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Modified Receptor Internalization upon Coexpression of 5-HT_1B Receptor and 5-HT_2B Receptors

Institut National de la Santé et de la Recherche Médicale, U839, Paris, France (V.S., L.M.); Université Pierre et Marie Curie-Paris 6, Institut du Fer à Moulin, Unité Mixte de Recherche S0839, Paris, France (V.S., L.M.); Centre National de la Recherche Scientifique UMR7104, Illkirch, France (A.J., M.D., S.G., L.M.); Assistance Publique-Hopitaux de Paris, Hôpital Lariboisière, Service de Biochimie, Paris, France (J.C., B.S., P.M., J.M.L.); EA3621, IFR139, Paris, France (J.C., B.S., P.M., J.M.L.)

Received November 14, 2006; accepted February 26, 2007

	Abstract

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Serotonin 5-HT_2B receptors are often coexpressed with 5-HT_1B receptors, and cross-talk between the two receptors has been reported in various cell types. However, many mechanistic details underlying 5-HT_1B and 5-HT_2B receptor cross-talk have not been elucidated. We hypothesized that 5-HT_2B and 5-HT_1B receptors each affect the others' signaling by modulating the others' trafficking. We thus examined the agonist stimulated internalization kinetics of fluorescent protein-tagged 5-HT_2B and 5-HT_1B receptors when expressed alone and upon coexpression in LMTK^– murine fibroblasts. Time-lapse confocal microscopy and whole-cell radioligand binding analyses revealed that, when expressed alone, 5-HT_2B and 5-HT_1B receptors displayed distinct half-lives. Upon coexpression, serotonin-induced internalization of 5-HT_2B receptors was accelerated 5-fold and was insensitive to a 5-HT_2B receptor antagonist. In this context, 5-HT_2B receptors did internalize in response to a 5-HT_1B receptor agonist. In contrast, co-expression did not render 5-HT_1B receptor internalization sensitive to a 5-HT_2B receptor agonist. The altered internalization kinetics of both receptors upon coexpression was probably not due to direct interaction because only low levels of colocalization were observed. Antibody knockdown experiments revealed that internalization of 5-HT_1B receptors (expressed alone) was entirely clathrin-independent and Caveolin1-dependent, whereas that of 5-HT_2B receptors (expressed alone) was Caveolin1-independent and clathrin-dependent. Upon coexpression, serotonin-induced 5-HT_2B receptor internalization became partially Caveolin1-dependent, and serotonin-induced 5-HT_1B receptor internalization became entirely Caveolin1-independent in a protein kinase C-dependent fashion. In conclusion, these data demonstrate that coexpression of 5-HT_1B and 5-HT_2B receptors influences the internalization pathways and kinetics of both receptors.

A general feature of GPCRs is the existence of complex intracellular regulatory mechanisms that modulate receptor responsiveness. Receptor desensitization and down-regulation are well described for various individual GPCR subtypes and are important homeostatic mechanisms. Homologous desensitization involves the desensitization of a particular receptor subtype upon activation of that receptor subtype. In contrast, heterologous desensitization involves the desensitization of receptor subtype(s) upon stimulation of a different receptor subtype. In view of the increasing number of reports of GPCRs participating in complexes via dimerization or scaffolding proteins, cross-talk between receptor subtypes may represent an important additional regulatory mechanism at modulating sensitivity and/or signal transduction. Agonist-induced internalization of GPCRs uses a pathway determined by a kinase that phosphorylates the receptor: for the 1-adrenergic receptor, protein kinase A (PKA)-mediated phosphorylation directs internalization via caveolae, whereas GPCR kinase (GRK)-mediated phosphorylation directs internalization through clathrin-coated pits (Rapacciuolo et al., 2003). The recruitment, activation, and scaffolding of cytoplasmic signaling complexes occurs via two multifunctional adaptor and transducer molecules, -arrestin-1 and -2 (Lefkowitz and Shenoy, 2005).

Among the described internalization mechanisms, 5-HT-induced receptor desensitization has been reported for receptors closely related to 5-HT_2B receptors (i.e., 5-HT_2A and 5-HT_2C receptors). Desensitization of 5-HT_2A receptor was shown to involve receptor internalization through caveolin1 (Cav1), a scaffolding protein enriched in caveolae, in a number of cell lines expressing exogenous 5-HT_2A receptors, and rat brain synaptic membrane preparations (Bhatnagar et al., 2004). There is also evidence for functional interactions among 5-HT_2A receptors and other plasma membrane microdomain proteins: 5-HT-induced 5-HT_2A receptor desensitization can also involve receptor internalization through a clathrin- and dynamin-dependent process (Hanley and Hensler, 2002). Internalization and desensitization of 5-HT_2A receptors in some cell types is -arrestin-independent (Gray et al., 2003). A direct interaction between PSD-95 and the 5-HT_2A receptor at a type I PSD-95, Dlg, ZO-1 (PDZ)-binding domain at the C terminus regulates the receptor's signal transduction and trafficking (Xia et al., 2003). For the 5-HT_2C receptor, constitutively active edited isoform is spontaneously internalized in an agonist-independent manner via the activity of a GRK/-arrestin (Marion et al., 2004).

Receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has also been shown to have implications for receptor trafficking, signaling, and pharmacology (George et al., 2002). Serotonin 5-HT_2C receptors were shown to exist as constitutive homodimers on the plasma membrane of living cells using a confocal-based fluorescent resonance energy transfer (FRET) method (Herrick-Davis et al., 2004). Inactive 5-HT_2C receptors can inhibit wild-type 5-HT_2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane (Herrick-Davis et al., 2005). The 5-HT_1B and 5-HT_1D receptor subtypes that share a high amino acid sequence identity have also been shown to exist as monomers and homodimers when expressed alone and as monomers and heterodimers when coexpressed (Xie et al., 1999). Heterodimerization between 5-HT₁ and 5-HT₂ receptors, and its functional consequences have yet to be investigated.

The mechanistic details of 5-HT_1B receptors internalization have not yet been determined. Despite the coexpression of 5-HT_1B and 5-HT_2B receptors in various tissues including endothelial and smooth muscle cells (Ullmer et al., 1995), and given the inhibitory effect of 5-HT_2B receptors on 5-HT_1B receptor signaling (Tournois et al., 1998), physical interaction between the two receptors seems plausible. The internalization of the 5-HT_2B receptor is faster than that of 5-HT_2A and 5-HT_2C receptors (Porter et al., 2001; Schaerlinger et al., 2003; Deraet et al., 2005), although the mechanism underlying this distinction has not been uncovered. In this work, we investigated potential 5-HT_1B/2B receptor interactions by examining the colocalization and internalization kinetics of 5-HT_1B and 5-HT_2B receptors expressed alone or together. Using cyan and yellow fluorescent protein (CFP and YFP) tagged receptors and confocal microscopy, we observed agonist-induced receptor endocytosis in real time. We also performed whole-cell radioligand binding studies as an additional means of measuring receptor internalization. Our results indicate that the stimulation of 5-HT_1B receptors affects the internalization dynamics of 5-HT_2B receptors and vice versa, with the effect of 5-HT_1B receptors on 5-HT_2B receptors being more pronounced than the effect of the latter receptor on the former. Furthermore, we used an antibody knockdown strategy to ascertain which pathways each receptor used for internalization. Our findings reveal that coexpression of 5-HT_1B and 5-HT_2B receptors affects both the kinetics of receptor internalization and the internalization pathway employed compared with either receptor expressed alone.

	Materials and Methods

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Reagents. RS127445 was kindly provided by Roche (Mannheim, Germany); CP93129, BW723C86, H-89, Gö 6850-Bisindolylmaleimide I, Gö 6976, and all other chemicals were reagent grade, purchased from usual commercial sources. The radioactive compounds 1-(2,5-dimethoxy-4-[¹²⁵I]iodophenyl)-2-aminopropane hydrochloride ([¹²⁵I]DOI; 81.4 TBq/mmol) and [¹²⁵I]serotonin-5-O-carboxymethyl-glycil-iodo-tyrosamine ([¹²⁵I]GTI; 81.4 TBq/mmol) were purchased from PerkinElmer Life and Analytical Sciences. Antibodies that were already specificity-tested were used: rabbit antisera against rodent Cav1 (H-97), clathrin (H-300), GRK2,3 (H-222), and GRK5,6 (C-20) and goat antiserum against rodent -arrestin-2 (D-18) and PKC (C-15) (Santa Cruz Biotechnology; Santa Cruz, CA).

Mutagenesis and 5-HT_2B Receptor Constructs. The fluorescent-tagged fusion proteins of the mouse 5-HT_2B and 5-HT_1B receptors were generated by PCR-based subcloning. The receptor coding regions were subcloned into pECFP, pEYFP, pPA-GFP (photo-activable GFP) vectors with the XFP fused to the N terminus of the receptors. The entire coding sequence of all constructs was verified by automated DNA sequencing.

Cell Culture. 5-HT_2B and 5-HT_1B receptor cDNAs were stably transfected into nontransformed murine fibroblast (LMTK^–) cells, which are devoid of endogenous 5-HT receptors because they do not exhibit a concentration-dependent rise in second messengers after 5-HT stimulation (Manivet et al., 2000). LMTK^– cell lines were routinely cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum and 40 µg/ml gentamicin. The stably expressing mouse 5-HT_1B and 5-HT_2B cell lines were generated by calcium phosphate transfection followed by selection with either G-418 or hygromycin and clonal isolation. Stable receptor-expressing lines were subcultured in serum free medium for at least 24 h before experiments. Stable cell lines with different combinations of receptor expression insured the absence of individual cell based effects.

Radioligand Binding Experiments. Radioligand binding experiments were performed using [¹²⁵I]DOI or [¹²⁵I]GTI either on intact cells or on membranes from stably transfected cells as previously detailed (Loric et al., 1995).

Cell Permeabilization. The cells were washed twice with phosphate-buffered saline containing 0.1% bovine serum albumin and exposed to 1 hemolytic unit of alveolysin/10⁶ cells at 22°C under agitation as described previously (Manivet et al., 2000).

Internalization Measurements by Confocal Microscopy. For confocal microscopic studies of living cells, stable or transiently transfected cell lines were plated 36 h before the analysis on 35-mm glass-bottomed dishes (MatTek, Ashland, MA) and grown in a 5.4% CO₂ incubator. Twelve hours before the experiment, cells were washed in Dulbecco's modified Eagle's medium without serum and maintained in this medium until the experiment. Confocal analysis were performed at 22°C unless otherwise mentioned in the text to extend the kinetic and 37°C was used to confirm the observed phenomenon at 22°C. Cells were visualized using a confocal microscope (SP2AOBS; Leica, Wetzlar, Germany) with laser excitation lines of 458 nm and 514 nm for CFP- and YFP-tagged receptors and transmitted light. Images of CFP and YFP emission were recorded simultaneously with the transmitted light images. The emission recording channels and the intensity of the excitation lasers were carefully chosen using single tag control linear unmixing technique (Leica) to avoid bleed-through. Images (in xyzt mode) were recorded sequentially every 5 min in two different emission intervals (462–500 nm for CFP and 520–600 nm for YFP) with two different excitation wavelengths (458 and 514 nm). To ensure consistency among z-plane images during time-lapse study, we took at least five z-plane images and manually selected only one plane for the time-lapse analysis. To visualize receptor internalization after agonist treatment, time-lapse series were taken every 5 min over a 30-min period. To calculate the internalization kinetics of the receptors, we selected 10 regions of interest (ROIs) on the plasma membrane per cell and followed the relative intensity changes of these ROIs by time, including at least three to five individual cells per experiment. Data represent more than four independent experiments. The kinetic curves were corrected for bleaching by using the intensity of the whole cell as a normalization factor. PA-GFP was activated with a 405 nm diode laser line pulse lasting 10 to 15 sec and visualized between 495 and 515 nm using excitation at 488-nm.

FRET Measured by Confocal Microscopy. For the colocalization of 5-HT_2B and 5-HT_1B receptors in living cells, sensitized emission fluorescent resonance energy transfer (FRET) was used. Two additional channels [the FRET channel (excitation, 458 nm; emission, 520–600 nm) and a control channel (excitation, 514 nm; emission, 462–500 nm)] were recorded along with the CFP and YFP channels as described above. The bleed-through of CFP and the direct excitation of YFP by the 458 nm laser light were subtracted from the FRET channel signal. To estimate these artifacts, we used cells transfected with either CFP- or YFP-tagged receptors. Calculation of corrected FRET was carried out on a pixel-by-pixel basis for the entire image. Indeed, positive FRET signal could be obtained using tagged proteins known to interact in similar experimental set-ups (data not shown).

Colocalization Calculation. We considered two proteins to be colocalized if the observed signals of the two corresponding labels were nonzero at the same pixel. The quantitative estimate of colocalization is given as the colocalization coefficients (Manders et al., 1992). To quantify the colocalized fraction of each receptor pair, a threshold value for each channel was estimated and subtracted. Bleed-through in each of the two detecting channels was subtracted using the linear unmixing method (Leica).

Data Analysis. Binding data were analyzed using the iterative nonlinear regression model (Prism 2.0; GraphPad Software, San Diego, CA). This allowed the calculation of dissociation constants (K_D) and the number of sites (B_max). All values represent the average of independent experiments ± S.E.M. (n = number of experiments as indicated in the text). Comparisons between groups were performed using Student's unpaired t test or analysis of variance and a Fischer test. Significance was set at p < 0.05.

	Results

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Radioligand saturation binding assays were performed on membranes from stable, clonal cell lines expressing either 5-HT_1B or 5-HT_2B receptors, bearing various N-terminal fluorescent protein tags as well as on cells coexpressing both receptors. The clones chosen for subsequent experiments were selected so as to approximate physiological receptor expression (B_max of approximately 100 fmol/mg of protein) (Tables 1 and 2). In control experiments with nontagged receptors, we verified that none of the N-terminal fluorescent tags affected radioligand affinity (Tables 1 and 2). In addition, whole-cell radioligand binding experiments on clones expressing either fluorescent protein-tagged or nontagged receptors yielded a B_max approximately 60% of that measured on membrane preparations, suggesting that the fluorescent tag did not perturb membrane targeting (Tables 1 and 2).

Kinetics of 5-HT-Induced Internalization of 5-HT_2B and 5-HT_1B Receptors. To establish a quantitative method to measure internalization kinetics of the tagged receptors, fluorescence intensity at the membrane was compared with that in the cytoplasm in cells treated with 100 nM 5-HT for 0 to 30 min. The time-dependent fluorescence intensity changes at the plasma membrane and in the cytoplasm both fit a one-phase exponential function (decrease for the plasma membrane, increase for the cytoplasm) and had similar half-lives (5-HT_2B receptor cytoplasm t_1/2 = 23.0 ± 3.0 min; membrane t_1/2 = 22.2 ± 3.3 min; 5-HT_1B receptor cytoplasm t_1/2 = 9.8 ± 2.2 min, membrane t_1/2 = 10.7 ± 1.4 min). Because the signal-to-noise ratio was higher for the measurement of decreases in plasma membrane fluorescence, this was chosen to measure endocytosis kinetics at the plasma membrane (Fig. 1, A and B). To further confirm that we were measuring receptor endocytosis, we performed experiments with photoactivatable (PA) GFP-tagged 5-HT_1B receptors and compared the rate of decrease in fluorescence intensity of the illuminated membrane region with that determined for plasma membrane YFP-tagged 5-HT_1B receptors. As before, for both GFP- and YFP-tagged 5-HT_1B receptors, the internalization kinetics fitted a one-phase exponential decay and were identical (5-HT_1B-PA-GFP t_1/2 = 9.8 ± 0.7 min; 5-HT_1B-YFP t_1/2 = 10.1 ± 1.2 min), further validating our image-based analysis of receptor internalization kinetics.

View larger version (26K): [in this window] [in a new window]   Fig. 1. 5-HT stimulus on 5-HT2B and 5-HT1B receptor, dose dependence. Confocal studies in living cells were performed on various LMTK–-transfected cell lines plated on glass-bottomed dishes in Dulbecco's modified Eagle's medium without serum. Quantitative internalization kinetics of the GFP-tagged receptors was assessed by measuring the fluorescence intensity disappearance at the membrane compared with the intensity increase in the cytoplasm of approximately 10 ROI per cell and followed their relative intensity changes. Both signals could be fitted with a single exponential function over time, giving the same half-time. A, series of single confocal plane images taken from living cells expressing 5-HT1B receptor-GFP by time lapse video were used to evaluate the internalization kinetics after stimulation by 100 nM 5-HT (right). Distribution of 5-HT1B receptors expressed at 0 (0) and 50 (50) min of 5-HT stimulation. B, series of single confocal plane images was taken from living cells expressing 5-HT2B receptor-GFP by time lapse video. Internalization kinetics after stimulation by 100 nM 5-HT stimulation (right). Distribution of 5-HT2B receptors expressed at 0 and 50 min of stimulation. These images are representative of more than three cells observed in each of at least four independent experiments. Scale bars, 2 µM. C, similar studies by confocal laser microscopy showed that 5-HT1B and 5-HT2B receptors internalized with a single exponential kinetics after stimulation with 100 nM, 1 µM, or 10 µM 5-HT, and their half-lives were concentration-dependent. D, study by confocal laser microscopy showed that when 5-HT2B receptors were coexpressed with 5-HT1B receptors (5-HT2B1B), the 5-HT2B receptor internalized with a single exponential kinetics after stimulation with 100 nM 5-HT, but its half-life became five times faster. E, when 5-HT1B receptors were coexpressed with 5-HT2B receptors (5-HT1B2B light gray bar), the 5-HT1B receptor internalized after stimulation with 100 nM 5-HT with a similar half-life to 5-HT1B receptor alone (black bar). F, independent radioligand binding experiments using [125I]DOI (5-HT2B receptor) or [125I]GTI (5-HT1B receptor) on intact living cells confirmed that [compared with 5-HT2B receptor expressed alone (dark gray bar)] when the 5-HT2B receptor was coexpressed with 5-HT1B receptors, stimulation with 100 nM 5-HT decreased the 5-HT2B receptor (5-HT2B1B white bar) half-life but not that of 5-HT1B receptors (5-HT1B2B light gray bar). Graphs display the mean± S.E.M. of at least three independent experiments.

Having established internalization kinetics for both receptors expressed alone, we set out to ascertain whether coexpression altered 5-HT-induced 5-HT_1B and 5-HT_2B receptor internalization rates. Coexpression with the 5-HT_1B receptor resulted in a 5-fold increase in the rate of 5-HT-induced 5-HT_2B receptor internalization (2B^1B)(t_1/2 = 4.0 ± 1.5 min versus 23.0 ± 3.0) (Fig. 1, D and E). On the other hand, coexpression with 5-HT_2B receptors had no effect on 5-HT-induced 5-HT_1B receptor internalization rate (1B^2B)(t_1/2 = 9.0 ± 1.0 min versus 9.8 ± 2.2) (Fig. 1, D and E). Kinetic whole-cell radioligand binding experiments corroborated our microscopy data, revealing the asymmetric effect of receptor coexpression on 5-HT-induced 5-HT_1B and 5-HT_2B receptor internalization rate (Fig. 1, E and F).

Agonist-Dependent 5-HT_2B Receptor Internalization. To determine whether the effect of 5-HT_1B receptors on 5-HT_2B receptor internalization kinetics involved activation of 5-HT_2B receptors, we stimulated cells coexpressing both receptors in the absence and presence of the highly selective 5-HT_2B receptor antagonist RS127445 (RS). In cells expressing 5-HT_2B receptor alone, 100 nM RS completely blocked 5-HT-induced 5-HT_2B receptor internalization. It was striking that RS had no effect on 5-HT-induced 5-HT_2B receptor internalization in cells coexpressing 5-HT_1B receptors (Table 3). RS did not significantly affect 5-HT-induced 5-HT_1B receptor internalization irrespective of 5-HT_2B receptor coexpression.

To investigate whether modulation of internalization kinetics was agonist-dependent, we stimulated with the preferential 5-HT_2B receptor agonist BW723C86 (BW). Treatment with 50 nM BW induced no 5-HT_1B receptor internalization but did stimulate 5-HT_2B receptor internalization (t_1/2 = 11.0 ± 1.5 min) in noncoexpressing cells (Fig. 2A; Table 3). Coexpression of 5-HT_1B and 5-HT_2B receptors did not significantly alter the effect of BW on the internalization of either receptor: the internalization kinetics of 5-HT_2B receptors changed only slightly in the presence of 5-HT_1B receptor (t_1/2 = 7.2 ± 1.1 min) (Fig. 2B; Table 3), whereas no internalization of 5-HT_1B receptors was observed (Fig. 2C)

View larger version (30K): [in this window] [in a new window]   Fig. 2. Ligand dependence of 5-HT1B receptor and 5-HT2B receptor internalization. Quantitative internalization kinetics of the GFP-tagged receptors was assessed by measuring the fluorescence intensity disappearance at the membrane of series of single confocal plane images taken from living transfected LMTK– cells by time lapse video. A–C, the fluorescence intensity disappearance has been fitted with a single exponential function over time and used to evaluate the internalization kinetics after stimulation by the preferential 5-HT2B receptor agonist BW723C86 (BW) at the final concentration of 50 nM that produced no effect on the internalization of 5-HT1B receptor alone. D–F, the fluorescence intensity disappearance has been fitted with a single exponential function over time and used to evaluate the internalization kinetics after stimulation by the selective 5-HT1B receptor agonist CP93129 (CP) at the final concentration of 75 nM that produced no effect on the internalization of 5-HT2B receptor alone. The x-axes display 0 to 30 min recording for fast internalization (A–C and E) and 0 to 60 min for slow internalization (D and F). Deduced half-life values are reported in Table 3 with statistics.

Temperature Dependence of 5-HT_1B and 5-HT_2B Receptor Internalization. To verify that agonist-induced receptor internalization was due to an energy-dependent endocytic process, receptor internalization assays were performed at various temperatures. As expected, the kinetics of 5-HT-induced receptor internalization were faster at higher temperatures for both receptors, irrespective of coexpression. In addition, the temperature dependence of agonist (5-HT or subtype selective)-induced receptor internalization rate appeared linear for 5-HT_1B receptors and biphasic for 5-HT_2B receptor when expressed alone (Fig. 3, A and B).

View larger version (26K): [in this window] [in a new window]   Fig. 3. Temperature dependence of the of 5-HT1B receptor and 5-HT2B receptor internalization. The receptors half-life (t1/2) were evaluated using transfected LMTK– cells expressing GFP-tagged receptors by measuring the fluorescence intensity disappearance at the membrane after fitting with a single exponential function over time. Upon 5-HT stimulation, the t1/2 values in single receptor transfected cells was evaluated at different temperatures (22°C to 25°C and 37°C) for 5-HT1B receptors (A), for 5-HT2B receptors (B), or alone or coexpressed, after stimulation by CP for 5-HT1B receptors (C–E), or for 5-HT2B receptor (D–E) after BW stimulation. NS, no significant statistical difference. *, P < 0.05, more than three cells were analyzed in at least four independent experiments.

Coexpression of both receptors did not seem to affect the effect of temperature on CP-induced 5-HT_1B receptor internalization (Fig. 3C). On the other hand, coexpression with 5-HT_1B receptors rendered the temperature dependence of BW-induced 5-HT_2B receptor internalization more linear (Fig. 3D). Furthermore, the temperature dependence of CP-induced 5-HT_2B receptor internalization rate was identical to that observed for 5-HT_1B receptors (Fig. 3E).

Microscopic Analysis Revealed No Receptor Colocalization. The apparent effect of receptor coexpression on agonist-induced 5-HT_2B and 5-HT_1B receptor internalization led us to hypothesized receptor heterodimerization. Therefore, we performed confocal microscopy on cells coexpressing CFP-5-HT_1B and YFP-5-HT_2B receptors. Cellular distribution analysis of the two receptors revealed approximately 20% colocalization in the plasma membrane before stimulation (Fig. 4). After 30 min of agonist stimulation, cytoplasmic colocalization of 5-HT_2B and 5-HT_1B receptors was still approximately 20%, irrespective of agonist. Using enhanced emission to measure FRET, we observed nearly no FRET signal at 5-HT_1B/5-HT_2B receptor colocalization points (Fig. 4). Thus, our image-based analysis of colocalization was not consistent with agonist-induced 5-HT_1B/5-HT_2B receptor complex formation.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Single-cell fluorescence measurements for quantitative analysis. The cellular distribution of CFP-tagged 5-HT2B receptors (2B, red) co-expressed with YFP-5-HT1B receptors (1B green) was video-recorded and is displayed on a single confocal plane before (0 min) and after (30 min) stimulation with different agonists. The corresponding colocalizing points (1B2B, yellow) and deduced FRET values are shown (right panels, white). A, distribution of 5-HT2B receptor expressed at 0 and 30 min of CP stimulation. B, distribution of 5-HT2B receptor expressed at 0 and 30 min of 5-HT stimulation. C, distribution of 5-HT2B receptor expressed at 0 and 30 min of BW stimulation. These images are representative of more than 3 cells observed in each of at least four independent experiments. Scale bars, 2 µM.

We observed that the internalization of 5-HT_1B receptors expressed alone was independent of GRK5,6 (data not shown) and clathrin but totally dependent on Cav1 and GRK2,3 when stimulated by 5-HT or CP (Fig. 5, A and B). The internalization of 5-HT_2B receptors expressed alone was independent of GRK5,6 (data not shown) and Cav1 but completely dependent on clathrin and -arrestin-2 when stimulated by 5-HT or BW (Fig. 5, A–C). These results established that 5-HT_1B and 5-HT_2B receptors, when expressed alone, used distinct internalization pathways in an identical cell background.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Whole-cell binding and antibody knockdown for quantitative analysis. The amount of membrane receptor sites in transfected LMTK– cells was assessed by radioligand binding experiments using [125I]DOI (5-HT2B receptor) or [125I]GTI (5-HT1B receptor) on intact living cells 0, 5, and 10 min after agonist stimulation (KD and Bmax). With unmodified KD (not illustrated), the time-dependent changes in Bmax allowed the approximation of the receptor half-life in response to various agonists (left). Exposure of alveolysin-permeabilized cells to antibodies against caveolin-1, clathrin, -arrestin-2, or GRK2,3 modifies the receptor half-life (expressed as -fold change over unexposed cells, right), upon agonist stimulation 5-HT (A), CP (B), and BW (C). Black bars, 5-HT1B receptors; light gray bars, 5-HT1B receptors coexpressed with 5-HT2B receptors (5-HT1B2B); dark gray bars, 5-HT2B receptors; white bars, 5-HT2B receptors coexpressed with 5-HT1B receptors (5-HT2B1B). Graphs display the mean ± S.E.M. of at least three independent experiments.

We next applied the antibody knockdown strategy to cells coexpressing 5-HT_1B and 5-HT_2B receptors. When stimulated by 5-HT or CP, but not BW, the internalization of 5-HT_2B receptors became partially sensitive to Cav1 antibodies. Furthermore, the internalization of 5-HT_2B receptors was entirely sensitive to Arrestin-2 antibodies when stimulated with 5-HT in the absence or presence of 5-HT_1B receptors, and with CP in the presence of 5-HT_1B receptors (Fig. 5, A and B). However, 5-HT_2B receptor internalization was only partially inhibited by anti--Arrestin-2 when coexpressed with 5-HT_1B receptors and stimulated with BW (Fig. 5C). This result further suggested that 5-HT_1B receptors could affect the internalization pathway of 5-HT_2B receptors. Finally, the agonist-induced internalization of 5-HT_2B receptors was partially dependent on GRK2,3 when coexpressed with 5-HT_1B receptors and stimulated with 5-HT, CP, or BW (Fig. 5). Thus, the effect of 5-HT_1B receptor coexpression on 5-HT_2B receptors caused a fraction of 5-HT_2B receptors to internalize via a Cav1- and GRK2,3-dependent pathway.

With respect to 5-HT_1B receptors, coexpression with 5-HT_2B receptors caused 5-HT-induced 5-HT_1B receptor internalization to become totally independent of Cav1 and GRK2,3. Furthermore, upon coexpression with 5-HT_2B receptors, 5-HT-induced 5-HT_1B receptor internalization was still independent of clathrin and Arrestin-2. In contrast, the Cav1/GRK2,3 dependence of CP-induced 5-HT_1B receptor internalization was not affected by coexpression with 5-HT_2B receptors. Thus, the effect of 5-HT_2B receptor coexpression on 5-HT_1B receptor internalization is to alter the 5-HT-induced internalization pathway from a fully Cav1-dependent pathway to one fully independent of both Cav1 and clathrin (Fig. 5).

Serotonin-Induced Stimulation of PKC by 5-HT_2B Receptors Regulates the Pathway of 5-HT_1B Receptor Internalization. To investigate the non–Cav1-dependent/non–clathrin-dependent internalization pathway used by 5-HT-stimulated 5-HT_1B receptors coexpressed with 5-HT_2B receptors, we tested the effect of various protein kinase inhibitors. The wide protein kinase inhibitor staurosporine (5 µM), which inhibits PKC, PKA, and protein kinase G, blocked 5-HT-induced 5-HT_1B receptor internalization when coexpressed with 5-HT_2B receptors (Fig. 6). H89 (5 µM), which inhibits PKA, protein kinase G, and PKCµ, but not other PKCs (Davies et al., 2000), had no effect on 5-HT_1B or 5-HT_2B receptor internalization. To further refine these results, we used PKC isotype-selective inhibitors to identify the PKC isozyme involved. We found that Gö 6850-Bisindolylmaleimide I (100 nM), a PKC inhibitor with high selectivity for PKC, -I, -II, -, -, and - isozymes, completely prevented 5-HT-induced 5-HT_2B receptor internalization or that of 5-HT_1B receptors in the presence of 5-HT_2B receptors. This blocking effect was not observed with Gö 6976 (100 nM), which selectively inhibits the Ca²⁺-dependent PKC and -I. Finally, the blocking effect of Gö 6850-Bisindolylmaleimide I was completely reproduced using PKC antibody knockdown (Fig. 6). These data indicate that the 5-HT stimulation of 5-HT_2B receptors triggers 5-HT_1B receptor internalization via a pathway that requires 5-HT_2B receptor dependent PKC activation.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Whole-cell binding and pharmacological treatment for quantitative analysis. The amount of membrane receptor sites in transfected LMTK– cells was assessed by radioligand binding experiments using [125I]DOI (5-HT2B receptor) or [125I]GTI (5-HT1B receptor) on intact living cells 0, 5, and 10 min after agonist stimulation (KD and Bmax). With unmodified KD (not illustrated), the time-dependent changes in Bmax allowed to approximate the receptor half-life in response to various protein kinase blockers: 5-HT (A), 5-HT + staurosporine (5 µM) (B), 5-HT + H89 (5 µM) (C), 5-HT + Gö 6976 (100 nM) (D), and 5-HT+Gö 6850-Bisindolylmaleimide I (100 nM) (E). Exposure of alveolysin-permeabilized cells to antibodies against PKC modifies the receptor half-life, upon 5-HT stimulation (F). Black bars, 5-HT1B receptors; light gray bars, 5-HT1B receptors coexpressed with 5-HT2B receptors (5-HT1B2B); dark gray bars, 5-HT2B receptors; white bars, 5-HT2B receptors coexpressed with 5-HT1B receptors (5-HT2B1B). Graphs display the mean ± S.E.M. of three independent experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The present results demonstrate that individually, 5-HT_1B and 5-HT_2B receptors expressed in nontransformed mouse fibroblast LMTK^– cells use typically described agonist-dependent internalization pathways. The differences in the kinetics and temperature dependence of internalization strongly support the notion that these two receptors—when expressed alone—use different endocytic pathways, each agonist leading to specific output. The antibody knock-down experiments validate these findings and demonstrate that 5-HT_1B receptors expressed alone internalize via a Cav1- and GRK2,3-dependent pathway, whereas 5-HT_2B receptors expressed alone internalize via a clathrin-, -arrestin-2-, and PKC-dependent pathway (Fig. 7A).

View larger version (48K): [in this window] [in a new window]   Fig. 7. Model of interactions between 5-HT2B and 5-HT1B receptors in their internalization pathways. A, when expressed as a single receptor in cells devoid of endogenous 5-HT receptors expression and upon 5-HT stimulation, 5-HT1B receptor internalizes via a Cav1- and GRK2,3-dependent pathway, whereas 5-HT2B receptors internalize via a clathrin and arrestin-2-dependent pathway. B, when coexpressed, the two receptors respond differently to selective agonists. A selective 5-HT2B receptor agonist, BW, does not trigger any internalization of coexpressed 5-HT1B receptor and acts as 5-HT on the 5-HT2B receptor internalization (not illustrated). By contrast, stimulation of 5-HT1B receptor by CP, a selective 5-HT1B receptor agonist, makes the 5-HT1B receptor internalizing via Cav1 and GRK2,3 as 5-HT. Although inefficient on cells expressing 5-HT2B receptor alone, CP triggers also the internalization of coexpressed 5-HT2B receptor via Cav1, GRK2,3, and -arrestin-2 imposing for these two receptors the same kinetic for internalization. GRK2,3 becomes a partner of this trans-internalization of 5-HT2B receptors. C, the costimulation of both receptors by 5-HT makes 5-HT2B receptors to internalize via its classic clathrin but also via 5-HT1B triggered Cav1-dependent pathways (large arrow), which imposes in return 5-HT1B receptors to adopt new internalization pathways independent of both clathrin and Cav1 but that becomes dependent on 5-HT2B receptor-induced PKC (small arrow).

Our experimental results implicate GRK2,3 in mediating the cross-regulation of 5-HT_2B receptor internalization by 5-HT_1B receptors. When 5-HT_2B receptors are coexpressed with 5-HT_1B receptors, CP activates 5-HT_1B receptors that in turn activate GRK2,3, which leads to agonist-independent, Cav1-dependent internalization of 5-HT_2B receptors. One likely possibility is that GRK2,3 phosphorylates 5-HT_2B receptors in a way that enables them to use a Cav1-dependent internalization pathway (Fig. 7B).

Activation of 5-HT_2B receptors by 5-HT was shown to lead to PKC activation (Cox and Cohen, 1995; Launay et al., 2006). Our data also support the notion that PKC is responsible for the effect of 5-HT_2B receptors on 5-HT-induced 5-HT_1B receptor internalization (Fig. 7C). Using a combination of pharmacological inhibitors and antibody knockdown, we have identified PKC as the isotype necessary for the 5-HT_2B receptor-dependent 5-HT_1B receptor internalization. PKC stimulation could phosphorylate—either directly or indirectly—the 5-HT_1B receptor, rendering it unable to internalize via a Cav1-dependent pathway. One inconsistency between our experimental data and the proposed model is that activation of 5-HT_2B receptors by BW does not affect the Cav1-dependent 5-HT_1B receptor internalization. One possible explanation for this discrepancy is that the partial 5-HT_2B receptor agonist BW poorly activates PKC. Alternatively, or additionally, both 5-HT-induced 5-HT_1B receptor GRK2,3 activation and 5-HT-induced 5-HT_2B receptor PKC activation are required for 5-HT_1B receptors to internalize via the Cav1- and clathrin-independent pathway (Fig. 7C).

This cross-talk, which affects receptor internalization mechanics, is likely to explain the previously observed Gi uncoupling of 5-HT_1B receptors by 5-HT_2B receptors. Activation of the 5-HT_2B/2C receptor has been shown to inhibit the 5-HT_1B receptor function in two independent studies:

1. Using 5-HT_2C and 5-HT_2A receptors, stably transfected Chinese hamster ovary cells, it has been reported that activation of 5-HT_2C receptors abolishes the endogenous 5-HT_1B receptor-mediated inhibition of forskolin-stimulated cAMP accumulation. In contrast, activation of 5-HT_2A receptors does not alter the 5-HT_1B response and 5-HT_2C receptor-mediated inhibition of 5-HT_1B receptor function was blocked when 5-HT_2A receptors were activated simultaneously (Berg et al., 1996).
   
2. Using the teratocarcinoma-derived cell line 1C11 that expresses 5-HT_1B and 5-HT_2B and then 5-HT_2A receptors endogenously and sequentially, Tournois et al. (1998) showed that at day 2 of differentiation, when 5-HT_1B and 5-HT_2B receptor expression is induced, 5-HT_2B receptors exert a dominant-negative regulation of the G_i-coupled 5-HT_1B receptor; at day 4, when functional 5-HT_2A receptors begin to be expressed, 5-HT_2A receptor activation prevents the negative regulation exerted by 5-HT_2B receptor on 5-HT_1B receptor function. Therefore, it is plausible that 5-HT_2B and 5-HT_2C receptors should share a common intracellular internalization and signaling pathway by which to control 5-HT_1B function, whereas 5-HT_2A receptors use alternate pathway(s).
   

This newly described internalization route fits with other evidence supporting independent intracellular trafficking of 5-HT_1B and 5-HT_2B receptors (i.e., internalization of 5-HT_2B receptors upon activation of 5-HT_1B receptors despite the apparent lack of agonist-induced colocalization). Upon coexpression, stimulating one or the other receptor or both generates different cellular responses; this fact is supported by completely independent techniques (i.e., confocal microcopy image analysis and antibody knock-down coupled with whole-cell radioligand binding studies). This work provides the first evidence that one receptor may adopt different internalization pathways within the same cells upon the presence and stimulation of another receptor. Identified interactions regulate receptor internalization and could explain the observed coordination between 5-HT_1B and 5-HT_2B receptor internalization. Our work suggests that indirect events in trans are mediating the 5-HT_1B/5-HT_2B receptor cross-regulation that affects their cellular distribution during the endocytic process. Given the wide clinical use of 5-HT_1B receptor agonists in the treatment of migraines, and the suspected prophylactic effect of 5-HT_2B receptor antagonists, these newly identified functional interactions may be involved in therapeutic effects of these compounds. The phenomenon may also be relevant to the design of novel antimigraine therapies.

	Acknowledgements

 
We thank P. Hickel for excellent technical assistance.

	Footnotes

 
This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Pierre et Marie Curie, the Université Louis Pasteur, and by grants from the Fondation de France, the Fondation pour la Recherche Médicale, the Association pour la Recherche contre le Cancer, the french ministry of research (ACI and ANR) and the European community. L.M.'s team is an "Equipe FRM."

*A.J. and M.D. contributed equally to this work

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.106.032656.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; GPCR, G protein-coupled receptor; GRK, GPCR kinase; PKA, protein kinase A; PDZ, postsynaptic density 95/disc-large/zona occludens (PSD-95, Dlg, ZO-1); CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; GFP, green fluorescent protein; RS, RS127445; RS-127445, 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine; CP, CP93129; CP93129, 1,4-dihydro-3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo{3,2-b}pyridin-5-one dihydrochloride; BW, BW723C86; BW723C86, 1-{5-(2-thienylmethoxy)-1H-3-indolyl}propan-2-amine hydrochloride; H-89, N-{2-((p-bromocinnamyl)amino)ethyl}-5-isoquinolinesulfonamide dihydrochloride; Gö 6850-Bisindolylmaleimide I, 2-{1-(3-dimethylaminopropyl)-1H-indol-3-yl}-3-(1H-indol-3-yl)-maleimide; Gö 6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; [¹²⁵I]DOI, [¹²⁵I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride; [¹²⁵I]GTI, [¹²⁵I]serotonin-5-O-carboxymethyl-glycil-iodo-tyrosamine; PKC, protein kinase C; ROI, regions of interest; FRET, fluorescent resonance energy transfer; PA, photoactivatable; Cav-1, caveolin-1.

Address correspondence to: Luc Maroteaux, INSERM, U616-U839, Hôpital Pitié-Salpetrière, Bat Pédiatrie, 47 Bd de l'Hôpital 75013 Paris, France. E-mail: luc.maroteaux{at}chups.jussieu.fr

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