGAAAC-30 (sense) and
50 -TCAGCTGTCCTTGCCTCCA-30 (antisense) primers for
NA14, 50 -CACTGAAGGAGCGTGTATC-30 (sense) and
50 -ACAGCAGAGCCCCACAAT-30 (antisense) primers for
TPRA40, or 50 -ACCACAGTCCATGCCATCAC-30 (sense)and 50 -TCCACCACCCTGTTGCTG-30 (antisense) primers
for G3PDH were used to observe the expression. The

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Fig. 1. Effect of short hairpin RNA-mediated knockdown of TPRA40 on cell division of mouse embryos. A,B: Short hairpin RNA expression
vectors, TPRA40-shRNA-I and -II, were transfected into COS7 cells with the FLAG-TPRA40 expression vector (A) or into H9c2 cells without the
FLAG-TPRA40 expression vector (B). Forty-eight hours after transfection, cell extracts were prepared and subjected to immunoblotting with ananti-FLAG antibody (A) or an anti-TPRA40 antibody (B). Similar results were obtained in two separate experiments. C,D: The GFP-TPRA40
expression vector and shRNA-I (C), -II (D), or shRNA-empty vector removed short hairpin sequences (C,D) as the indicated, were
simultaneously injected into mouse embryos 1 day after fertilization, were incubated in a drop of M16 medium, and were observed at 48 h after the
injection under a fluorescence microscopy (left parts). Similar results were obtained in three separate experiments. The embryos were also
observed under a bright field (right parts). E: A short hairpin RNA expression vector, TPRA40-shRNA-I, TPRA40-shRNA-II, or shRNA-empty
vector removed the short hairpin sequence, was injected into mouse embryos 1 day after fertilization and the embryos were incubated in a drop of
M16 medium. Forty-six (shRNA-empty vector), 50 (TPRA40-shRNA-I), 53 (TPRA40-shRNA-II) embryos were used in each group (MP < 0.05 forshRNA-empty vector; Mann–Whitney’s U-test). F: The GFP-TPRA40 expression vector and shRNA-II vector, mutation 9 with a point mutation at
the position 9 of the targeting sequence for shRNA-II, or mutation 10 with a point mutation at the position 10 were simultaneously injected into
mouse embryos 1 day after fertilization, were incubated in a drop of M16 medium, and were observed at 48 h after the injection under a fluorescencemicroscopy (upper parts). The embryos were also observed under a bright field (lower parts). Similar results were obtained in three
separate experiments. G: TPRA40-shRNA-II, mutation 9 or mutation 10, was injected into mouse embryos 1 day after fertilization and the embryos
were incubated in a drop of M16 medium. Fifty-three (shRNA-II for TPRA40), 50 (mutation 9), 50 (mutation 10) embryos were used in each group(MP < 0.05 for TPRA40-shRNA-II; Mann–Whitney’s U-test). The development of the embryos was observed at the indicated times under a brightfield microscope during the 4 days after fertilization.

JOURNAL OF CELLULAR PHYSIOLOGYREGULATION OF EARLY EMBRYOGENESIS BY TPRA40

Fig. 1.

amplified fragments were separated by electrophoresis on a
1.8% agarose gel.
Results
Knockdown of TPRA40 by RNA interference
accelerates cell division of early mouse embryos

To investigate the physiological role of TPRA40, we first tried
to generate transgenic mice that express a shRNA of TPRA40.
Two candidate sequences for the shRNA were selected fromthe region in which the nucleotide sequences are perfectlyconserved in the mouse and rat TPRA40 cDNAs. The effects ofthese shRNAs on TPRA40 expression were examined byimmunoblotting using anti-FLAG and anti-TPRA40 antibodies
for exogenous and endogenous TPRA40 expression. As shownin Figure 1A, both vectors (TPRA40 shRNA-I and -II)
suppressed FLAG-TPRA40 expression more thanapproximately 50% in COS7 cells. In H9c2 cells, endogenous
JOURNAL OF CELLULAR PHYSIOLOGY(Continued )TPRA40 expression was also suppressed by these shRNAs(Fig. 1B). To confirm that shRNAs work in mouse early
embryos, TPRA40 shRNA-I or -II was injected into the embryoswith GFP-TPRA40. ShRNAs for TPRA40 inhibited the
GFP-TPRA40 expression, indicating the shRNAs were usefulfor the embryos (Fig. 1C,D). We next examined the effect of
microinjecting the shRNA expression vector on thedevelopment of mouse embryos. Of 46 embryos injected withshRNA-empty vector, 14 embryos developed to the 8-cell stage
by 4 days after fertilization (Fig. 1E). On the other hands, of
50 embryos and 53 embryos injected the TPRA40 shRNA-I andshRNA-II vector, respectively, more than 26 (56%) embryos
developed to the 8-cell stage or more by the same time
(Fig. 1E, P < 0.05 vs. vector control). To confirm the findings, weprepared the mutant 9 and 10 with a point mutation at theposition 9 and 10 of the target sequence for shRNA-II,
respectively. The both mutants for shRNA-II entirely restored
in GFP-TPRA40 expression to control level (Fig. 1F), and the5

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cell divisions in early embryos injected the mutants wereconsiderably slow as compared with those injected shRNA-II
for TPRA40, and were similar to the data in the embryos
injected shRNA-empty vector (Fig. 1G). These results suggestthat TPRA40 negatively regulates the cell division of the earlymouse embryos.
The C-terminal region of TPRA40 is required for the
regulation of mouse embryogenesis

To determine the region essential for the effect of TPRA40 oncell division, the proliferation of HeLa cells expressing
FLAG-TPRA40 or its C-terminal deletion mutant,
FLAG-TPRA40DC (Fig. 2A), was examined by counting cellnumbers for 4 days. As shown in Figure 2B, TPRA40 significantly
decreased the proliferation of HeLa cells compared with thecontrol ( P < 0.05 vs. vector control) at days 2 and 3. The
proliferation of cells expressing TPRA40DC was unchanged for
4 days compared with the control (Fig. 2B). These results
suggest that TPRA40 regulates HeLa cell proliferation throughits C-terminal region. To determine at which stage of the cell
cycle is involved in the proliferation by TPRA40, we performed
the cell cycle analysis upon serum stimulation in HeLa cells
expressing TPRA40. As shown in Figure 2C,D, overexpressionof FLAG-TPRA40 induced a decrease in cell population in G2/M
phase and an increase in cell population in G1 phase comparedwith HeLa cells expressing empty vector. In contrast, there wasno difference in profile of cell population between empty
vector- and TPRA40DC-expressing cells (Fig. 2C,E). These
results suggests that overexpression of TPRA40 may
participates in G2/M arrest. We next examined the effects of

TPRA40 and TPRA40DC on the development of mouseembryos. Of the TPRA40-injected 32 embryos, 11 (35%)
developed to the 8-cell stage by 4 days after fertilization (Fig. 3),
while approximately 51% of the control vector-injected
embryos developed to the 8-cell stage (Fig. 3). Theseobservations indicate that the development in the embryos
injected FLAG-TPRA40 cDNA was delayed as compared withthat in FLAG-empty vector, consistent with the results in HeLa
cell transfected with FLAG-TPRA40. The TPRA40DC-injected
embryos developed at the same rate as vector control by 4 days
after fertilization (Fig. 3). These observations indicate that
TPRA40 regulates the proliferation/division of cells through its
C-terminal region.
Screening of TPRA40-interacting proteins by
the yeast two-hybrid system

To identify binding proteins for the putative C-terminal
cytoplasmic tail of TPRA40, we carried out yeast two-hybrid
screening with a cDNA library derived from human kidney using
the C-terminal fragment of TPRA40 as bait. Of 200,000
transformants, 31 clones that grew in the absence of histidine,
tryptophan, adenine and leucine were isolated. Among them,
3 clones turned out to be positive in the a-galactosidase
spot assay (data not shown). Clone 6 encodes an
N terminal-deleted partial fragment of human NA14 (Nuclear
Autoantigen for Sjögren’s syndrome of 14 kDa), a protein
identified as an antigen recognized by the autoimmune serumof a patient with Sjögren’s syndrome (Ramos-Morales et al.,1998). The characterizations of the other clones are now inprogress.

Fig. 2. Effects of TPRA40 on the proliferation of HeLa cells (A) Schematic representation of the structures of rat TPRA40 and its C-terminal
deletion mutant. B: HeLa cells were transfected with the indicated vectors in addition to pMACS4.1, which encodes a truncated human CD4 surface
molecule, and selected with anti-CD4 antibody and G418. Cells were counted under a bright field microscope for 4 days after selection(mean W SE, n U 3; MP < 0.05 vs. control vector). C–E: HeLa cells transfected with the indicated vectors, were stimulated by serum for 20 h after theserum removal for 24 h, and subjected to FACS analysis as described under Experimental Procedures. Similar results were obtained in three
separate experiments.

JOURNAL OF CELLULAR PHYSIOLOGYREGULATION OF EARLY EMBRYOGENESIS BY TPRA40

Fig. 3. Effects of TPRA40 on the cell division of mouse embryos 1 day after fertilization, embryos were injected with the indicatedvectors and incubated in a drop of M16 medium. The embryos were observed at the indicated times under a bright field microscope. Thirty-nine(FLAG-empty), 34 (FLAG-TPRA40), or 38 (FLAG-TPRA40DC) embryos were used for each group (MP < 0.05 for FLAG-empty; Mann–Whitney’s
U-test). Typical images of embryos (part A) and summarized data of the developmental stages (part B) are shown.

TPRA40 interacts with NA14 in vitro and in vivo

To verify the binding of TPRA40 to NA14 in vitro, we
performed GST pull-down assays with mixtures of COS7 celllysates transfected with either FLAG-TPRA40 or GST-NA14.
As shown in Figure 4A, a band with a molecular mass
corresponding to approximately 40 kDa was detected only inthe lysate transfected with FLAG-TPRA40 (Fig. 4A, lane 4), but
not in control lysate (Fig. 4A, lane 1), indicating the expressionof FLAG-TPRA40. Cell lysates transfected with FLAG-TPRA40
or GST-NA14 were mixed, pulled-down with GST-NA14, andanalyzed by immunoblotting with an anti-FLAG-antibody.
FLAG-TPRA40 was detected in the precipitate pulled-downwith GST-NA14 (Fig. 4A, lane 6), but not in the precipitate
pulled-down with GST (Fig. 4A, lane 5). FLAG-TPRA40 was
undetected in precipitates not transfected with FLAG-TPRA40(Fig. 4A, lanes 2 and 3). These results indicate that TPRA40
binds to NA14 in vitro. To test the interaction between
TPRA40 and NA14 in vivo, FLAG-TPRA40 and GFP-NA14
were co-transfected into COS7 cells. Immunoblotting analyses
using an anti-FLAG-antibody showed that comparable amountsof FLAG-TPRA40 were detected only in cell lysates transfectedwith FLAG-TPRA40 (Fig. 4B, lanes 1–4). Comparable
expressions of GFP and GFP-NA14 were also confirmed using
JOURNAL OF CELLULAR PHYSIOLOGYan anti-GFP antibody (Fig. 4B, lanes 5–8). Then, the cell lysates
were immunoprecipitated with an anti-FLAG antibody andanalyzed by immunoblotting using an anti-GFP antibody.
GFP-NA14 was detected only in the precipitate transfectedwith FLAG-TPRA40 and GFP-NA14 (Fig. 4B, lane 12). No bands
were observed in the precipitates transfected with FLAGTPRA40 and GFP (Fig. 4B, lane 11), or in the precipitatestransfected with FLAG-empty (Fig. 4B, lanes 9 and 10). These
results indicate that TPRA40 interacts with NA14 in vivo.
To access the specificity of the binding between NA14 andTPRA40, we examined whether FLAG-GPR30, another orphan
receptor, also binds to NA14. GST-NA14 and either
FLAG-TPRA40 or FLAG-GPR30 were co-transfected into
COS7 cells. FLAG-TPRA40 was detected in the precipitate
pulled-down with GST-NA14 while FLAG-GPR30 was notdetected (Fig. 4C, lanes 5 and 6). The expressions of
FLAG-TPRA40 and FLAG-GPR30 in COS7 cells were almost
the same (Fig. 4C, lanes 1–3). These results indicate that
TPRA40 specifically interacts with NA14.
NA14 co-localizes with TPRA40 in HeLa cells

We first examined the subcellular localizations of NA14 with
distinct tag-polypeptides, FLAG-NA14 and GFP-NA14, in HeLa

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and GFP-TPRA40 was distributed largely on the endoplasmic
reticulum (data not shown). Therefore, FLAG and GFP as
tag-polypeptides for TPRA40 and NA14, respectively, wereused for further experiments. GFP-NA14 and FLAG-TPRA40
were co-transfected to HeLa cells and the localizations of these
proteins were analyzed 12, 24, 36, and 48 h after transfection.
Both GFP-NA14 and FLAG-TPRA40 showed punctuatedlocalization in the cytoplasm (Fig. 5B, e–g and I–k) and partiallyco-localized during 12–36 h after transfection as indicated by
the arrowheads (Fig. 5B, m–o). Forty-eight hours after
transfection, FLAG-TPRA40 was largely transported to theplasma membrane, and small amounts of FLAG-TPRA40 wereobserved in the cytoplasm (Fig. 5B, h). These results indicatethat FLAG-TPRA40 is transported to the plasma membrane in atime-dependent manner, and co-localizes with GFP-NA14
during the process of plasma membrane transport.
Plasma membrane transport of TPRA40 isinhibited by NA14DN

Fig. 4. Binding of TPRA40 and NA14 in vitro and in vivo (A) COS7
cells were transfected with the GST-NA14 or FLAG-TPRA40
expression vector for 48 h, and extracted as described inExperimental Procedures Section. Then the cell lysates wereincubated at 4-C over night with glutathione-immobilized beads,
precipitated, and subjected to immunoblotting with an anti-FLAG
antibody (lanes 1–6). Similar results were obtained in three separate
experiments. B: COS7 cells were co-transfected with the indicatedvectors for 48 h and extracted. Cell lysates were subjected toimmunoblotting with both anti-FLAG (lanes 1–4) and anti-GFP (lanes
5–8) antibodies to confirm the expressions of the proteins.
Immunoprecipitation was performed with anti-FLAG antibodyimmobilized beads, and the precipitated proteins were subjected toimmunoblotting with an anti-GFP antibody (lanes 9–12). Similar
results were obtained in three separate experiments. C: COS7 cells
were co-transfected with GST-NA14 in combination with
FLAG-TPRA40 or FLAG-GPR30 for 48 h and extracted. The celllysates were subjected to immunoblotting with an anti-FLAG
antibody to confirm the expressions of the proteins (lanes 1–3). The
cell lysates were also incubated with glutathione-immobilized beads
at 4-C overnight, and subjected to immunoblotting with an anti-FLAG
antibody (lanes 4–6). Similar results were obtained in three separate
experiments.

cells to select useful tag-polypeptides for immunocytochemical
studies. As shown in Figure 5A, GFP-derived fluorescence(green) and anti-FLAG antibody-derived fluorescence (red)were largely merged (yellow), suggesting that both FLAG- and
GFP-tags do not affect the subcellular localization of NA14.
When FLAG-TPRA40 and GFP-TPRA40 were co-expressed in
HeLa cells, FLAG-TPRA40 inserted into the plasma membrane
JOURNAL OF CELLULAR PHYSIOLOGY

Since NA14 was previously reported to be associated withmicrotubules (MTs), we examined whether NA14 is involved in
the transport of TPRA40 to the plasma membrane throughMTs. NA14 contains a typical coiled-coil motif, which is
speculated to be necessary for the association with MTs, at its
N-terminus. The effect of N-terminal-deleted NA14
(NA14DN) on the plasma membrane transport of TPRA40 was
examined. Immunolocalization analysis showed that GFPNA14DN (Fig. 6A, o and u) was diffused widely throughout thecytoplasm in contrast to GFP-NA14 (Fig. 6A, n and t), indicatingthat the N-terminal coiled-coil motif of NA14 is required for its
association with MTs. Co-transfection of GFP-NA14DN
inhibited the plasma membrane transport of FLAG-TPRA40(Fig. 6A, i and u). However, when GFP or GFP-NA14 wasco-transfected, FLAG-TPRA40 was transported to the plasma
membrane (Fig. 6A, g, h, s, and t). These results indicate that
NA14DN, which cannot binds to MTs, inhibits the plasma
membrane transport by dissociating FLAG-TPRA40 from MTs.
FLAG-TPRA40DC was not transported to the plasma
membrane if co-transfected with GFP, GFP-NA14, or
GFP-NA14DN (Fig. 6A, j, k, l, v, w, and x), indicating that theinteraction with NA14 is necessary for TPRA40 to betransported to the plasma membrane.
Biotinylation analysis of cell surface proteins

The involvement of NA14 in the plasma membrane transport of
TPRA40 was further examined by biotinylation assays of cellsurface proteins. As shown in Figure 6B, biotinylated
FLAG-TPRA40 was detected by streptavidin-conjugatedperoxidase, indicating the plasma membrane expression of
FLAG-TPRA40. Amounts of biotinylated FLAG-TPRA40 wereclearly increased when the FLAG-TPRA40 was co-transfectedwith GFP-NA14 than when it was con-transfected with GFP(Fig. 6B, lanes 1 and 2). In the cells transfected with
GFP-NA14DN, no biotinylated FLAG-TPRA40 was detectedunder our experimental conditions (Fig. 6B, lane 3). The
amounts of FLAG-TPRA40 were almost the same in all samples(Fig. 6B, lanes 4–6). These results indicate that NA14 regulates
plasma membrane transport of TPRA40 in a positive mannerthat requires the N-terminal region of NA14.
Knockdown of NA14 by RNA interference inhibits
TPRA40 transport

To confirm that NA14 is involved in the membrane transport of
TPRA40, we next examined the effect of knockdown of NA14
by RNA interference. To test the effect of a small interference
RNA (siRNA) mixture on NA14, a siRNA mixture wasco-transfected with GFP-NA14, and GFP fluorescence inthe living cells was observed 48 h after transfection. When

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REGULATION OF EARLY EMBRYOGENESIS BY TPRA40

Fig. 5. Localization of TPRA40 and NA14 in HeLa cells FLAG-NA14 and GFP-NA14 expression vectors (A) or FLAG-TPRA40 and GFP-NA14
expression vectors (B) were transfected into HeLa cells, and the cells were incubated for 48 h (A) or the indicated time (B). The cells were fixed in 4%
paraformaldehyde and permeabilized with 0.5% Triton X-100. A: Nuclei were stained with 1 mg/ml DAPI (blue). FLAG-NA14 was stained with ananti-FLAG antibody and then by incubation with a Cy3-conjugated anti-mouse IgG antibody (red). GFP-NA14 was visualized by the native
fluorescence of GFP (green). A merged image is also shown. Final magnification 1,000T. Similar results were obtained in a separate experiment.
B: Nuclei were stained with 1 mg/ml DAPI (DAPI; a–d, blue). FLAG-TPRA40 was stained with an anti-FLAG antibody followed by incubation with a
Cy3-conjugated anti-mouse IgG antibody (TPRA40; e–h, red). GFP-NA14 was visualized by the native fluorescence of GFP (NA14; i–l, green).
Merged images (merge; m–p) are also shown. The arrowheads indicate structures positive to both FLAG-TPRA40 and GFP-NA14. Finalmagnification 1,000T. Similar results were obtained in three separate experiments.

co-transfected with GFP-NA14, the siRNA mixture
dramatically reduced the fluorescence derived from GFPNA14 (Fig. 7A, g and h). SiRNA had no effect on GFP expression(Fig. 7A, e and f). These observations suggest that siRNA
specifically down-regulates NA14. We then examined theeffect of the siRNA mixture on the plasma membrane transportof FLAG-TPRA40. Although the transfection of FLAG-TPRA40
alone resulted in plasma membrane transport (Fig. 7B, c), cotransfection of the siRNA mixture and FLAG-TPRA40
suppressed plasma membrane transport (Fig. 7B, d), suggestingthat NA14 is involved in the transport of TPRA40.
TPRA40/NA14 regulates cell division ofearly mouse embryos

To examine whether the interaction of TPRA40 with NA14
were caused in the early embryos, we determined the gene
expression in the embryos using RT-PCR. NA14 and TPRA40
were abundantly expressed in the embryos as well as mousebrain (Fig. 8A), indicating that the interaction observed in HeLa
cells can be present in the mouse early embryos. We examined
whether NA14 is involved in the regulation of mouseembryogenesis by TPRA40. Embryos injected with GFP and
FLAG-TPRA40 developed according to the time after injection,and more than half of the embryos developed to the morula/
blastcyst stage by 4 days after fertilization (Fig. 8B,C). However,most embryos injected with GFP-NA14 and FLAG-TPRA40
remained at only the 2-cell stage for 4 days (Fig. 8B,C).
GFP-NA14DN restored the development of mouse embryos
to that observed in the presence of GFP (Fig. 8B,C).
Immunoblotting analysis using anti-FLAG antibody indicatesthat the expression level of FLAG-TPRA40 in the injected
embryos remained almost constant in each group (data not
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shown). The expression levels of GFP, GFP-NA14, and
GFP-NA14DN in the injected embryos were confirmed byfluorescence microscopy although the GFP proteins could beundetected by westernblotting. These results show that
TPRA40 regulates mouse embryogenesis through NA14.
Discussion

In this study, we have shown that an orphan receptor, TPRA40,
regulates cell division of early mouse embryos. As shown in
Figure 1C, shRNA-mediated knockdown of TPRA40 facilitatescell division of early mouse embryos, suggesting the
involvement of TPRA40 in early mouse embryogenesis. The
effect of shRNA was observed during the period 2–4 days after
injection, indicating that TPRA40 is likely to be involved inembryogenesis from the first cell division. Further evidence forthe involvement of TPRA40 comes from the observation that
injection of the TPRA40 expression vector suppressed cell
division of mouse embryos at day 4. As shown in Figure 3,
approximately 10% of embryos injected with the TPRA40
expression vector developed to the morula/blastocyst stage by
day 4, while approximately 40% of control embryos developed
to the morula/blastocyst stage. At least 2 days after transfection
are required for the functional plasma membrane transport of
the TPRA40 protein in HeLa cells, and 1 day for the completionof cell division. These results indicate that the suppression ofcell division by exogenous TPRA40 cDNA might be observed
3 days after injection, a supposition that is consistent with ourobservations as shown in Figure 3.
For many GPCRs, dimerization is required for their exit fromthe ER because it masks ER-retension signals or hydrophobic
patches that keep the proteins in the ER (Petaja-Repo et al.,
2000; Petaja-Repo et al., 2001). TPRA40DC has the deletion of

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AKI ET AL.

Fig. 6. Effects of NA14 and NA14DN on the plasma membrane transport of TPRA40 in HeLa cells (A) FLAG-TPRA40 and FLAG-TPRA40DC
expression vectors were co-transfected with GFP, GFP-NA14, or GFP-NA14DN expression vectors in HeLa cells, and the cells were incubated for
48 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with an anti-FLAG antibody followed byincubation with a Cy3-conjugated anti-mouse IgG antibody. Nuclei were stained with 1 mg/ml DAPI. DAPI-derived fluorescence (DAPI; a–f, blue),Cy3-derived fluorescence (TPRA40; g–l, red), and the native fluorescence of GFP (NA14; m–r, green) were observed under a fluorescencemicroscope. Merged images (merge; s–x) are also shown. Final magnification 1,000T. Similar results were obtained in four separate experiments.
B: Cell surface biotinylation assay. HeLa cells were transfected with FLAG-TPRA40 and GFP, GFP-NA14, or GFP-NA14DN for 48 h, andbiotinylated on the cell surface by incubating with biotinylation reagents as shown in the upper part. Then, the cells were extracted and subjected toelectrophoresis. Total FLAG-TPRA40 in the cell lysates was visualized by an anti-FLAG antibody (lanes 4–6). To detect biotinylated
FLAG-TPRA40, the cell lysates were precipitated with anti-FLAG antibody-immobilized beads, and the precipitated proteins were subjected toelectrophoresis. Biotinylated FLAG-TPRA40 was visualized by streptavidin-peroxidase (lanes 1–3). Similar results were obtained in a separate
experiment.

the putative cytoplasmic region of the C-terminus, and is
scarcely transported to the plasma membrane even 48 h after
transfection in HeLa cells as shown in Figure 6A. TPRA40DC
suppresses early embryogenesis, as shown in Figure 3,
suggesting that TPRA40DC interferes with the function ofendogenous TPRA40, and works as a dominant negative forwild-type TPRA40. Dominant negative effects of
C-terminus-deleted mutants were also observed for other
GPCRs. For example, the naturally occurring deletion mutantof the C-terminal cytoplasmic region of the CCR5 chemokine
JOURNAL OF CELLULAR PHYSIOLOGY

receptor is not transported to the plasma membrane and isretained in the ER (Benkirane et al., 1997; Schulz et al., 2000).
When the mutant CCR5 and wild-type CCR5 are coexpressed, the plasma membrane transport of wild-type CCR5
is inhibited by its retention in the ER (Shioda et al., 2001). A
splice variant of the dopamine D3 receptor, D3nf, has a deletionof the C-terminal portion of receptor and is localized to an
intracellular compartment (Schmauss et al., 1993; Karpa et al.,2000). The D3 receptor also mislocates from the plasma
membrane to the intracellular compartment in the presence ofCOLORREGULATION OF EARLY EMBRYOGENESIS BY TPRA40

Fig. 7. Effect of siRNA-mediated knockdown of NA14 on the plasma
membrane transport of TPRA40 in HeLa cells (A) GFP and
GFP-NA14 expression vectors were transfected with or withoutsiRNA mixture (4 nM) into HeLa cells. After 48 h of transfection, the
GFP fluorescence in live cells was observed under a fluorescencemicroscope. BF indicates bright field images. Final magnification
200T. Similar results were obtained in three separate experiments.
B: FLAG-TPRA40 with (R) or without () an siRNA mixture (4 nM)
were transfected into HeLa cells, and the cells were incubated for
48 h. The cells were fixed with 4% paraformaldehyde, permeabilizedwith 0.5% Triton X-100, and incubated with an anti-FLAG antibody,followed by incubation with a Cy3-conjugated anti-mouse IgG
antibody. Nuclei were stained with 1 mg/ml DAPI. DAPI-derived
fluorescence (DAPI; a and b), and Cy3-derived fluorescence(TPRA40; c and d) were observed under fluorescence microscope.
Merged images (merge; e and f) are also shown. Final magnification
1,000T. Similar results were obtained in three separate experiments.

D3nf (Karpa et al., 2000). The results obtained using
TPRA40DC indicate that the C-terminal cytoplasmic region of
TPRA40 plays an essential role in both transport to the plasma
membrane and the regulation of cell division.
We searched for molecules that bind to the C-terminal
cytoplasmic region of TPRA40. Unexpectedly, Sjögren’s
syndrome-associated protein NA14 was identified as a binding
protein for TPRA40. Sjögren’s syndrome (SS) is an autoimmune
disorder clinically characterized by dry mouth and eye caused
by decreased function of the lacrimal and salivary glands(Thomas et al., 1998). Although NA14 was found from among
the autoantigens recognized by serum from an SS patient(Ramos-Morales et al., 1998), the relationship between NA14
and the clinical expressions of SS have been poorly elucidated.
NA14 is composed of 119 amino acids, and has an N-terminal
acidic coiled-coil region and a C-terminal basic domain(Ramos-Morales et al., 1998). We have observed the dotted
localization of NA14 in HeLa cells. Immunocytochemical
analyses using an antibody against a-tubulin has suggested thatthese dotted structures are distributed along microtubules(data not shown). In contrast, NA14DN (amino acids 23–119),
which is deleted from the coiled-coil region (amino acids 8–22),
was diffused throughout the cytoplasm. These observations
indicate that NA14 associates with MTs through its N-terminal
coiled-coil region. Consistent with these data, Pfannenschmid
JOURNAL OF CELLULAR PHYSIOLOGYet al. (2003) reported that NA14 is associated withmicrotubule (MT) structures, basal bodies in human sperm andcentrosomes in HeLa cells. The binding region for NA14 to
TPRA40 might be located near its C-terminal regions (aminoacids 23–119) since we obtained a C-terminal fragment of
NA14 (NA14DN) as a binding protein for TPRA40. Therefore,
NA14 might bind to MTs through its N-terminal region (aminoacids 1–22) and to TPRA40 at its C-terminal region (amino acids
23–119).
As demonstrated by both immunocytochemical andbiochemical (cell surface biotinylation) assays, TPRA40 istransported to the plasma membrane in HeLa cells (Fig. 6A,B).
NA14 is required for the transport of TPRA40 because
knockdown of NA14 by siRNA inhibited the transport, asshown in Figure 7B. NA14DN also inhibited the plasma
membrane transport of TPRA40 when the two were coexpressed in HeLa cells, suggesting that NA14DN functions as adominant-negative molecule for NA14. This can be explained asfollows. NA14DN contains binding region for TPRA40, andmight compete with NA14 for binding to TPRA40. TPRA40 thatbinds to NA14DN cannot bind to MTs since NA14DN isdeleted from the binding region for MTs. Therefore, the binding
to NA14DN results in the retention of TPRA40 within thecytoplasm. A possible role for NA14 in MT dynamics has also
been suggested by Errico et al. (2004). They showed that NA14
interacts with spastin, an AAA family ATPase that interacts withMTs, and is involved in MT dynamics (Errico et al., 2004). NA14
is required for the MT-serving activity of spastin since deletionof the NA14-binding region of spastin results in the dissociationof spastin from MTs (Errico et al., 2004). Impairment of spastin
activity underlies the pathogenesis of hereditary spastic
paraplegia, which is characterized by the degeneration of motor
axons with the impairment of intracellular transport (Errico
et al., 2002; McDermott et al., 2003; Fink, 2003a,b). Theseobservations indicate that NA14 is involved in intracellularcargo transport as an essential adaptor molecule for interaction
between the cargo and MTs.
What is the physiological significance of the suppression ofearly mouse embryogenesis by TPRA40? Other than TPRA40,
there are a few factors that are reported to inhibit
preimplantation embryogenesis (Stewart and Cullinan, 1997;
Hardy and Spanos, 2002). TNF-a (Pampfer et al., 1997; Wuu
et al., 1999) and IFN-g (Haimovici et al., 1991) are factors thatsuppress early mouse embryogenesis; however, in contrast to
TPRA40, the effects of TNF-a and IFN-g appear only in theblastocyst stage. TNF-a mainly inhibits the proliferation of theinner cell mass (Stewart and Cullinan, 1997) while IFN-g inhibits
trophoblast outgrowth by inducing the degeneration ofthese cells (Haimovici et al., 1991). Therefore, TPRA40 isinvolved in the negative regulation of embryogenesis in a celllineage-nonspecific manner, while TNF-a and IFN-g are
involved in the cell lineage-specific inhibition of embryogenesis.
Early mouse embryogenesis is regulated not only by cytokines
but by the environments in which it takes place (Fleming et al.,2004). It has been reported that maternal diabetes andhyperglycemia suppress early mouse embryogenesis during
preimplantation (Moley et al., 1991, 1998a,b; Chang et al.,2005). Since TPRA40 expression is up-regulated in diabetic
mice (Yang et al., 1999) and under hyperglycemic conditions in
cultured cells (Fujimoto et al., 2001), TPRA40 may be a
mediator of the retardation of early mouse embryogenesis
under these conditions.
In conclusion, the results of the present study demonstrate
one of the physiological functions of an orphan receptor,
TPRA40. In addition, the present study also shows that NA14 isinvolved in the plasma membrane transport of TPRA40.
The unexpected connection between an orphan receptor andthe autoantigen of SS might shed light on the pathophysiology
of SS.

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Fig. 8. Effect of TPRA40 and NA14 on cell division of mouse embryos (A) mRNA was extracted from C57/BL6 mouse brain and embryos. RT was
performed, and the product of the RT reaction was amplified by PCR using the primers for NA14, TPRA40, and G3PDH. The RT-PCR products
were electrophoresed in agarose gels, and stained with ethidium bromide. The figure shows representative data obtained from three independent
experiments. B,C: One day after fertilization, embryos were injected with the indicated combinations of vectors, and cell divisions wereobserved at the indicated times under a bright field microscope. 54 (FLAG-TPRA40 R GFP-empty vector), 52 (FLAG-TPRA40 R GFP-NA14), or
51 (FLAG-TPRA40 R GFP-NA14DN) embryos were used in each group (MP < 0.05 for FLAG-TPRA40 R GFP-empty vector; Mann–Whitney’s
U-test). Typical images of embryos (upper part) and summarized data on the developmental stages (lower part) are shown.

Acknowledgments

We thank Prof. M. Okabe (Osaka University) for providing the
pCX-EGFP vector plasmid and Dr. M Tabuchi (Kagoshima
University) for assistance with the yeast two-hybrid assay.
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13

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