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J. Biol. Chem., Vol. 277, Issue 23, 20812-20819, June 7, 2002

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5-Hydroxytryptamine 4(a) Receptor Is Coupled to the G Subunit of Heterotrimeric G₁₃ Protein^*

Evgeni G. Ponimaskin, Jasmina Profirovic^§, Rita Vaiskunaite^§, Diethelm W. Richter, and Tatyana A. Voyno-Yasenetskaya^§¶

From the  Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany and ^§ Department of Pharmacology, University of Illinois, Chicago, Illinois 60612

Received for publication, December 20, 2001, and in revised form, March 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter that regulates multiple events in the central nervous system. Many of the 5-HT functions are mediated via G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins, including G_s, G_i, and G_q subfamilies (Martin, G. R., Eglen, R. M., Hamblin, M. W., Hoyer, D., and Yocca, F. (1998) Trends Pharmacol. Sci. 19, 2-4). Here we show for the first time that the 5-hydroxytryptamine 4(a) receptor (5-HT_4(a)) is coupled not only to heterotrimeric G_s but also to G₁₃ protein, as assessed both by biochemical and functional assays. Using reconstitution of 5-HT_4(a) receptor with different G proteins in Spodoptera frugiperda (Sf.9) cells, we have proved that agonist stimulation of receptor-induced guanosine 5'-(3-O-thio)triphosphate binding to G₁₃ protein. We then determined that expression of 5-HT_4(a) receptor in mammalian cells induced constitutive- as well as agonist-promoted activation of a transcription factor, serum response element, through the activation of G₁₃ and RhoA. Finally, we have determined that expression of 5-HT_4(a) receptor in neuroblastoma × glioma NIE-115 cells cause RhoA-dependent neurite retraction and cell rounding under basal conditions and after agonist stimulation. These data suggest that by activating 5-HT_4(a) receptor-G₁₃ pathway, serotonin plays a prominent role in regulating neuronal architecture in addition to its classical role in neurotransmission.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

5-Hydroxytryptamine (5-HT¹ or serotonin) is a neuromodulator involved in a wide range of physiological functions via activation of a large family of receptors. With the exception of the 5-HT₃ receptor, which is a cation channel, all other 5-HT receptors are members of the superfamily of seven transmembrane-spanning G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins. The 5-HT₄ receptor is expressed in a variety of tissues, including brain, gastrointestinal tract, and heart (2). In the mammalian brain, the 5-HT₄ receptor contributes to the control of dopamine secretion and regulates learning and long term memory (3, 4). Furthermore, 5-HT₄ receptors are thought to be involved in various central and peripheral disorders, including neurodegenerative disease (5). Therefore, understanding of the signaling pathways regulated by 5-HT₄ receptor could provide novel insight into physiological and pathophysiological processes in which 5-HT₄ receptor is involved. The wide distribution of 5-HT₄ receptors is paralleled by the existence of many 5-HT₄-splicing variants. In mouse, four 5-HT₄ receptor isoforms, 5-HT_4(a), 5-HT_4(b), 5-HT_4(e), and 5-HT_4(f), have been cloned (6).

G₁₃ protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface by activating multiple signaling pathways. Prominent downstream effectors in G₁₃-mediated signaling are members of the Rho family of small GTPases (Rac, Cdc42, and Rho). RhoA, the founding member of the Rho subfamily, has been shown to play a critical role in regulating the actin cytoskeleton required for neurite retraction (7-9). In neuronal cells, activation of G_12/13-coupled receptors causes RhoA-dependent formation of a cortical shell of F-actin that mediates cytoskeletal contraction, which is thought to underlie growth cone collapse, retraction of developing neurites, and rounding of the cell body (10, 11). The Rho-specific guanine nucleotide exchange factor, p115 RhoGEF, has been also shown to directly interact with G₁₃ protein (12, 13). Most recent studies show that G₁₃ protein also interacts with and regulates the function of the ERM (ezrin/radixin/moesin) family of proteins (14). ERM proteins function as cross-linkers between the actin cytoskeleton and the plasma membrane to regulate the organization of cortical actin (15). Another novel and functionally important partner of G₁₃ is a protein kinase A-anchoring protein, AKAP110, which is expressed in distinct locations including granule neurons of cerebellum (16). The AKAP proteins are characterized by a functional "anchoring domain," which binds to RII (regulatory subunit of protein kinase AII), and a "targeting domain," which directs the subcellular localization of the protein kinase A-AKAP complex through its association with structural proteins, membranes, or cellular organelles (17). Although a principal function of AKAPs is to target PKA, some AKAPs can simultaneously bind more than one signaling molecule with different actions, such as kinases and phosphatases (17). Thus AKAPs serve as scaffold proteins that either prevent cross-talk among related signaling pathways or facilitate signal transduction by preforming multimolecular complexes that can be rapidly activated by incoming signals. It is likely that the diversity of signaling proteins that can interact with G₁₃ is a molecular basis for the complex cell functions regulated by G₁₃.

The molecular interactions that occur between the receptor and G proteins are fundamental to the transduction of signals into specific cellular responses. Using the baculovirus expression system, we have recently provided direct biochemical evidence of the coupling between 5-HT_4(a) receptors and G_s (18). In the present study, we demonstrate the coupling between 5-HT_4(a) receptor and G₁₃ by measuring directly the activation of G proteins introduced into Sf.9 cells. We then verified that the expression of 5-HT_4(a) receptor in mammalian cells induced constitutive activation of an serum response element (SRE) transcription factor, and this response involves activation of G₁₃ and RhoA. In addition, we show that expression of 5-HT_4(a) receptor in the mouse NIE-115 neuronal cell line causes basal as well as agonist-promoted cell rounding, suggesting that serotonin could regulate neuronal morphology by activation of RhoA through 5-HT_4(a) receptor-G₁₃ signaling pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- SRE.L luciferase reporter plasmid was provided by Paul Sternweis. Myc-tagged G₂ and G₁ subunits were provided by Janet Robishaw. N19RhoA was obtained from the Guthrie Research Institute. The RGS domain of p115 RhoGEF was provided by Tohru Kozasa. Wild type G_s, G_i2, G_q, transducin, and RGS4 were provided by Henry Bourne. [³⁵S]GTPS (1300 Ci/mmol) was purchased from PerkinElmer Life Sciences. Enzymes used in molecular cloning were obtained from New England Biolabs (Germany). 5-HT, lysophosphatidic acid, and protein A-Sepharose CL-4B beads were obtained from Sigma. Myo[³H]inositol was from Amersham Biosciences. BIMU8 was kindly provided by Boehringer Ingelheim (Germany). TC-100 insect cell medium, Dulbecco's modified Eagle's medium, fetal calf serum, and LipofectAMINE 2000 reagents were purchased from Invitrogen. Rho antibody was purchased from Santa Cruz Biotechnology. G_i, G_s, G₁₃, and G_q antibodies were from Santa Cruz Biotechnology. The polyclonal antiserum AS9459, raised against the C-terminal peptide of m5-HT_4(a) receptor as well as G₁₂ antibody AS1905 were described previously (19, 20).

Recombinant DNA Procedures-- The construction of recombinant baculovirus coding for the murine 5-HT_4(a) receptor has been described previously (19). For the expression in NIE-115 cells, the murine 5-HT_4(a) cDNA was cleaved from pFastBac plasmid (Invitrogen) with XbaI and HindIII endonucleases to yield the 1.1-kb fragment containing the entire coding sequence. The fragment was treated with T4 DNA polymerase to create the blunt ends and then ligated to the PmeI site in the multiple cloning site of the pTracer-CMV2 donor plasmid (Invitrogen). The 5-HT_4(a) mutants with the substitution of serine for cysteine 328/329, 346, 386, 328/329 + 346, 328/329 + 386, and 346 + 386 were performed in pFastBac/5-HT_4(a) plasmid using an oligonucleotide containing the mutation(s) corresponding to the above substitutions by standard PCR protocols using the overlap extension technique. The recombinant baculoviruses encoding for 5-HT_4(a) mutants were constructed, purified, and amplified as described previously (21). All mutants were verified by double-stranded dideoxy DNA sequencing at the level of the final plasmid.

Assay for [³⁵S]GTPS Binding in Membranes of Sf.9 Cells-- Agonist-promoted binding of [³⁵S]GTPS to different G proteins induced by stimulation of 5-HT_4(a) receptor was performed according to the method described previously (22). Briefly, membranes from Sf.9 cells expressing the 5-HT_4(a) receptor or thrombin PAR1 receptor and G subunits of G_i2, G_s, G_q, G₁₂, and G₁₃ together with G₁₂ subunits were resuspended in 55 µl of 50 mM Tris/HCl, pH 7.4, containing 2 mM EDTA, 100 mM NaCl, 3 mM MgCl₂, and 1 µM GDP. After adding [³⁵S]GTPS (1300 Ci/mmol) to a final concentration of 30 nM, samples were incubated for 5 min at 30 °C in the absence or presence of BIMU8. The reaction was terminated by adding 600 µl of 50 mM Tris/HCl, pH 7.5, containing 20 mM MgCl₂, 150 mM NaCl, 0.5% Nonidet P-40, 200 µg/ml aprotinin, 100 µM GDP, and 100 µM GTP and incubating on ice for 30 min. The samples were incubated for 20 min with 150 µl of a 10% suspension of Pansorbin cells (Calbiochem) preincubated with non-immune serum to remove nonspecific bound proteins. Samples were agitated for 1 h at 4 °C with 5-10 µl of appropriate G subunit-directed antiserum preincubated with 100 µl of 10% suspension of protein A-Sepharose. Immunoprecipitates were washed three times and boiled in 0.5 ml of 0.5% SDS, and radioactivity was measured by scintillation spectrometry.

Reporter Gene Assays-- SRE-mediated gene expression was determined by SRE.L reporter system (Stratagene, La Jolla, CA). Briefly, NIH3T3 cells at 90% confluency grown on 24-well plates were transfected with the following plasmids (per well): 50 ng of pSRE.L (for SRE-dependent gene expression), 50 ng of pCMV--Gal (transfection efficiency control plasmid), and 100 ng of the indicated plasmid, balanced with pCMV vector alone. The day before the experiment, cells were serum-starved in 0.2% calf serum overnight. The cells were washed twice with phosphate-buffered saline and lysed in protein extraction reagent, and the cleared lysates were assayed for luciferase and -galactosidase activity using the corresponding assay kits (Promega, Madison, WI). To account for differences in the transfection efficiency, luciferase activity of each sample was normalized to -galactosidase activity and expressed as percent of the maximal response to G subunit stimulation. The activity of -galactosidase was relatively constant.

Measurement of Rho Activity-- pGEX-2T containing rhotekin-Rho binding domain was provided by Dr. M. A. Schwartz (Scripps Research Institute, La Jolla, CA). Bacterial-expressed glutathione S-transferase-rhotekin-Rho binding domain protein was purified from isopropyl-1-thio-D-galactopyranozide (1 mM)-induced DH5 cells previously transformed with the appropriate plasmid. Confluent NIH3T3 cells (100-mm dishes) were transfected with 5 µg of the 5HT_4(a) receptor or the constitutively active mutant of G₁₃, G₁₃Q226L, for 48 h. Cells were serum-starved 24 h before the experiment. Cells were quickly washed with ice-cold Tris-buffered saline and lysed in lysis buffer (50 mM Tris, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl₂, 10 µg/ml each aprotinin and leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 14,000 × g at 4 °C for 2 min, and equal volumes of cell lysates were incubated with glutathione S-transferase-rhotekin-Rho binding domain beads (15 µg) at 4 °C for 1 h. The beads were washed 3 times with wash buffer (50 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl₂, 10 µg/ml each aprotinin and leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride), and bound Rho was eluted by boiling each sample in Laemmli sample buffer. Samples eluted from the beads and the total cell lysate were then electrophoresed on 12.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, blocked with 5% nonfat milk, and analyzed by Western blotting using a polyclonal anti-Rho-A antibody. The amount of rhotekin-Rho binding domain-bound Rho was normalized to the total amount of Rho in cell lysates for quantitation of Rho activity in different samples using scanning densitometry.

Transfection and Morphological Analysis of NIE-115 Cells-- NIE-115 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C under 5% CO₂. For transient transfection, cells were seeded at low density (5 × 10⁵) into 35-mm dishes and transfected with 1 µg of pTracer or pTracer/5-HT_4(a)-wild type using LipofectAMINE 2000 reagent (Invitrogen). At 14-16 h post-transfection, the cells were washed and incubated for 24-36 h in serum-free Dulbecco's modified Eagle's medium to induce morphological differentiation. In some experiments, 1 µg of Myc-tagged dominant-negative RhoA, N19RhoA, was cotransfected into the cells. Agonist-induced changes in shape were monitored using the LSM510 (Zeiss) confocal microscope with appropriate green fluorescence protein filter setting. Experiments were performed at 37 °C in bicarbonate/CO₂-buffered Dulbecco's modified Eagle's medium. Cells were either scored rounded, flattened, or flattened with neurites the length of at least twice the cell body diameter ("neurite-bearing"). For each transfection, the percentage of rounded, flattened, and neurite-bearing cells was calculated from at least 400 green cells counted. The experiments were performed in duplicate per transfection, and morphologies were scored without prior knowledge of the dish identities. An average percentage was calculated from at least three independent experiments.

Inositol Phosphate Accumulation-- Inositol phosphate accumulation was determined as described earlier (23). Briefly, NIH3T3 cells were seeded onto 24-well plates and transfected with the indicated constructs. Twenty-four hours after transfection, cells were labeled with myo[³H]inositol (2 µCi/ml) for 24 h. They were then washed once with HEPES-buffered Dulbecco's modified medium containing 5 mM LiCl. Total inositol phosphate accumulation was assayed using Dowex columns, and results were expressed as counts/min of [³H]inositol phosphate (10³) divided by the sum of the counts/min in both the [³H]inositol phosphate and [³H]inositol fractions.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

5-HT_4(a) Receptor Specifically Activates G₁₃ Subunit-- Because Sf.9 cells have virtually no background expression of most mammalian receptors, co-expression of receptor and G protein in insect cells followed by measurements of agonist-promoted binding of [³⁵S]GTPS to the G subunit provides a useful experimental approach for assessing the selectivity of receptor-G protein coupling (24). Coupling of 5-HT_4(a) receptor and G proteins was evaluated by a [³⁵S]GTPS binding assay, which determines the GDP-GTP exchange on the G subunit. Membranes from Sf.9 cells co-infected with combinations of baculoviruses encoding mammalian G protein subunits ( with G₁₂ in all cases) and 5-HT_4(a) receptor were incubated in the presence of [³⁵S]GTPS. Thereafter, G-specific affinity-purified antibodies were used to immunoprecipitate G subunits from the detergent extracts. The amount of [³⁵S]GTPS in the immunoprecipitates was used as a measure of G subunit activation.

Using this system we found that 5-HT_4(a) receptor is coupled to the G₁₃ subunit. Fig. 1 shows a set of experiments in which Sf.9 cell membranes containing G_i2, G_s, G₁₃, G₁₂, and G_q were analyzed for [³⁵S]GTPS binding. Western blotting analysis confirmed co-expression of the 5-HT_4(a) receptor with G subunits (data not shown). To determine whether receptor activation could increase GTPS binding by a specific G subunit, cells were incubated with [³⁵S]GTPS in the presence or in the absence of BIMU8, a specific 5-HT_4(a) receptor agonist (2). Agonist stimulation of 5-HT_4(a) receptor did not promote the GTPS binding to G_i2, G₁₂, and G_q subunits; however, it significantly enhanced GTPS binding to G_s and G₁₃ subunits (Fig. 1A). Omission of the receptor from the assay demonstrated that G_s or G₁₃ alone did not bind [³⁵S]GTPS (data not shown). Activation of G_s by a 5-HT_4(a) receptor was consistent with our recently reported data (18). Activation of G₁₃ by a 5-HT_4(a) receptor was a novel observation that required further investigation.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   The 5-HT4(a) receptor communicates with G13 subunit. Membranes were prepared from Sf.9 cells expressing recombinant proteins as indicated and then incubated with [35S]GTPS. [35S]GTPS binding was assessed after incubation with or without 100 nM BIMU8 (A) or in the presence of either vehicle (H2O) or 30 µM thrombin receptor activator (SFLLRNPNDKYEPF) (B). Immunoprecipitations were performed with the appropriate antibodies directed against indicated G subunits. Data points represent the mean ± S.E. from at least three independent experiments. A statistically significant increase between values obtained in A for non-stimulated and agonist-stimulated probes expressing G13 is noted (*, p < 0.05). WT, wild type.

In control experiments we co-expressed thrombin receptor PAR1 beside the different G protein -subunits. Activation of PAR1 with thrombin resulted in a significant increase of [³⁵S]GTPS incorporation in G_q, G₁₂, and G₁₃ (Fig. 1B). These results confirm our previous data (20, 22) and demonstrate that both G_q and G₁₂ can be activated in the presence of suitable receptor.

5-HT_4(a) Receptor Constitutively Activates Serum Response Element-- Because G₁₃ subunit regulates gene expression by transcriptional activation of distinct transcriptional control elements such as SRE (25, 26), we attempted to determine if 5-HT_4(a) receptor could stimulate SRE activity. In SRE-mediated transcription of a luciferase reporter gene, an altered c-fos SRE, SRE.L, was placed in front of luciferase gene (27). SRE.L binds only to the transcription factor SRF and not to tertiary complex factor (TCF). NIH3T3 cells were transiently transfected with 5-HT_4(a) receptor and SRE-driven luciferase reporter. NIH3T3 cells were chosen because the cell line was extensively characterized for G₁₃-induced cell responses and also because this cell line does not contain SV40 large T antigen that can cause overexpression in transfected cells. To correct variations in transfection efficiency, an expression vector coding for -galactosidase was co-transfected with the above constructs, and the expressed  -galactosidase activity was used for normalization of SRE luciferase data. Twenty-four hours after transfection, cells were serum-starved for an additional 16 h. Thereafter, cells were stimulated with the indicated concentrations of serotonin for an additional 6 h. Then, cells were collected, and induced SRE luciferase reporter activity was measured.

Data showed that serotonin induced a dose-dependent SRE activation in the cells transfected with 5-HT_4(a) receptor with an EC₅₀ of ~100 nM (Fig. 2A). SRE activation was dependent on 5-HT_4(a) receptor because serotonin did not induce SRE activation in the cells transfected only with SRE luciferase reporter (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Agonist-induced activation of SRE via 5-HT4(a) receptor. A, dose-dependent activation of SRE by serotonin in the NIH3T3 cells expressing 5-HT4(a) receptor. NIH3T3 cells seeded onto 24-well plates were transfected with 100 ng of 5-HT4(a) receptor, 50 ng of pSRE.L, and 50 ng if pCMV--Gal. Twenty-four hours after transfection, cells were serum-starved for 16 h and stimulated with indicated concentrations of serotonin for 6 h. Thereafter, the activity of SRE was determined as described under "Experimental Procedures". Presented data are the mean ± S.E. from three independent experiments. B, agonist-induced activation of SRE via wild type (WT) and palmitoylation-deficient mutants of 5-HT4(a) receptor. 100 ng of each construct was transfected to SRE measurement. The presented data are the mean ± S.E. from three independent experiments. The activity of -galactosidase was relatively constant. To account for differences in transfection efficiency, luciferase activity of each sample was normalized to -galactosidase activity and expressed as fold of the maximal response induced by agonist. Samples from parallel transfection were used for Western blotting analysis with 5-HT4(a) receptor-specific antibody.

We previously showed that 5-HT_4(a) receptor undergoes dynamic palmitoylation and that receptor stimulation by agonists increases the rate of palmitate turnover (19). Because palmitoylation of 5-HT_4(a) receptor occurs on two different sites (Cys-328/Cys-329 and Cys-386) (18), we determined agonist-induced SRE activation by independent individual mutants. Data showed that stimulation of the wild type and all acylation-deficient mutants with 10 µM serotonin resulted in SRE activation (Fig. 2B). Palmitoylation-deficient mutants were able to stimulate SRE as well as wild type receptor. Western blotting analysis showed that both wild type and mutant receptors were expressed at similar levels (Fig. 2B), thus ruling out a possible difference in the protein expression. Agonist dose dependence of different receptor mutants was virtually superimposable with that of wild type receptor (data not shown), suggesting that potency of palmitoylation-deficient mutants of 5-HT_4(a) receptor was also not affected.

Because we have previously shown that 5-HT_4(a) receptor constitutively activates adenylyl cyclase (18), we studied whether 5-HT_4(a) receptor could activate SRE in an agonist-independent manner. Our data showed that expression of wild type 5-HT_4(a) receptor induced an increase in SRE activity in a range from 2- to 12-fold above base line and correlated with the amount of input receptor cDNA (from 20 to 800 ng) (Fig. 3A). We have also determined previously that 5-HT_4(a) receptor possesses the constitutive activity that resulted in the agonist-independent cAMP accumulation and that C328S/C329S mutant stimulates cAMP accumulation more potently than its wild type counterpart (18). Therefore, we determined whether constitutive activity of the palmitoylation-deficient mutants to induce SRE was influenced. Data showed that all 5-HT_4(a) receptor mutants were able to stimulate SRE under basal conditions (Fig. 3B). Interestingly, the potency of C328S/C329S mutant to stimulate SRE without agonist was slightly reduced, whereas C386S and C328S/C329S/C386S mutants consistently stimulated SRE to a higher degree (Fig. 3B).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   5-HT4(a) receptor activates SRE in a ligand-independent manner. A, dose-dependent activation of SRE by 5-HT4(a) receptor in NIH3T3 cells. The indicated concentrations of 5-HT4(a) receptor cDNA were transfected into NIH3T3 cells, and SRE activity was determined as described earlier. B, constitutive activation of SRE by wild type (WT) and palmitoylation-deficient mutants of 5-HT4(a) receptor. 100 ng of indicated plasmids were transiently transfected into NIH3T3 cells, and SRE activity was determined as described earlier. The activity of -galactosidase did not vary with increasing amounts of the receptor cDNA. Each sample was normalized to -galactosidase activity and expressed as the fold of the maximal response induced by receptor compared to vector. The presented data are the mean ± S.E. from three independent experiments.

5-HT_4(a) Receptor Activates Serum Response Element via G₁₃ Subunit and Rho GTPase-- Because our data showed that the stimulation of the 5-HT_4(a) receptor resulted in the activation of G_s and G₁₃, we examined the identity of the G proteins that mediate the 5-HT_4(a) -dependent activation of SRE. To determine whether 5-HT_4(a) receptors also functionally couple to G_i/o proteins, we treated the transfected cells with pertussis toxin (PTX, 100 ng/ml for 24 h). This treatment did not reduce 5-HT_4(a)-induced SRE activation (Fig. 4A). PTX also did not affect the serotonin-induced SRE activation in the cells transfected with 5-HT_4(a) receptor (Fig. 4A). To test for the ability of PTX to inhibit G_i-mediated pathway, NIH3T3 cells were transfected with endothelin-1 receptor, ETa, which was coupled to G_q/11, G_12/13, and G_i heterotrimeric G proteins (26, 28-30). ETa receptor was also shown to be able to stimulate SRE (26). Cells were stimulated with 100 nM endothelin-1 (ET1) for 6 h before and after pretreatment with PTX. Data showed that PTX pretreatment resulted in ~45% reduction of ET1-induced SRE activation (Fig. 4A). Treatment with PTX, 5-HT, or ET1 of the cells transfected with only the vector did not affect SRE activity (data not shown). Together, these data suggested that pertussis toxin-sensitive G proteins are not involved in the 5-HT_4(a)-induced activation of SRE.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   5-HT4(a)-induced SRE activation could be specifically inhibited by RGS domain of p115 RhoGEF. A, NIH3T3 cells were transfected with 100 ng of 5-HT4(a) receptor, 100 ng of ETa receptor, 50 ng of pSRE.L, and 50 ng of pCMV--Gal. After serum starvation, cells were treated with PTX, 100 ng/ml, for 24 h. Thereafter, cells were stimulated with 10 µM 5-HT or 100 nM ET1 for 6 h, and SRE activity was determined as described. B, NIH3T3 cells were transfected with 50 ng of pSRE.L, 50 ng pCMV--Gal, 100 ng of 5-HT4(a) receptor, and 200 ng of either RGS4 or RGS domain of p115RhoGEF as indicated. Equal amounts of cDNA were maintained in all transfections using empty pcDNA3 vector. C, inositol phosphate (IP) accumulation in NIH3T3 cells transfected with 100 ng of bradykinin receptor or 5-HT4(a) receptor and 200 ng of either RGS4 or RGS domain of p115RhoGEF as indicated. Cells were transfected and labeled with myo[3H]inositol as described under "Experimental Procedures." Thereafter, they were washed once with HEPES-buffered Dulbecco's modified medium containing 5 mM LiCl and stimulated with 1 µM bradykinin (BK) or 10 µM 5-HT for 30 min as indicated. Inositol phosphate accumulation was determined as described. The presented data are the mean ± S.E. from three independent experiments.

We next used regulators of G protein signaling (RGS) as tools to dissect pertussis toxin-insensitive G proteins in the receptor-effector coupling (32). RGS proteins often function as GTPase-activating proteins that accelerate deactivation of trimeric G proteins both in vitro and in vivo (33). RGS4 exhibits GAP activity and inhibits in vivo functions of G_i- and G_q-type of G proteins (34). RGS homology domain of Rho-specific guanine nucleotide exchange factor p115RhoGEF has been shown to exhibit GAP activity and inhibit functions of G₁₂ and G₁₃ proteins (13, 35). Therefore, to validate the functional coupling between 5-HT_4(a) and G₁₃ protein, SRE activation was determined in the cells that were transfected with 5-HT_4(a) receptor and either the RGS4 or RGS domain of p115RhoGEF. The RGS domain of p115 significantly inhibited the 5-HT_4(a)-induced SRE activation, whereas RGS4 did not affect it (Fig. 4B).

To control for the functional activity of RGS4, an in-parallel set of experiments determined that RGS4 inhibited and the RGS domain of p115RhoGEF did not affect the bradykinin-stimulated synthesis of inositol phosphates, confirming the conclusion that RGS4 inhibits G_q-mediated pathway (Fig. 4C). Together, these data suggested that G₁₃ protein is likely to mediate the 5-HT_4(a)-induced activation of SRE.

It was determined that co-expression of a given G protein that couples to the receptor further enhances the receptor-mediated function (36). Thus, to examine the involvement of the specific G proteins in the 5-HT_4(a) signaling, 5HT_(4a) receptor was co-transfected with wild type subunits of G₁₃, G₁₂, G_i2, G_q, and G_s. Alone, G₁₃, G₁₂, and G_q induced moderate activation of SRE, which was consistent with previously reported data (26). G_i2 and G_s did not affect SRE activity. Co-transfection of the 5-HT_4(a) receptor with G₁₃ resulted in a marked potentiation of the SRE activity (Fig. 5A), whereas co-transfection of the 5-HT_4(a) receptor with other G subunits did not further modulate SRE activity (Fig. 5A). Moreover, potentiation of 5-HT_4(a)-induced SRE activation by G₁₃ was dependent on the amount of transfected G₁₃ (Fig. 5B). In control experiments, thrombin-induced SRE activation was strongly potentiated in the cells expressing wild type G_q, G₁₂, or G₁₃ (Fig. 5C). These data (i) confirm the conclusion that thrombin receptor activates multiple G proteins, including G_q, G₁₂, and G₁₃ (see also Fig. 1B) and (ii) support the observation that 5-HT_4(a) receptor preferentially couples to G₁₃ for SRE activation.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   G13 is functionally coupled to 5-HT4(a) receptor to induce SRE activation. A, G13 specifically potentiated 5-HT4(a)-induced SRE activation. NIH3T3 cells were transfected with 50 ng of pSRE.L, 50 ng of pCMV--Gal, 100 ng of 5-HT4(a) receptor, and 200 ng of the indicated G subunit. SRE activity was determined in three independent experiments performed in triplicate. B, dose-dependent potentiation by G13 of the 5-HT4(a)-induced SRE activity. NIH3T3 cells were transfected with 50 ng of pSRE.L, 50 ng of pCMV--Gal, 100 ng 5-HT4(a) receptor, and the indicated concentrations of G13 subunit cDNA. SRE activity was determined in three independent experiments performed in triplicate. C, SRE activation induced by thrombin was potentiated by Gq, G12, and G13 subunits. NIH3T3 cells were transfected with 50 ng of pSRE.L, 50 ng of pCMV--Gal, and 200 ng of the indicated G subunit. Serum-starved cells were stimulated with 1 unit/ml thrombin for 6 h, and SRE activity was determined. The presented data are the mean ± S.E. from three independent experiments.

Because G₁₃ is known to stimulate SRE via Rho GTPase, we next determined whether 5-HT_4(a)-induced SRE stimulation is also dependent on Rho activity. For this purpose, we co-transfected the C3 component of botulinum toxin that ADP-ribosylates and specifically inactivates Rho proteins (37). C3 toxin completely inhibited 5-HT_4(a)-induced SRE activation (Fig. 6A).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   5-HT4(a) receptor stimulates Rho protein. A, NIH3T3 cells were transfected with 50 ng of pSRE.L, 50 ng of pCMV--Gal, 100 ng of 5-HT4(a) receptor, and 200 ng of either C3 or transducin cDNA. B, NIH3T3 cells were transfected with 50 ng of pSRE.L, 50 ng of pCMV--Gal, 100 ng of G13Q226L, 100 ng of each G1 and G2 subunits, and 200 ng of C3 construct. SRE activity was determined in three independent experiments performed in triplicate. C, Rho activation induced by 5-HT4(a) receptor. NIH3T3 cells were transfected with 5 µg of either 5-HT4(a) receptor or G13Q226L and serum-starved for 24 h. Rho activity was determined as the indicated by the amount of rhotekin-Rho binding domain-bound Rho (top) normalized to the amount of Rho in whole cell lysates (bottom). Western blots from a representative experiment showing activation of Rho in response 5-HT4(a) receptor or G13Q226L. The experiment was performed four times with similar results.

Receptor-mediated activation of heterotrimeric G proteins results in a dissociation of G from G subunits, which can directly stimulate several downstream effectors (38). To test the involvement of G subunits, 5-HT_4(a) receptor was co-transfected with a G scavenger, transducin. Expression of transducin resulted in an inhibition of 5HT_4(a)-induced SRE activity (Fig. 6A) but did not affect SRE activation induced by a mutationally active G₁₃, G₁₃Q226L (Fig. 6B). That C3 toxin inhibited SRE activation completely whereas transducin inhibited it only partially could indicate that both G and G subunits were released by a constitutively active 5-HT_4(a) receptor signal to Rho GTPase. To test this hypothesis, G₁₃Q226L and G₁₂ were co-transfected in the presence or absence of C3 toxin. Data showed that both G₁₃Q226L and G₁₂ induced SRE activation, although G activation was less pronounced. In both cases, this activation was completely blocked by a C3 toxin (Fig. 6B).

To evaluate the effect of 5-HT_4(a) receptor on Rho activation, we finally used Rho binding domain of Rho effector, rhotekin, to affinity-precipitate active Rho as a direct readout for Rho activation. NIH3T3 cells were transfected with either 5-HT_4(a) receptor or an active mutant of G₁₃, G₁₃Q226L. Data showed that 5-HT_4(a) receptor induced a 3-4-fold increase in Rho activity, similar to that induced by G₁₃Q226L (Fig. 6C). Stimulation of the receptor with 10 µM serotonin apparently did not induce further increase of Rho activation (data not shown). It is possible that the apparent lack of the agonist-dependent Rho stimulation was due to the nature of the Rho binding assay, which is typically less sensitive than SRE activation assay. These data provided the direct evidence of RhoA activation by 5-HT_4(a) receptor in mammalian cells.

5-HT_4(a) Receptor Induces Neurite Retraction in the N1E-115 Cells-- The Rho GTPase family, which regulates the actin cytoskeleton, has been shown to be involved in processes of neurite outgrowth. Neuroblastoma NIE-115 cells express multiple G protein-coupled receptors, and these cells show G₁₂/G₁₃-mediated activation of RhoA with subsequent growth cone collapse and neurite retraction in response to lysophosphatidic acid and thrombin (11, 39). The NIE-115 cells acquire a flattened morphology and begin to extend neurites after serum removal. Given that 5-HT_4(a) receptor couples to G₁₃ subunits, we used NIE-115 cells as a model to analyze the role of 5-HT_4(a) receptor in the regulation of neuronal morphology.

To analyze the possible role of 5-HT_4(a) receptor in controlling neurite behavior, NIE-115 neuroblastoma cells were transiently transfected with 5-HT_4(a) receptor subcloned into pTracer vector. This vector allows scoring the transfected cells only by the parallel expression of the green fluorescence protein. Correct protein expression of the transfected cDNAs was verified by Western blotting analysis (not shown). Data showed that in non-stimulated NIE-115 cells, ~80% of the cells were flattened, 10% were rounded, and another 10% displayed neurite outgrowth. As shown in the Fig. 7, A and B, expression of the 5-HT_4(a) receptor induced significant changes in the cell shape (11.3 ± 2.7% of rounded cells after transfection with pTracer and 51.8 ± 3.3% after transfection with the 5-HT_4(a) receptor), suggesting that the native 5-HT_4(a) receptor expressed in these cells possesses a high G₁₃-mediated basal constitutive activity. To assess whether RhoA is required for the contractility in neuronal cells induced by 5-HT_4(a)-dependent activation of G₁₃, we co-transfected dominant-negative RhoA (N19) together with 5-HT_4(a) receptor. As shown in Fig. 7B, N19RhoA significantly reduced cell rounding, suggesting that 5-HT_4(a) operated via RhoA to induce cytoskeletal contraction. Apparently incomplete inhibition of the cell rounding by N19RhoA could be partially attributed to the toxicity displayed by N19RhoA at higher concentrations and/or incomplete inhibition of the endogenous Rho guanine nucleotide exchange factors by this construct.

View larger version (68K): [in this window] [in a new window]   Fig. 7.   Regulation of neuronal cell shape by 5-HT4(a) receptor in NIE-115 cells. NIE-115 cells were transiently transfected with either a control (Tracer) vector or with Tracer vector encoding for 5-HT4(a) receptor. Cells were cultured in serum-free medium overnight, and morphologies were assessed. A, phase contrast fluorescence micrographs of serum-deprived NIE-115 cells transfected with Tracer vector or 5-HT4(a) receptor. B, effect of 5-HT4(a) receptor on the cell rounding (left panel). Dominant-negative Rho, N19RhoA, inhibited cell rounding induced by 5-HT4(a) receptor (right panel). The data points represent the mean ± S.E. from at least three independent experiments performed in duplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study we have demonstrated for the first time that the serotonin 5-HT_4(a) receptor is coupled both biochemically and functionally to the G₁₃ subunit of heterotrimeric G protein. It is widely accepted that native as well as heterologously expressed 5-HT₄ receptors couple positively to adenylyl cyclase, catalyzing cAMP production (40, 41). We have recently shown by reconstitution of 5-HT_4(a) receptor and different G proteins in Sf.9 cells that the recombinant 5-HT_4(a) receptor directly communicates with G_s but not with G_i, G_q, or G₁₂ subunits. Additional assays including agonist-promoted cAMP production and activation of cAMP-gated ion channels also confirmed that the 5-HT_4(a) receptor operates through G_s (18). Using a baculovirus expression system, we have now demonstrated that 5-HT_4(a) receptor also stimulates the G₁₃ subunit (Fig. 1). We further determined that 5-HT_4(a) receptor-mediated stimulation of G₁₃ resulted in SRE-mediated activation of gene transcription (Fig. 2) and induction of neurite retraction and cell rounding in neuronal cells (Fig. 7).

The SRE activation is crucial for the transcription of many immediate early genes (42). The regulation of the activity of the SRE is mediated by two different signaling pathways, TCF-dependent and TCF-independent pathways. It was convincingly determined that TCF-independent regulation is modulated by members of the Rho family of small GTPases, RhoA, Rac1, and Cdc42Hs (27). Indeed, stimulation of the transcriptional activity of SRE induced by serum, lysophosphatidic acid, and AlF (an activator of heterotrimeric G proteins) is signaled by RhoA in NIH3T3 cells, since the expression of the C3 component of the Clostridium botulinum toxin efficiently blocks this effect. It was also determined that the G₁₂ subfamily of heterotrimeric G protein subunits is able to induce the SRE activity by a RhoA-dependent pathway (25). Therefore, to study 5-HT_4(a)-induced SRE activation, we have used an altered c-fos SRE, SRE.L, which binds only to the transcription factor SRF (serum response factor) but not to TCF. The involvement of G₁₃ and RhoA in 5-HT_4(a)-induced SRE activation was evidenced by the following observations. 1) Expression of wild type G₁₃ enhanced 5-HT_4(a)-induced SRE activation (Fig. 5); 2) G₁₃ was inhibited by the RGS domain of p115RhoGEF, which is specific for G₁₂ and G₁₃ (Fig. 4); 3) C3 exoenzyme, a specific inhibitor of Rho proteins, inhibited SRE activation by 5-HT_4(a) (Fig. 6); 4) the above-mentioned inhibitors and dominant-negative constructs also effectively inhibited SRE activation induced by G₁₃; 5) finally, direct measurement of Rho activity using a rhotekin binding assay showed that the 5-HT_4(a) receptor is able to activate Rho (Fig. 6C).

A precise pattern of neuronal connections is essential for the function of the adult nervous system. During embryonic development, neuronal growth cones navigate along specific pathways delineated by multiple molecular guidance cues to reach their appropriate distant targets. Neurite outgrowth and growth cone motility are among the many aspects of neuronal development that can be affected by specific neurotransmitters. Serotonin (5-HT) is one of neurotransmitters that may affect the neurite outgrowth besides its well established role in neuronal communication. However, the effect of 5-HT varied substantially among the cell types and systems that were analyzed. For instance, the addition of 5-HT to the growing neurites from the snail (Helisoma) neurons caused an abrupt cessation of their elongation (43, 44). Depletion of 5-HT in the snail Achatina fulica resulted in axonal sprouting of buccal ganglion neurons (45). Similarly, decreasing 5-HT levels increased distal axon outgrowth and branching in the embryonic ENC1 neurons from Helisoma, whereas increasing the 5-HT level reduced the number of neurite branch points (46). Other experiments demonstrate that application of 5-HT induces growth cone collapse in chick dorsal root ganglion as well as in cerebral giant cells of Lumnaea stagnalis (47, 48). In addition, 5-HT inhibited neurite outgrowth from retinal neurons of goldfish (49). Although all of the above data are consistent with the assumption that 5-HT acts to decrease or arrest neuritic outgrowth, several other experiments provide opposing results. For example, in the sphinx moth exposure to 5-HT enhances neurite growth from antennal lobe neurons (50). Likewise, in the rat brain, application of 5-HT increased the dendritic differentiation of calretenin-positive neurons in the cerebral cortex (51) and promoted the neurite outgrowth from thalamic neurons (52).

Whereas the growth-promoting effect of 5-HT seems to be mediated by the activation of 5-HT_1B, 5-HT_1D, and 5-HT₂ receptors (31, 53, 54), the molecular mechanisms underlying the inhibitory effects of 5-HT on neurite outgrowth are poorly understood. It has been reported that activation of the small GTPase RhoA through a G_12/13-initiated pathway induce growth cone collapse and neurite retraction in neuronal cells (11). Similarly, expression of constitutive active G₁₂ and G₁₃ induced RhoA-dependent neurite retraction in PC12 cells (9). Our present data showing that 5-HT_4(a) receptor directly activates G₁₃ could, therefore, provide the molecular basis for serotonin-induced RhoA-dependent inhibition of neurite outgrowth. In this way, G₁₃-mediated signaling by 5-HT may play an important role in the development and plasticity functions of the nervous system.

It is also of considerable interest that expression of the 5-HT_4(a) receptor in NIH3T3 and in NIE-115 neuronal cells results in activation of SRE (Fig. 3) and in a significant elevation of neurite retraction, even without agonist stimulation (Fig. 7B), suggesting a high level of agonist-independent constitutive 5-HT_4(a) receptor activity mediated by G₁₃. It is well established that G protein-coupled receptors can reach their active state even in the absence of agonist as a result of a natural shift in the equilibrium between their inactive (R) and active conformations (R*) (31). Meanwhile, the capacity of a native receptor to spontaneously isomerize to an active form represents a very important pharmacological characteristic because this may explain part of its physiological and possible pathological behavior. Although the agonist-independent signaling has been observed for a wide variety of G protein-coupled receptors, molecular constraints involved in the regulation of receptor constitutive activity remain poorly understood. The future challenge will be the understanding of the 5HT_4(a)-G₁₃ pathway in the physiological and pathophysiological systems.

	FOOTNOTES

^* These studies were supported by National Institutes of Health Grant GM56159 (to T. V.-Y.), by funding from the Medical School at the University of Göttingen, and by Deutsche Forschungsgemeinschaft Grant PO 732/1-1 (to E. G. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology (MC 868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-996-9823; Fax: 312-996-1225; E-mail: tvy@uic.edu.

Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M112216200

	ABBREVIATIONS

The abbreviations used are: 5-HT, 5-hydroxytryptamine or serotonin; 5-HT_4(a), mouse 5-hydroxytryptamine 4(a) receptor; BIMU8, (endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide; SRE, serum response element; p115 RhoGEF, Rho-specific guanine nucleotide exchange factor; PTX, pertussis toxin; RGS, regulators of G protein signaling; Sf.9, S. frugiperda cells; GTPS, guanosine 5'-(3-O-thio)triphosphate; AKAP, protein kinase A-anchoring protein; TCF, tertiary complex factor; Luc, luciferase; -Gal, -galactosidase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES