A human brain cDNA library in the GAL4 activation domain vector pACT2 was purchased from BD biosciences (Palo Alto, CA). Full-length non-tagged and HA-tagged human 5-HT₆R cDNA in pcDNA3.1 were purchased from UMR cDNA Resource Center (Miner Circle Rolla, MO). The Myc-tagged 5-HT₆R plasmid was constructed by recloning the human 5-HT₆R cDNA into pCMV-Tag3B vector (Stratagene, La Jolla, CA). cDNA fragment encoding the carboxyl terminus of 5-HT₆R (6RCT) was subcloned into pGBKT7 (Clontech, Palo Alto, CA). GST-6RCT was kindly provided by Dr. Y. G. Yu (Kookmin Univ., Korea). His-tagged 6RCT was constructed by subcloning the 6RCT into pET28a (+) vector. The CTs of 5-HT₄R and 5-HT_7BR (4RCT and 7BRCT) were cloned into pGEX4T-1 (Amersham, Piscataway, NJ) to express them in a GST-fusion form. The primer sequences used were as follows: 4RCT (Fw, 5′-GAATTCCCGAGACGTGCCTTCCTC TC-3′; Rv, 5′-CTCGAGAGTGTCACTGGGCTGAGCAG-3′) and 7BRCT (Fw, 5′-GAATTCCCGGACCTGAGGACCACCTATC-3′; Rv, 5′-CTCGAGCAGCACAAACTCAGGTC-3′). Full length human Nova-1 in pCMV6-XL5 was purchased from Origene (Rockville, MD). Flag-tagged Nova-1 was constructed by recloning into pCMV-Tag2B (Stratagene). GST-Nova239-419 (239–419 amino acids of mouse Nova-1) and GST-KH3 (KH3 domain, 420–507 amino acid of mouse Nova-1) were constructed by subcloning from full length Nova-1 into pGEX4T-1 vector. Nova-1 full length, Nova-1-ΔKH1 and Nova-1-ΔKH1/2 constructs were generated by fusing a HA tag to either full length or partially truncated human Nova-1 constructs.

The yeast two-hybrid assay was performed using the Matchmaker GAL4 two-hybrid system 3 (Clontech). The bait plasmid, pGBKT7/CT of 5-HT₆R, was stably expressed in yeast strain AH109 and did not have a self-transcriptional activity. The prey plasmid, human brain cDNA library/pACT2, was transformed into yeast strain Y187. All yeast two-hybrid screening was performed as described previously [17].

GT1-1, NIH3T3, and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (GenDEPOT) and 100 units/ml penicillin/streptomycin (Gibco) in a humidified atmosphere containing 5% CO₂ at 37℃. HEK293 cell lines stably expressing the human 5-HT₆R (HEK293/6R cells) were selected and maintained with 800 µg/ml and 400 µg/ml of G418, respectively. For the transient transfection, cells were plated in 6-well plates and grown to 70–90% confluence in 1 day. The cells were transfected with plasmid DNA using Lipofectamine PLUS reagent (Invitrogen). Six hours after transfection, the medium was changed with complete medium. For the transfection into GT1-1 cells, Lipofectamine reagent was treated for 4 h. The cells were analyzed after 24 h of recovery in normal medium.

GST-mediated pull-down assay was performed using the Profound Pull-down GST Protein:Protein Interaction kit (Pierce, Rockford, IL). GST and GST-6RCTs plasmids were transformed into bacterial strain BL21 (DE3) and their protein expressions were induced by adding 0.5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG, Sigma) at 25℃ during mid-log phase. GST and GST-6RCTs proteins were immobilized by glutathione gel. To prepare prey protein, Flag-Nova-1 plasmid was transfected into HEK293 cells and cell lysates were harvested after 24 h of transfection. For GST pull-down assay using rat brain lysates, the lysates were prepared from age- and weight controlled adult male SD rats (60–70 days old, 230–260 g) as previously described [17]. Prepared cell or brain lysates were incubated with immobilized GST proteins. Bound proteins were eluted by boiling for 10 min at 95℃ in SDS sample buffer followed by immunoblotting with anti-Nova-1 (Upstate Biotechnology Inc., Lake Placid, NY) and anti-GST antibodies (Novagen, Madison, WI).

Co-immunoprecipitation was performed as described by Yun et al. [18]. Soluble HEK293 cell lysates were precleared with 50 µl of ImmunoPure immobilized protein G Plus (Pierce) and 2 µg of rabbit normal IgG for 1 h. Precleared lysates were incubated with 4 µg of anti-Myc, anti-HA (Cell Signaling Technology, Beverly, MA), or anti-Flag (Sigma-Aldrich, St. Louis, MO) antibodies overnight at 4℃. Then the lysates were added with 50 µl of ImmunoPure immobilized Protein G Plus and incubated for 4 h at 4℃ and washed three times with lysis buffer. Immune complexes were eluted by boiling for 5 min at 95℃ in SDS sample buffer, followed by Western blot analysis. For co-immunoprecipitation using rat brain lysates, the lysates were immunoprecipitated with 10 µg of anti-5-HT₆R antibody (GeneTex Inc., San Antonio, TX).

Total RNA was extracted using Easy-spin total RNA extraction kit (Intron biotechnology, Korea). To eliminate possible DNA contamination, 1 µg of RNA samples were treated with 0.2 units of DNase I (NEB, Beverly, MA) for 10 min at 37℃. Then the RNA was heated for 10 min at 75℃ to inactivate DNase I and reverse-transcribed using random hexamers. The primers used for RT-PCR were as follows: 6R E1-F, 5′-TGTCTGCTTTGGACCGCCTT-3′; 6R IA-F, 5′-GCTTCAGCTGGGGATCAGTCAA-3′; 6R E2-R, 5′-ATGCTGGCCACAAAGAAGGG-3′. The primers for GnRH E1-up, IA-up, and E3-dn were synthesized referring to the sequences previously described [32]. The primer sequences used are as follows: E1-up, 5′-GGAAGACATCAGTGTCCCAGA-3′; IA-up, 5′-TACCTCTGCAGTTTCTGTGA-3′; E3-dn, 5′-GAAGTGCTGGGGTTCTGC-3′. GAPDH was used as an internal control for semi-quantitative RT-PCR. The GAPDH primers were forward primer 5′-CTCTCCAGAACATCATCCCTG-3′ and reverse primer 5′-CACCCTGTT GCTGTAGCCAAA-3′.

For immunoblotting, proteins were resolved on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane (Millipore, Bedford, MA). The membrane was blocked with Tris-buffered saline containing 5% skim milk (BD Difco) and 0.1% Tween 20 (Sigma) for 1 h at room temperature (RT). Then, the membranes were incubated with the respective primary antibodies overnight. After three washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 h at RT. The immune complexes were visualized with an ECL detection kit (Millipore). The following antibodies were used for western blot: anti-5-HT₆R (GeneTex Inc., San Antonio, TX); Anti-Nova-1 (Novus Biologicals, USA); anti-HA (Cell Signaling Technology, Beverly, MA); anti-Actin (sigma); anti-β-Tublin (Cell Signaling Technology); anti-histone H3 (Cell Signaling Technology); anti-phospho-ERK (Cell Signaling Technology); and anti-ERK (Cell Signaling Technology).

HEK293, GT1-1 cells or cortical neurons were grown on coverslips (Corning, Corning, NY). Twenty four hours after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 20 min at RT. The cells were washed with PBS and permeabilized with 0.2% Triton X-100 (Sigma) for 15 min. Then the cells were blocked with 3% bovine serum albumin (GenDEPOT) for 1 h at RT and incubated overnight with rabbit anti-HA (Sigma) and mouse anti-Flag antibodies (1:500) at 4℃. After three washes, the cells were incubated with anti-rabbit IgG (FITC) and anti-mouse IgG (rhodamine) secondary antibodies (1:400; Jackson ImmunoResearch) for 2 h at RT. The cells were washed three times and counter-stained with DAPI (Sigma) for 10 min. After mounting on glass slide using ProLong Antifade reagent (Invitrogen), the cells were examined with a FluoView® Confocal Laser Scanning Microscope (Olympus, Japan).

Twenty four hour after transfection, HEK293 cells were harvested by centrifugation at 600 × g for 5 min at 4℃. Nuclear and cytoplasmic fractions were separated using the Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA), following the manufacturer's protocol.

5-HT₆R activity was measured using an FDSS6000 96-well fluorescence plate reader (Hamamatsu Photonics, Japan) as previously described [18,33]. Briefly, HEK293/6R stable cells were transiently transfected with Gα₁₅ protein using Lipofectamine Plus (Invitrogen). After 6 h, the cells were transferred into 96-well black wall/clear bottom plate and cultured overnight. The cells were loaded with Ca²⁺ indicator dye Fluo-4-AM (5 µM) and 0.001% Pluronic F-127 (Molecular Probes, Eugene, OR) and incubated in a HEPES-buffered solution (150 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl₂, 10 mM HEPES, 13.8 mM glucose and 2 mM CaCl₂, pH 7.4) for 1 h at 37℃. After three washes, 10 µM of 5-HT (Sigma) was added to the cells and Ca²⁺ response was measured at 480 nm. All data were collected and analyzed using the FDSS system and related software (Hamamatsu Photonics).

The intensity of bands was measured with Image J software (National Institute of Health, Bethesda, MD) and analyzed using the GraphPad Prism Version 4 (GraphPad Software Inc., San Diego, CA). All numeric values are represented as the mean±S.E.M. Data were analyzed by Student's t-test, one-way analysis of variance (ANOVA) followed by Duncan's post-hoc tests or two-way ANOVA followed by Newman-Keuls post hoc tests.

Our group reported previously that Fyn, Jab1 and MAP1B directly interact with 5-HT₆R in vitro and in vivo and play roles in 5-HT₆R-mediated signal transduction [17,18,19]. In this study, we also identified Nova-1 as a new binding protein of 5-HT₆R, through a yeast two-hybrid screening assay using the C-terminal (CT) region of human 5-HT₆R (bait) and the human brain cDNA library. We confirmed this specific interaction by a separate yeast two-hybrid assay in which the CT of 5-HT₆R (bait) and full-length Nova-1 cDNA (prey) were transformed in the AH109 and Y187 yeast strains, respectively (Fig. 1A), and a blue color colony was detected after mating the two strains (Fig. 1B). GST pull-down assay also verified the direct binding between Nova-1 and 5-HT₆R. GST-fused to the CT of 5-HT₆R (GST-6RCT) bound to the Flag-tagged-Nova-1 (Flag-Nova-1), but GST protein did not (Fig. 1C), indicating that 5-HT₆R directly binds to Nova-1 via the CT region. However, given that this interaction occurred with partial fragment of 5-HT₆R outside cells, we attempted to determine whether full-length 5-HT₆R binds to Nova-1 in a specific manner in mammalian cells using co-immunoprecipitation. After full-length Myctagged 5-HT₆R (Myc-5-HT₆R) and Flag-Nova-1 or empty vector (Flag-V) were transiently transfected into HEK293 cells, cell lysates were prepared, immunoprecipitated with anti-Flag antibodies, and subsequently immunoblotted with anti-Myc antibodies. Myc-5-HT₆R band was visible only in the HEK293 lysates containing Flag-Nova-1 protein (Fig. 1D). When co-immunoprecipitation was performed in reverse with anti-Myc antibodies followed by immunoblotting with anti-Flag antibodies, Myc-5-HT₆R immunoprecipitated with Flag-Nova-1 protein (Fig. 1E). These results confirm the direct interaction between Nova-1 and 5-HT₆R in mammalian cells.

Next, we examined whether the interaction between 5-HT₆R and Nova-1 occurs in vivo using the rat brain lysates. As previously reported, 5-HT₆R was widely expressed in the brain while Nova-1 was preferentially expressed in the brainstem and hypothalamus (Fig. 1F). Similarly to the GST pull-down assay in HEK293 cells, GST-6RCT pulled down Nova-1 in the rat brain lysate, but GST protein did not (Fig. 1G). We further confirmed this interaction in vivo using co-immunoprecipitation assay. Rat whole brain lysates were immunoprecipitated with anti-5-HT₆R antibody and subsequently immunoblotted with anti-Nova-1 antibody. As shown in Fig. 1H, endogenous Nova-1 signal was selectively detected in the sample immunoprecipitated with anti-5-HT₆R. These results consistently showed that 5-HT₆R interacts with Nova-1 under physiological conditions. To prove the specific binding between Nova-1 and 5-HT₆R, we also examined whether Nova-1 interacts with other types of serotonin receptors. Among several 5-HT receptors, we chose 5-HT₄R and 5-HT_7BR given that these both receptors belong to the Gα_s-family similar to 5-HT₆R, and used their intracellular domain CT regions as bait proteins for GST pulldown assay. As in Fig. 1I, CT of 5-HT₄R (4R) and CT of 5-HT_7BR (7BR) did not bind to Nova-1 protein, while CT of 5-HT₆R (6RCT) showed the distinct binding signal to Nova-1. Taken together, these results suggest that the 5-HT₆R selectively and directly binds to Nova-1 in vitro and in vivo.

Then, we tried to identify the binding sites of Nova-1 protein that interact with 5-HT₆R. Nova-1 has three KH domains that recognize and bind to target RNA [24,25,26]. On the basis of KH domains, three different Nova-1 constructs were generated in HA- and venus C-terminal (VC)-tagged form; full length Nova-1 (amino acid 1–507), Nova-1-ΔKH1 (amino acid 118~507, including KH2 and KH3 domains), and Nova-1-ΔKH1/2 (amino acid 239~507, including KH3 domain) (Fig. 2A). These constructs were transiently transfected into HEK293 cells and then their expressions were analyzed by immunoblotting with anti-HA antibody (Fig. 2B). To identify which domain binds to 5-HT₆R, HEK293 cell lysates containing HA-VC-tagged full length or truncated Nova-1 proteins were incubated with GST or GST-6RCT, and then GST pull-down assay was performed. As shown in Fig. 2C, CT of 5-HT₆R interacted with all three different Nova-1 proteins (full length and two kinds of truncated Nova-1 proteins). Also, when immunoprecipitation was carried out using anti-HA antibody, full length and two truncated Nova-1 proteins pulled down GST-6RCT protein (Fig. 2D). To verify the interaction between three different Nova-1 constructs and 5-HT₆R in vivo, immunoprecipitation with anti-5-HT₆R antibody was performed between rat brain and HEK293 lysates containing full length or truncated Nova-1 proteins. As seen in Fig. 2E, endogenous 5-HT₆R in the brain bound to all HA-VC-tagged full length and truncated Nova-1 proteins, whereas control IgG showed no binding signal. Taken together, these results suggest that 5-HT₆R is likely to interact with KH3 domain or upstream flanking region of KH3 because these domains are present in all of three Nova-1 constructs.

To further narrow down the specific binding site of Nova-1, 239–507 aa of Nova-1 was divided into two fragments, 239–419 aa and KH3 domain (420–507 aa) in a GST-fusion form (Fig. 2F, left). Then, we examined which domain binds to the 5-HT₆R using GST pull-down assay. GST-fused Nova-1 protein fragments and His-tagged 6RCT were expressed in E. coli and protein-protein interaction was examined. As a result of GST pull-down, CT of 5-HT₆R bound to only KH3 domain of Nova-1 while no signal was detected in control GST and GST-239-419 (Fig. 2F, right). These results indicate that 5-HT₆R binds to Nova-1 via KH3 domain.

Based on evidence for a physical interaction between 5-HT₆R and Nova-1 and, we next examined whether the function of Nova-1 is affected by 5-HT₆R. Nova-1 is a neuron-specific RNA binding protein and regulates RNA splicing [27]. Therefore, we investigated whether there is any change in splicing activity of Nova-1 when 5-HT₆R is overexpressed, using gonadotropin-releasing hormone (GnRH) transcript that was reported as a splicing target of Nova-1 [32]. GnRH minigene and Nova-1 DNA were transiently transfected into NIH3T3 cells, and after 24 h, competitive RT-PCR was performed using three primers to evaluate the relative amount of spliced intermediates of target pre-mRNA (Fig. 3A). As described previously [32], Nova-1 facilitated the rate of intron A excision of GnRH pre-mRNA (Fig. 3B). However, when 5-HT₆R DNA was co-transfected with Nova-1, splicing activity of Nova-1 was significantly suppressed (8.0±1.1% of control at 1 µg of 5-HT₆R DNA, Fig. 3C). Meanwhile, truncated 5-HT₆R (5-HT₆RΔCT) lacking the CT region, a binding domain of 5-HT₆R with Nova-1, did not affect Nova-1-mediated intron A excision of GnRH, implying that binding of two proteins via CT region regulates the splicing activity of Nova-1 (Fig. 3D). In GT1-1 cells in which GnRH and Nova-1 are endogenously expressed, a consistent pattern of splicing intermediates was observed. As seen in Fig. 3E and F, 5-HT₆R overexpression suppressed the splicing activity of Nova-1 (25.7±10.4% of control at 2 µg of 5-HT₆R DNA), but truncated 5-HT₆R (5-HT₆RΔCT) did not affect the intron A excision rate of GnRH. Taken together, these results suggest that the increased interaction between Nova-1 and 5-HT₆R induced by overexpression of 5-HT₆R hampers the splicing activity of Nova-1, and the CT region of 5-HT₆R plays a critical role in mediating this interaction as well as the regulation of Nova-1 function.

We next examined how interaction with 5-HT₆R could affect the splicing function of Nova-1. At first, we assessed whether total protein levels of Nova-1 were changed by 5-HT₆R expression in GT1-1 cells, and found that the expression levels of Nova-1 remained unchanged, regardless of 5-HT₆R expression levels (Fig. 4A). Interestingly, however, Nova-1 was translocated from nucleus to cytoplasm when co-expressed with 5-HT₆R. In contrast, Nova-1 remained in nucleus when the control empty vector was co-transfected (Fig. 4B). This translocation of Nova-1 by 5-HT₆R expression was observed in HEK293 cells as well (Fig. 4C). Moreover, nuclear/cytoplasmic fractionation result also demonstrated that Nova-1 was mainly located in nucleus (183.4±2.3% compared by cytosol), but when co-expressed with 5-HT₆R, the cellular distribution of Nova-1 was significantly changed from nucleus to cytoplasm (nucleus, 38.9±10.2% compared by cytosol, Fig. 4D). Taken together, these results suggest that overexpression of 5-HT₆R interferes with the proper subcellular localization of Nova-1 to execute its function.

We then examined whether overexpression of Nova-1 has an effect on 5-HT₆R activity. We previously developed the assay system to assess the activity of 5-HT₆R [34]. In this assay system, co-transfected promiscuous Gα₁₅ along with 5-HT₆R facilitates coupling of Gα_S–coupled receptors to phospholipase C and subsequent intracellular Ca²⁺ release, which is detected using an FDSS6000 96-well fluorescence plate reader [34]. Using this system, the effect of interaction with Nova-1 on the activity of 5-HT₆R was examined. Twenty-four hours after transfection of 5-HT₆R and promiscuous Gα₁₅ into HEK293 cells, 5-HT-induced Ca²⁺ increases via 5-HT₆R were measured with FDSS6000 system. Fluorescence peaked within 20–30 s after 5-HT treatment and gradually decreased with time. Compared to control, when Nova-1 was co-expressed, 5-HT-induced Ca²⁺ signal was significantly reduced (48.5±7.2% of control, n=10, Fig. 5A, B). This reduction of Ca²⁺ release by Nova-1 co-expression was not observed when 5-HT₄R or 5-HT_7BR was co-expressed with Nova-1 (Fig. 5C). As an alternative method to detect 5-HT₆R activity, we measured 5-HT-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) via 5-HT₆R in HEK293/6R stable cells, given that activated 5-HT₆R phosphorylates ERK1/2 to mediate signal transduction [17,33]. 5-HT-induced ERK1/2 phosphorylation levels were decreased as the levels of Nova-1 were increased (38.7±3.4% of control at 4 µg on Nova-1 DNA, Fig. 5D). In addition, the expression levels of 5-HT₆R were inversely correlated with Nova-1 expression levels both in HEK293/6R cells and in GT1-1 cells (HEK293/6R cells, 14.5±2.5% of control at 4 µg of Nova-1 DNA; GT1-1 cells, 4.5±0.5% of control at 4 µg of Nova-1 DNA, Fig. 6A, B). Thus, these results suggest that the activity and expression levels of 5-HT₆R are inversely related to the expression of Nova-1. To examine whether the reduction in 5-HT₆R levels upon overexpression of Nova-1 is mediated by proteasome-mediated protein degradation, its total protein levels were measured in the presence of proteasome inhibitor, MG132. Interestingly, the reduction of 5-HT₆R in the presence of Nova-1 overexpression was rescued by MG132, indicating that overexpression of Nova-1 seems to make 5-HT₆R unstable and vulnerable to degradation via proteasome (Fig. 6C). Interestingly, Nova-1 is also subject to proteasomal degradation. However, upon the degradation of 5-HT₆R, Nova-1 is likely to shuttle back to the nucleus from cytoplasm (Fig. 4D), thus potentially making Nova-1 less sensitive to proteasome activity. Taken together, these results suggest that the Nova-1 causes the 5-HT₆R degradation and overall decrease of 5-HT₆R function.