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The Serotonin Receptor Subtype 5b Specifically Interacts with Serotonin Receptor Subtype 1A

Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht5b) in a mouse model of Rett syndrome (RTT). 5-ht5b has not been extensively studied, so we set out to characterize it in more detail. Unlike common cell surface receptors, 5-ht5b displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht5b possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht5b receptors can specifically interact with 5-HT1A receptors and retain them in endosomal compartments. This interaction reduces 5-HT1A surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht5b receptors regulate the activity of other 5-HT receptor.


INTRODUCTION
G-protein coupled receptors (GPCRs) make up ∼2% of all cellular proteins and constitute one of the most important pharmaceutical drug targets. With all their diversity, GPCRs share some defining features, e.g., they are composed of seven trans-membrane domains (TMD) with three extracellular and four intracellular loops, and many receptors need to form homo-or heterodimers to become functional active (Gurevich and Gurevich, 2008;Schwenk et al., 2010;Pellissier et al., 2011). The receptors relate extracellular signals by activating G-proteins acting on either one of the main effectors calcium (G q via Phospholipase C) or cyclic adenosine monophosphate (cAMP). cAMP can be up-or down-regulated via stimulatory (G s ) or inhibitory (G i /G 0 ) G-proteins who activate or inhibit the adenylate cyclase, respectively. Increases in cAMP may directly activate cyclic nucleotide gated (CNG) ion channels (Kaupp and Seifert, 2002) or protein kinase A (PKA; Meinkoth et al., 1993). Intracellular sensors for cAMP like exchange protein activated by cAMP (EPAC; Bos, 2006) or popeye domain-containing proteins (Popdc, Simrick et al., 2013) function through small GTPases.
5-ht 5b is expressed in many regions throughout the murine brain like bulbus olfactorius, hippocampus, or cerebellum at low levels. However, 5-ht 5b was found to be upregulated under the control of ATF-7 in mice undergoing social stress (Maekawa et al., 2010). Also, 5-ht 5b dysregulation was described in mouse models of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by mutations in the transcription factor MeCP2. We found 5-ht 5b to be developmentally regulated in the brainstem of Mecp2 −/y mice, where it showed 75-fold upregulation (Vogelgesang et al., 2017), while 5-ht 5b was also increased in astrocytes of Mecp2 308/y mice (Delépine et al., 2015). We showed that a full length as well as a truncated protein of 5-ht 5b can be detected by Western blot in murine brain lysates (Vogelgesang et al., 2017). Truncation of receptors has been described many times, but previously identified truncated GPCRs were predominantly retained in the endoplasmic reticulum (ER; Karpa et al., 2000;Leung et al., 2007;Gonzalez et al., 2011).
5-ht 5b 's massive dysregulation in a disease model for RTT prompted us to more closely investigate the reasons for and the physiological significance of its subcellular localization.

Ethical Statement
The experimental procedures were performed in accordance with European Community (EU Directive 2010/63/EU for animal experiments) and National Institutes of Health guidelines for the care and use of laboratory animals. The study was approved by the Georg-August-University, Göttingen and the approval ID T12/18 was assigned to this work.

Animals
C57BL/6J mice were kept in a temperature-and humiditycontrolled 12 h light-dark cycle and had free access to water and standardized pellet food. The Mecp2 knockout mouse (Mecp2 −/y ), strain B6.129P2(C)-Mecp2 tm1-1Bird (Guy et al., 2001) obtained from The Jackson Laboratory (Bar Harbor, ME, USA) was maintained on a C57BL/6J background. For this work we used wild-type and hemizygous Mecp2 +/− females.

Cell Culture, Transfection and Plasmids
Murine neuroblastoma cell line N1E-115 was obtained from the American Type Culture collection (ATCC). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37 • C under 5% CO 2 .
For transient transfection, cells were seeded in cell culture dishes and transfected with indicated plasmids using Lipofectamine2000 Reagent (Invitrogen) according to the manufacturer's instruction.
Primary neurons were obtained from dissociated hippocampi as detailed in Revelo et al. (2014). In brief, hippocampi were extracted from 1 day-old animals. They were incubated for 1 h in enzyme solution (10 ml DMEM, 2 mg cysteine, 100 mM CaCl 2 , 50 mM EDTA and 25 U papain, equilibrated with carbogen for 10 min, and sterile filtered). Before mechanical dissociation, cells were washed thoroughly with HBSS (Invitrogen, Waltham, MA, USA) and were incubated for 15 min in inactivating solution (2 mg albumin and 2 mg trypsin inhibitor in 10 ml FCS-containing DMEM medium). Sterilized coverslips were coated overnight with 1 mg/ml poly-L-lysine and were incubated with plating medium (MEM supplemented with 10% horse serum, 3.3 mM glucose and 2 mM glutamine). Neurons were plated at a concentration of ∼30,000/cm 2 and were left to adhere for 1-4 h. After adhesion, the medium was changed to Neurobasal-A medium (Gibco, Life Technologies, Carlsbad, CA, USA) containing 1:50 B27 supplement (Gibco) and 1:100 GlutaMAX (Gibco). Neurons were kept in culture at 37 • C and 5% CO 2 .
Expression constructs for 5-HT receptors 5-HT 1A and 5-HT 7 with either immuno-(HA/myc) or fluorescent tags have been described previously (Renner et al., 2012), while 5-HT receptors 5-HT 2C , 5-HT 4 , 5-ht 5a , 5-ht 5b and 5-HT 6 were generated from murine corresponding deoxyribonucleic acid (cDNA). Brain tissue was explanted and used for total ribonucleic acid (RNA) isolation with the OLS RNA kit (OLS, Germany) according to the manufacturer's instructions. The total RNA was used in one-step RT-PCR (Invitrogen) and resulting PCR fragment was cloned into pTarget expression vector (Promega). Primer sequences are given in Table 1. To obtain a C-terminal fluorophore fusion construct, the receptor and the fluorophores CFP, GFP, YFP or mRFP/mCherry were amplified individually, and fusion PCR was used to combine the cDNAs. The resulting PCR fragment was cloned into the pTarget expression vector (Promega). To test the hypothesis of a truncated 5-ht 5b receptor, we amplified the unlabeled and the mCherry fusion construct and cloned the fragments into pTarget. The truncated 5-ht 5b comprises the nucleotides from position 520 to 1113. As a control, a truncated 5-HT 7 was prepared in the same way. The truncated 5-HT 7 comprises the nucleotides from position 520 to 1347. Correct sequences of all constructs were determined by sequencing.

Generation of Glycosylation-Deficient 5-HT 1A Constructs
Wild type 5-HT 1A can be glycosylated at four positions (N10, N11, N24, N30) at the N-terminus. Following the glycosylation experiments on 5-ht 5a , we used site-directed mutagenesis (Quickchange II, Agilent) to change each individual Asparagine to Serine (Dutton et al., 2008). To generate clones with multiple exchanges, we performed subsequent rounds of mutagenesis. Primers used for mutagenesis are given in Table 1 and correct sequences of all constructs were determined by sequencing.

Generation of Hybrid Receptors
The modular structure of the GPCRs facilitates easy exchange of domains between receptors, which has previously been used successfully to generate versions with completely new characteristics (Conklin et al., 2008). 5-HT receptors are composed of seven TMD, which are connected by three extra-(ECL) and three intra-cellular loops (ICL). The extra-cellular N-terminus takes part in ligand attraction, while the intracellular C-terminus often, but not always features a PDZ-like domain and takes part in membrane anchoring or protein binding. The problem with this approach is the difficulty to predict domain boundaries, as incorrectly chosen domains may prevent correct folding and therefore render the new hybrid receptor functionless. As we employed this strategy multiple times, we took care to validate the correct function of the new hybrid receptors. Our first quality check was their production of a distinct staining, be it intracellular or membrane bound. All variants that showed a diffuse staining were assumed to be incorrectly folded and were not used for further studies. As long as the receptor showed at least some membrane localization, we also tested the hybrid receptor for its reaction to its natural ligand 5-HT. DNA sequences coding for TMDs were identified using TMHMM server 2.0 (Krogh et al., 2001). Hybrid receptors were generated using fusion PCR. Therefore, the DNA sequences to be fused were separately amplified by PCR using specific primers, introducing a small overlapping DNA region. After purification of both PCR products by gel extraction, 10 ng of each DNA was used as template for a subsequent fusion PCR using primers specific for the full-length hybrid open reading frame. The primers used for generation of different variants of Htr1a-Htr5b hybrid receptor variants are given in Table 1.

RT-PCR
The total RNA of homogenized tissue from specific brain regions was isolated using the Trizol method according to manufacturer's instructions (GibcoBRL) and its concentration was determined using the nanodrop ND-1000 spectrophotometer followed by its quality and integrity measurement by electrophoresis on RNA 6000 LabChip kit (Agilent 2100 Bioanalyzer). The RNA was transcribed into the cDNA using the iScript cDNA Synthesis Kit (BioRad). The primer pairs used for RT-PCR are the same used previously for quantitative RT-PCR (Vogelgesang et al., 2017).
To analyze interaction of HA-5-HT 1A and 5-ht 5b mCherry in N1E-115 neuroblastoma cells, double-transfected live cells were first stained at low temperatures with rabbit-anti-HA-tag primary antibodies for 5 min and anti-rabbit atto647-conjugated secondary antibodies for 5 min to avoid internalization. Cells were then fixed and permeabilized before they were stained again with mouse anti-HA-tag primary antibodies and anti-mouse atto488-conjugated secondary antibodies.
Co-localization was observed using Zeiss LSM 510 Meta system. Quantitative analysis of co-localization was carried out by calculating Pearson's correlation coefficients using LSM 510 software.

Confocal Laser-Scanning Microscopy
Distribution of recombinant receptors, organelle markers and immuno-fluorescent staining was analyzed by microscopy using a confocal laser scanning microscope LSM 510 Meta (Carl Zeiss, Jena, Germany) equipped with a 63× plan-apochromatic phasecontrast water-immersion objective, NA 1.4. Fluorophores were excited either with light at 458 nm, 488 nm or 514 nm from an Argon laser, at 543 nm from a HeNe laser or at 633 nm from a second HeNe laser. Emission of the fluorophores was recorded using the Meta detector. Analysis, e.g., intensity measurements, were performed by importing the images into ImageJ (Schneider et al., 2012). For plate assembly, images were digitally adjusted for brightness and contrast if necessary.

Förster Resonance Energy Transfer (FRET) Acceptor Photobleaching
Cells were transfected with equimolar amounts of 5-HT 1A -GFP and 5-ht 5b -mCherry or with GFP-rab5 and mRFP-rab5 (a gift from Ari Helenius (Addgene plasmid # 14437, Vonderheit and Helenius, 2005) or with single plasmids for negative control and fixed using 2% paraformaldehyde for 20 min prior to imaging. After collecting three images in both the GFP and the mRFP channels, the mRFP in the regions of interests (ROIs) were photobleached by scanning 50 times at high laser power. After photobleaching, again three images of both channels were taken. The obtained images were averaged to calculate the GFP before , GFP after, mRFP before and mRFP after values. The apparent Förster Resonance Energy Transfer (FRET) efficiency was calculated using the following formula: Apparent FRET efficiency = ((GFP after − GFP before ) × mRFP before ) × ((GFP after × mRFP before ) − (GFP before × mRFP after )) −1 (van Royen et al., 2009). Image analysis was performed in ImageJ (Schneider et al., 2012).

Fluorescence Activated Cell Sorting (FACS) Analysis
To determine to what extent 5-ht 5b expression affects translocation of 5-HT 1A to intracellular organelles, we performed QD-mediated fluorescence activated cell sorting (FACS) analysis to measure the amount of surface expression of HA-5-HT 1A . Cells expressing HA-5-HT 1A and 5-ht 5b at equimolar ratios were compared to cells expressing HA-5-HT 1A transfected with the same amount of DNA together with an equimolar amount of unspecific cDNA to compensate for transcriptional effects. Cells were fixed in 2% paraformaldehyde/PBS for 15 min and labeled with excess of primary antibody against rabbit-anti-HA-tag (Santa Cruz, sc-805) and with a secondary antibody coupled to quantum dots (655 nm, Invitrogen). Avoiding cell permeabilization as well as the bulk of the QDs ensured that no intracellular proteins were labeled. Samples were run on a modified LSR II instrument from Becton and Dickinson using 405 nm excitation and a 660/40 nm emission filter.

Quantification of cAMP Signaling
The ability of 5-HT to modulate cAMP levels in cells under pharmacological treatment was assessed with the cAMP-Glo assay (Promega) according to the manufacturer's instructions. cAMP amounts were determined by fitting the results from triplicate experiments to the standard curve. The phosphodiesterase inhibitor IBMX (50 mM) was present during this treatment.

Statistical Analysis
Statistical significance of the data was tested by one-way-ANOVA with Bonferroni's post-test for multiple comparisons using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA) on a Microsoft Windows PC. A p-value of less than 0.05 was considered significant. Data are presented as mean ± standard error of the mean (SEM).

The 5-ht 5b Receptor Shows an Unusual Intracellular Localization
When expressed in N1E-115 cells, most 5-HT receptors produced a clear membrane-localized signal with minor staining of the Golgi apparatus due to trafficking ( Figure 1A). With 5-HT 4 and 5-HT 6 , membrane staining was strong, however a moderate amount of punctate clusters were visible in transfected cells. Only 5-ht 5a and 5-ht 5b showed a strong, punctate intracellular localization with little (5-ht 5a ) or no (5-ht 5b ) membrane staining at all. We have previously described that 5-ht 5b is retained in intracellular organelles with the most significant co-localization found with endosomes and fluorescence immunohistochemistry on mouse brainstem sections detected 5-ht 5b positive cells (Figure 1B01). High magnification images of this area revealed a clustered sub-cellular localization for 5-ht 5b in these cells (Figures 1B02,03). 5-ht 5b shows massive dysregulation in a mouse model of RTT (Vogelgesang et al., 2017). To follow up on the previously published results, we tested the sub-cellular localization of 5-ht 5b in primary hippocampal neurons ( Figure 1C) and could verify the results from N1E-115 cells with the highest co-localization of 5-ht 5b with endosomal marker rab5 (0.867 ± 0.016), while markers for membrane (0.035 ± 0.007), mitochondria (0.538 ± 0.06), ER (0.273 ± 0.17) and lysosome (0.471 ± 0.11) showed little overlap.

N-Terminal Glycosylation of 5-ht 5 Receptors Controls Intracellular Localization
Based on literature and the truncation experiments, the most likely mechanism responsible for sorting proteins between membrane and intracellular organelles other than distinct localization signals is glycosylation of the N-terminus (Vagin et al., 2009;Guangyu, 2015). An influence of glycosylation on the trafficking of 5-ht 5a was also shown previously (Dutton et al., 2008), with two glycosylation sites identified at N6 and N21 ( Figure 2B). Incidentally, the intracellular clusters of 5-ht 5a also co-localize strongest with endosomal markers (data not shown). Analyzing the protein sequences of 5-HT receptors, we found that strictly plasma membrane localized 5-HT 1A receptor possess four N-terminal glycosylation sites (Figure 2A) at positions N10, N11, N24 and N30, while 5-ht 5b receptors ( Figure 2C) possess only one glycosylation site at position N5. As 5-ht 5b can be detected in Western blots of murine brain both as a full length as well as a truncated protein (Vogelgesang et al., 2017), we tested if the truncation causes intracellular localization. An artificially truncated 5-HT 7-trunc produced a similar staining with completely absent membrane localization and fluorescence concentrated in intracellular organelles ( Figure 2D). However, 5-HT 7-trunc co-localized strongest with the lysosomal marker (data not shown). Thus, the artificial truncation led to a defective receptor that either folds incorrectly or is otherwise marked for degradation and is sent to the lysosome.
In contrast, the number of glycosylation sites corresponds well with the strength of membrane expression with no discernable membrane staining in case of 5-ht 5b (Figure 2C), weak staining for 5-ht 5a ( Figure 2B) and strong staining for 5-HT 1A (Figure 2A).
Successive removal of glycosylation sites in 5-HT 1A by site-directed mutagenesis led to increasing loss of surface expression. While removing one glycosylation site had no visible effect (data not shown), removal of two glycosylation sites produced only weak surface expression and strong endosomal accumulation of the receptor (Figure 2E). Removal of three or more glycosylation sites led to a total loss of surface expression (data not shown). Complete, yet unspecific de-glycosylation of 5-HT 1A receptors with 1 µM Tunicamycin ( Figure 2F) blocked cell surface localization of the receptor indicating a glycosylation dependence of its trafficking.
fusion of the N-terminus of 5-ht 5b to 5-HT 1A abolishes membrane localization ( Figure 2G). As it had already been confirmed for 5-ht 5a (Dutton et al., 2008), we concluded that N-terminal glycosylation also determines the membrane trafficking of 5-ht 5b .
We co-expressed a representative member of each 5-HT receptor subgroup tagged with mCherry against the same set of 5-HT receptors now tagged with GFP ( Figure 3A). However, we excluded 5-HT 3 R in this matrix because it is a multimeric ion channel (Uetz et al., 1994;Boyd et al., 2002). Quantification of the correlation coefficients revealed three separate populations ( Figure 3B). Here, one population corresponds to the receptor pairs that co-localize to the plasma membrane while the other corresponds to receptor pairs that remain separated between membrane and intracellular organelles. The third population was comprised of the receptor pairs that showed possible interaction. Of this group, the receptor pair of 5-HT 1A and 5-ht 5b displayed the most unusual behavior, with 5-ht 5b not showing enhanced surface expression, but instead retaining 5-HT 1A in the endosomal compartments with select specificity (Figure 3A04). This change of 5-HT 1A 's sub-cellular localization could also be reproduced in primary neurons ( Figure 3C).
After observing the retention of 5-HT 1A on intracellular organelles by 5-ht 5b , we looked for the mechanism. With GPCRs known to form oligomers, direct interaction was an obvious answer, but overloading of the transport pathway was also possible. To differentiate between both mechanisms, we performed a series of experiments while always using equal amounts of transfected DNA. An early indicator for interaction was the specificity of interaction. The change of subcellular localization only happens between 5-HT 1A and 5-ht 5b , but not between 5-HT 1A and any other receptor or 5-ht 5b and any other receptor.
First, we tested direct interaction using acceptorphotobleaching FRET (abFRET) microscopy ( Figure 4A) using GFP-and mRFP-labeled constructs (see Materials for details). Apparent FRET efficiency (F app ) of the two 5-HT receptors was 29.74% ± 1.6, which was even slightly higher than two rab5's expressed together (25.58% ± 4.6). Here, a strong FRET signal is expected as the proteins localize to the same organelles and are therefore in close proximity. Testing unspecific interaction by co-expressing 5-ht 5b with rab5 showed a significantly lower F app of 9.56% ± 1.1. Here, some FRET might be expected, as 5-ht 5b co-localizes with endosomes ( Figure 1C), but the FRET signal was significantly smaller compared to the 5-HT 1A :5-ht 5b signal. The protein pair of 5-ht 5b and Lamp1 produced a very low F app of 0.02% ± 0.7, as the proteins are on separate organelles (Vogelgesang et al., 2017) and only interact very little.
Then, to quantify the surface expression of 5-HT 1A in the absence and in the presence of 5-ht 5b , we used fluorescence microcopy ( Figure 4B). First, we stained only surface-expressed FIGURE 3 | 5-ht 5b specifically interacts with 5-HT 1A to change the latter's subcellular localization. (A) We transfected a representative member of each 5-HT receptor subgroup tagged with mCherry against the same set of 5-HT receptors tagged with GFP. Each image of the matrix shows N1E-115 cells co-expressing two 5-HT receptors. The images are pseudo-colored purple for the receptor indicated horizontally and pseudo-colored green for the receptor indicated vertically. Co-localization is shown in yellow. 5-ht 5b specifically interacts with 5-HT 1A (04) with the normally strictly membrane-bound 5-HT 1A receptors partly retained intracellularly. In addition, 5-ht 5a seems to interact with 5-HT 4 (12) and 5-HT 6 seems to weakly interact with 5-ht 5a (17) and 5-ht 5b (19), although 5-HT 6 's surface expression remains strong. No other 5-HT receptors seem to interact with each other in a way that changes subcellular localization. Scale bars in all images are 10 µm. (B) For quantification, we separately calculated Pearson's correlation coefficients for the fluorescence signals at the membrane and at intracellular organelles by manually applying regions of interest (ROI). Plotting the organelle correlation coefficient over the membrane correlation coefficient for each receptor pair resulted in three populations of receptors. One population displays a high correlation at the membrane, but not at intracellular organelles. The second population displays correlation in neither compartment while the third population displays high correlation at intracellular compartments. (C) To verify interaction of 5-HT 1A and 5-ht 5b , we transfected primary hippocampal neurons with expression constructs of both receptors. 5-HT 1A (green) localizes to the cell membrane, while 5-ht 5b (purple) localizes to intracellular organelles, while co-expression of both receptors shows intracellular co-localization with no discernable membrane staining by 5-HT 1A . Scale bars in all images are 20 µm. 5-HT 1A (green) in live cells, as antibodies cannot penetrate through the membrane. Then we fixed and permeabilized the cells and stained 5-HT 1A again with different primary and secondary antibodies, this time staining both surface-expressed as well as internal receptors (purple). Cells co-expressing 5-ht 5b were identified by its inherent mCherry-fusion fluorescent signal (red). We quantified the fluorescence intensity of surface-expressed 5-HT 1A (green) in cells with and without 5-ht 5b signal and found a reduction of 5-HT 1A surface expression by 52.3% ± 8.05 in cells co-expressing 5-ht 5b .
To quantify 5-HT 1A in a larger amount of cells, we used FACS ( Figure 4C). Cells expressing 5-HT 1A together with an equal amount of filler DNA was used to determine 5-HT 1A surface expression under control conditions. Co-expression of 5-HT 1A with an equal amount of 5-ht 5a did not change the degree of surface expression. This indicates that the mere presence of another plasmid or receptor did not affect the membrane localization of 5-HT 1A . However, the surface expression of 5-HT 1A was reduced by 48.8% ± 17.4% when it was expressed with an equal amount of 5-ht 5b (Figure 4C). The reduction of surface-expressed 5-HT 1A was FIGURE 4 | Analysis of receptor interaction. (A) Acceptor-photobleaching Förster Resonance Energy Transfer (abFRET) microscopy was used to determine the extent of protein:protein-interaction. Protein pairs of 5-HT 1A :5-ht 5b , rab5:rab5, 5-ht 5b :rab5 and 5-ht 5b :Lamp1 were co-expressed in N1E-115 cells at equimolar concentrations and the apparent FRET efficiency (F app ) was measured. The color-coded image shows an exemplary experiment of 5-HT 1A :5-ht 5b , while the bar diagram shows the apparent FRET efficiencies of 5-HT 1A :5-ht 5b (29.74 ± 1.6), rab5:rab5 (25.58 ± 4.6), 5-ht 5b :rab5 (9.56 ± 1.1) and 5-ht 5b :Lamp1 (0.02 ± 0.7). Asterisks indicate significance ( * * * p ≤ 0.001; one-way-ANOVA with Bonferroni's post-test for multiple comparisons). (B) Immunohistochemical analysis of distribution and equal expression levels. Non-permeabilized cells were stained for 5-HT 1A (green), then fixed and permeabilized and stained again for 5-HT 1A (purple). 5-ht 5b is co-expressed in some cells as a fusion protein with a fluorescent tag (red). The fluorescence intensity of the first 5-HT 1A staining was then measured in cells expressing 5-HT 1A and in cells expressing 5-HT 1A and 5-ht 5b . The cartoon shows the experimental procedure. The bar diagram shows the mean fluorescence intensities of 5-HT 1A reactivity in cells expressing only 5-HT 1A and in cells expressing 5-HT 1A (1.0 ± 0.06) and 5-ht 5b (0.52 ± 0.08) ( * * * p ≤ 0.001; unpaired t-test). (C) The effect of 5-HT 1A :5-ht 5b co-expression on 5-HT 1A surface localization was quantified by fluorescence activated cell sorting (FACS) analysis of N1E-115 cells expressing HA-5-HT 1A alone or together with 5-ht 5b . Expressing 5-HT 1A together with an equal amount of filler DNA was used to determine 5-HT 1A surface expression under control conditions (blue). Co-expression of 5-HT 1A with an equal amount of non-interacting 5-ht 5a did not change the degree of surface expression (green). Co-expression of 5-HT 1A with an equal amount of 5-ht 5b decreased the cell surface expression of 5-HT 1A by 48.8% ± 17.4%. The cartoon shows the experimental procedure. Labeling was directed at HA-tag on 5-HT 1A with quantum dot-coupled secondary antibodies. To avoid skewing results by transcription level effects in the control measurement, an unspecific filler plasmid (mock) was co-transfected in place of the second receptor to maintain the same total amount of transfected plasmid. consistent with the reduction seen with immunohistochemistry ( Figure 4B).
While the translocation of 5-HT 1A to a different sub-cellular localization was obvious (Figure 3A04), the amount of surface availability could also be affected by different expression levels caused through co-transfection. As expression of all receptor constructs is CMV driven and all construct have roughly the same size, we regard this as unlikely. Nevertheless, we analyzed whether expression levels of 5-HT 1A were affected by co-transfection by comparing the fluorescence intensity of 5-HT 1A -GFP in cells expressing equal amounts of 5-HT 1A and a filler DNA or cells expressing 5-HT 1A and 5-ht 5b . Although expression levels of both receptors fluctuated in individual cells, we found no significant overall differences (Supplementary Figure S1) that could explain the 2-fold reduction in surface expression seen above.
With direct interaction being the most likely mechanism, we cannot rule out indirect interaction. It has been shown previously that transport of 5-HT 1A in neurons is controlled by co-factor Yif1b (Carrel et al., 2008), but the involvement of adaptor proteins will be the topic of further studies.
While the change in subcellular localization was most pronounced for 5-HT 1A by 5-ht 5b , for completeness we would like to mention additional possible interactions that were revealed by plotting the correlation coefficients ( Figure 3B). We found that 5-ht 5a co-localized with 5-HT 4 ( Figure 3A12) and that both 5-ht 5 receptors showed moderate co-localization with 5-HT 6 (Figures 3A17 and 3A19). Although intracellular aggregation became more pronounced upon co-expression, both 5-HT 4 and 5-HT 6 showed already minor intracellular clusters when expressed alone ( Figure 1A). In these cases, overloaded trafficking is a more likely mechanism, but analysis is more complicated and will be done at a later time.

Co-Expression of Serotonin Receptors in the Mouse Brain
We used RT-PCR to determine the expression of all receptors in different regions of the brain (Figure 5). RT-PCR confirmed wide-spread 5-HT receptor expression in the murine brain with multiple receptors being expressed in basically every region analyzed. Among these, the hippocampal formation showed the largest diversity of all regions analyzed. Specifically, for 5-HT 1A and 5-ht 5b , we found regional co-expression in the bulbus olfactorius, the hippocampal formation and the brainstem.

Identification of Interaction Domains
Having prepared some hybrid receptors for the study of glycosylation and trafficking, we continued this strategy to try and define domains which would explain the specific interaction between 5-HT 1A and 5-ht 5b (Figure 6A).
Replacing the PDZ-like C-terminal domain of 5-ht 5b with that of 5-HT 1A had no influence on subcellular localization or interaction (HRV-15, Figure 6B).
A hybrid receptor (HRV-17) consisting of 5-HT 1A (from start up to TMD V) and 5-ht 5b (intracellular loop 3 (ICL 3 ) and onwards) can interact with wild type 5-HT 1A , although not as strong as seen between both wild-type receptors. This indicated that ICL 3 contains at least parts of the interaction domain ( Figure 6C).
Exchanging TMD IV and V of 5-HT 1A in the previous hybrid clone by TMD IV and TMD V of 5-ht 5b , thus creating HRV-18, fully restored the interaction to levels seen for the wild-type receptors ( Figure 6D).

Physiological Consequences of 5-ht 5b Expression
The strong and far-ranging expression of 5-ht 5b (Figure 5) indicates some physiological significance. However, given its intracellular expression, 5-ht 5b differs from classical GPCRs. With at best sparse surface expression, 5-ht 5b seems to contribute FIGURE 6 | Identification of possible interaction domains between 5-HT 1A (green) and 5-ht 5b (purple). N1E-115 cells were transiently transfected with two hybrid receptor variants labeled with eGFP or mCherry. (A) Co-expression of full length 5-HT 1A and full length 5-ht 5b lead to strong internalization of 5-HT 1A . (B) Replacing the C-terminal domain of 5-ht 5b with the C-terminal domain of 5-HT 1A (HRV-15) did not change the amount of interaction. (C) Expressing 5-HT 1A together with a hybrid 5-HT 1A where the 3rd intracellular loop and the last two TMDs are from 5-ht 5b (HRV-17), the interaction is greatly reduced or even abolished. (D) Expressing 5-HT 1A together with a hybrid receptor composed of the first three TMDs of 5-HT 1A and the last four TMDs of 5-ht 5b (HRV-18) show interaction as the two full length receptors.
little to intracellular signaling. However, a more likely function of 5-ht 5b could be the modulation of its interaction partner by affecting its surface availability.

DISCUSSION
In this study we described the behavior of 5-HT receptor 5-ht 5b , which manifests in intracellular localization to endosomal compartments and specific interaction with another select member of the 5-HT receptor family. 5-ht 5b localizes to endosomes (Figure 1C), which was also seen for 5-ht 5a (data not shown) and therefore might present a defining feature of this sub-group of 5-HT receptors. It might also indicate a physiological significance, as other truncated receptors are retained mainly in the ER (Karpa et al., 2000;Leung et al., 2007;Gonzalez et al., 2011). It is known that many GPCRs FIGURE 7 | Physiological consequences of 5-HT 1A :5-ht 5b co-expression. To test whether translocation of 5-HT 1A by 5-ht 5b effects cyclic adenosine monophosphate (cAMP) signaling, we measured the cellular cAMP level under various conditions. To measure the decrease of cAMP, 50 nM Forskolin was added to all cells before the start the experiment to increase cAMP levels. Mock-transfected cells treated with Forskolin and stimulated with 1 µM 5-HT served as control and were set to 100%. In all experiments, cells were transfected with an equimolar amount of DNA. To keep the amount of DNA the same in experiments with only one receptor, a filler plasmid was co-transfected. Expression of 5-ht 5b without stimulation with 5-HT decreased cAMP by −7.75% ± 1.012 (I), while addition of 5-HT did not enhance the reaction (−9.46% ± 1.296; II). Expressing 5-HT 1A in cells had no effect on cAMP levels without stimulation (−2.49% ± 1.366; III), but adding 5-HT decreased cAMP by −28.12% ± 1.096 (IV). Co-expression of 5-HT 1A and 5-ht 5b together without stimulation by 5-HT lead to a drop in cAMP by −10.37% ± 1.029 (V). Adding 5-HT to co-expressing cells dropped the cAMP level by −19.91% ± 2.219 (VI). Asterisks indicate significance ( * * p ≤ 0.01; * * * p ≤ 0.001; one-way-ANOVA with Bonferroni's post-test for multiple comparisons). naturally show constitutive activity or that constitutively active mutants exist (Tao, 2008). GPCRs were even described to signal from endosomal vesicles after internalization (Irannejad et al., 2013). Internal signaling might be indicated by the fact that we could measure a minor decrease of cAMP upon expression of 5-ht 5b without application of a ligand (Figure 7).
In the literature, several mechanisms, including glycosylation (Dutton et al., 2008), TMD size (Sharpe et al., 2010) or N-terminal signal peptides (Jahnsen and Uhlen, 2012) are discussed to control receptor trafficking to the membrane. The unusual intracellular localization of 5-ht 5a and 5-ht 5b seems to be caused in large part by glycosylation of the N-terminus as artificially increasing the number of glycosylation sites of 5-ht 5b increases its surface localization (Figures 2H,I). This form of glycosylation was also found to control membrane localization of 5-ht 5a (Dutton et al., 2008).
Formation of hetero-oligomers has been described for many GPCRs (Schwenk et al., 2010) including 5-HT receptors (Pellissier et al., 2011;Renner et al., 2012), although their interaction is subtle and its physiological significance remains to be shown. Here, 5-ht 5b 's affinity to 5-HT 1A was so strong that it changes the subcellular localization of 5-HT 1A . As both 5-HT receptors were found co-expressed in the brain (Figure 5), it is feasible to assume a physiological role for this specific interaction. The interaction mapping is still only rudimentary, but efforts to identify the binding interfaces of class A GPCRs have been ongoing for years, with most studies leaning towards TMD IV and TMD V as dimerization interface (González-Maeso et al., 2008;Johnston et al., 2011;Hu et al., 2012). These were also identified here as a possible location for the interaction interface. With the strong interaction between 5-HT 1A and 5-ht 5b and an easy read-out through translocation, further studies to fine-map the interaction surface might profit.
The mostly intracellular 5-ht 5b receptor probably does not relay signals in the conventional way, but by retaining 5-HT 1A in intracellular compartments, 5-ht 5b reduces its surface expression significantly, thus effectively reducing 5-HT 1A 's contributions to cellular signaling (Figures 4, 7). In alternate modes of action, 5-ht 5b might modify intracellular signaling by acting on the cAMP level, either through constitutive activity and/or by scavenging G-proteins which would no longer be available for other signal cascades.
It is difficult to define a role for the 5-HT 1A :5-ht 5b interaction in RTT, as the syndrome displays very complex genetic imbalances and many different and sometimes opposing therapies seem to improve symptoms in RTT. Within the serotonergic system alone, two previous observations in animal models of RTT might be linked to 5-HT 1A :5-ht 5b . Application of 5-HT 1A agonists was described to have beneficial effects on the respiratory phenotype in mice (Abdala et al., 2010) and might be linked to the reduction of surface-expressed 5-HT 1A by 5-ht 5b . In contrast, treatment with 5-HT 7 agonists was reported to improve behavior (De Filippis et al., 2014, 2015a and might counteract the constitutive down-regulation of cAMP by 5-ht 5b .
However, we would like to advise some caution interpreting these results. Here, we transfected recombinant cDNA at equimolar concentrations into cell lines and measured the effect of receptor activation on cAMP. In vivo, the situation is most likely much more complicated, as receptor expression levels may not be equal, co-factors might be present and signaling cascades are more complex. For example, we did not measure effects on CNG ion channels or on small GTPases. Of interest regarding the physiological function of 5-ht 5b is the fact that 5-HT 1A also becomes internalized upon activation, after which it seems to activate the Erk-1/2 signaling pathway (Renner et al., 2012). This is especially interesting as 5-ht 5b initially came to our attention when analyzing a mouse model of RTT (Vogelgesang et al., 2017) and modulation of small RhoGTPase activity was shown to have beneficial effects on RTT (De Filippis et al., 2012, 2015b.