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J. Biol. Chem., Vol. 276, Issue 48, 44663-44668, November 30, 2001

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RNA Editing of the Human Serotonin 5-HT_2C Receptor Alters Receptor-mediated Activation of G₁₃ Protein^*

Raymond D. Price, David M. Weiner^§, Mike S. S. Chang, and Elaine Sanders-Bush^¶

From the  Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6600 and ^§ ACADIA Pharmaceuticals and the Departments of Neurosciences and Psychiatry, University of California, San Diego, La Jolla, California 92121

Received for publication, July 18, 2001, and in revised form, September 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The recent completion of the human genome predicted the presence of only 30,000 genes, stressing the importance of mechanisms that increase molecular diversity at the post-transcriptional level. One such post-transcriptional event is RNA editing, which generates multiple protein isoforms from a single gene, often with profound functional consequences. The human serotonin 5-HT_2C receptor undergoes RNA editing that creates multiple receptor isoforms. One consequence of RNA editing of cell surface receptors may be to alter the pattern of activation of heterotrimeric G-proteins and thereby shift preferred intracellular signaling pathways. We examined the ability of the nonedited 5-HT_2C receptor isoform (INI) and two extensively edited isoforms, VSV and VGV, to interact with various G-protein  subunits. Two functional assays were utilized: the cell-based functional assay, Receptor Selection/Amplification Technology^TM, in which the pharmacological consequences of co-expression of 5HT_2C receptor isoforms with G-protein  subunits in fibroblasts were studied, and 5HT_2C receptor-mediated rearrangements of the actin cytoskeleton in stable cell lines. These studies revealed that the nonedited 5-HT_2C receptor functionally couples to G_q and G₁₃. In contrast, coupling to G₁₃ was not detected for the extensively edited 5-HT_2C receptors. Thus, RNA editing represents a novel mechanism for regulating the pattern of activation of heterotrimeric G-proteins, molecular switches that control an enormous variety of biological processes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The monoamine 5-hydroxytryptamine (serotonin; 5-HT)¹ interacts with a large family of receptors to induce signal transduction events important in the modulation of neurotransmission (1). The 2C subtype of serotonin receptor (5-HT_2CR) is a member of the G-protein-coupled receptor superfamily and interacts with G_q to stimulate phospholipase C, resulting in the production of inositol phosphates and diacylglycerol (2). RNA transcripts encoding the human 5-HT_2CR undergo adenosine-to-inosine RNA editing events at five positions, termed A, B, C, D, and E (Fig. 1A), altering the amino acid coding potential within the putative second intracellular loop of the protein (3, 4). We and others have demonstrated a decrease in agonist potency at the human edited VSV and VGV isoforms (named for the amino acids at positions 156, 158, and 160) compared with the nonedited isoform, which codes for INI at these positions. This decrease in agonist potency is reflected as a rightward shift in the dose-response curve for inositol phosphate accumulation (5, 6) and calcium release (7). The decreased agonist potency was proposed to result from a reduced G_q-protein coupling efficiency induced by the introduction of these novel amino acids into the second intracellular loop, a region known to be important for G-protein coupling (8-16). Another consequence of RNA editing may be to alter the specificity of activation of heterotrimeric G-proteins and thereby shift intracellular signaling pathways. To test this hypothesis, we examined the ability of three 5-HT_2CR isoforms to functionally couple with the  subunits of various heterotrimeric G-proteins of the G_q family (G_q, G₁₁, G₁₄, G₁₅, and G₁₆) and the G₁₂ family (G₁₂ and G₁₃) using the cell-based functional assay, Receptor Selection/Amplification Technology (R-SAT).

Previous R-SAT studies of the co-expression of muscarinic receptors with G_q/11 (17), G₁₂ (16), G₁₃ (18), and G₁₄ and G₁₅² demonstrate that efficient receptor/G-protein coupling is paralleled by leftward shifts in agonist dose-response curves. Therefore, we have examined the effects of raising G-protein concentrations on the pharmacology of serotonergic ligands. Since VSV is the predominant 5-HT_2CR isoform expressed in human brain and VGV has the most prominent phenotypic differences (5, 7), the present R-SAT study focuses on these two edited receptors. In addition, the ability of both the nonedited isoform (INI) and the fully edited isoform (VGV) to dynamically regulate rearrangements of the actin cytoskeleton downstream of G₁₃ activation was examined.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

R-SAT-- NIH-3T3 cells were plated into 96-well plates 1 day before transfection at a density of 7500 cells/well. Cells were transfected with 0.5-25 ng of nonedited (INI) or edited (VSV, VGV) human 5-HT_2CR; G subunits (G_q, G₁₁, G₁₂, G₁₃, G₁₄, G₁₅, G₁₆); and 25 ng of -galactosidase-pSI (Promega)/well using Superfect (Qiagen) as a DNA carrier. One day after transfection, cells were cultured in ligand mixed in Dulbecco's modified Eagle's medium supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 2% Cyto-SF3 (Kemp Laboratories) to a final volume of 200 µl. After 5 days of incubation, the medium was removed from the wells, and 200 µl of phosphate-buffered saline (136.9 mM NaCl, 2.68 mM KCl, 8.09 mM Na₂HPO₄, and 1.47 mM KH₂PO₄, pH 7.4) supplemented with 3.5 mM o-nitrophenyl--D-galactopyranoside (ONPG) and 0.5% Nonidet P-40 (Sigma) was added to each well. Plates were then incubated at room temperature for up to 5 h, and optical density was recorded at 405 nm using a spectrophotometric plate reader (Biotek Instruments, Inc.). Curves were generated by least-squares fits with Prism (GraphPad Software) using the equation,

(Eq. 1)

where X represents the logarithm of concentration, y is the response, and "top" and "bottom" describe values for the asymptotic top and bottom of the fitted dose-response curve. Statistical analysis of EC₅₀ values was conducted using one-way analysis of variance with Bonferonni post-tests.

In the absence of receptor activity, NIH-3T3 cells grow to a monolayer, but in the presence of receptor activity, cells overcome contact inhibition and proliferate. A quantitative measure of cellular proliferation is obtained by measuring the levels of -galactosidase, a marker enzyme constitutively expressed by the transfected cells. R-SAT assays resolve partial agonists from full agonists as well as inverse agonists, ligands that reduce constitutive activity. Results obtained using this assay compare well with results obtained using inositol phosphates, GTPase, and whole tissue assays for a variety of other receptors (19, 20).

5-HT_2C Receptor and G-protein  Subunit Cloning-- 5-HT_2CRs and G-protein  subunits were cloned using PCR-based methodologies. To generate template for the 5-HT_2CR, RNA from human cerebellum was isolated utilizing the FastTrack mRNA kit from InVitrogen Inc. (Carlsbad, CA). Poly(A)⁺ RNA was reverse transcribed utilizing an 18-mer oligo(dT) primer and Superscript II from Life Technologies, Inc., per the manufacturer's protocol. Oligodeoxynucleotide primers used were as follows (all primers are reported in 5'-3' orientation): 5'-CTG ACG GGA TCC TTC AAA AAC AAC TAA AGG ATG and 3'-CAC TTT TCT AGA CAG CAA TAT TTA CAT TAG TTA. PCR conditions employed 100 ng (~125 pmol) of each primer, 250 µM dNTPs, 5% Me₂SO, 25 ng of cDNA, 1× Pfu cloned buffer, and 2.5 units of Pfu Turbo from Stratagene (San Diego, CA). The cycling conditions were as follows: 94 °C for 5 min and then 40 cycles of 94 °C for 30 s, 50 °C for 10 s, and 72 °C for 1 min/kilobase. The resultant PCR product was subcloned into the TOPO 2.1 vector from Invitrogen Inc. (Carlsbad, CA) as per the manufacturer's protocols. The sequence of the nonedited INI clone corresponds to GenBank^TM accession number U49516. These initial efforts also yielded 5-HT_2C clones encoding the VNV and VGV isoforms. All receptors were subcloned into the mammalian expression vector PSi from Promega Inc. (Madison, WI) for R-SAT studies. Using the PSi-based 5-HT_2C VNV construct as a template, the VSV isoform was generated by the QuikChange site-directed mutagenesis kit from Stratagene Inc. (San Diego, CA) according to the manufacturer's protocol. Primers used were 5'-GCA GTG CGT AGT CCT GTT GAG CAT AGC and 3'-CTC AAC AGG ACT ACG CAC TGC TAC ATA. PCR conditions, with 30 ng of template plasmid DNA, were as follows: 4 min at 94 °C, followed by 20 cycles of 30 s at 94 °C, 30 s at 55 °C, and 18 min at 72 °C. All receptor constructs were fully sequence-verified. Cloning of the -subunits of G_q and G₁₁ (17), G₁₂ (16), and G₁₃ (18) was described previously. G₁₄ was cloned by PCR from cDNA generated from NIH-3T3 cell total RNA using oligodeoxynucleotide primers: 5'-TTC GAG AAG CGT TAG CCT AGA GAT CCG AGC and 3'-AAG CAC TTG TAG ATC AGG CAG GAA GGG CTC with a 48 °C annealing temperature. Sequencing of multiple clones revealed that all differed from the published sequence (GenBank^TM accession number NM_008137) by a cg nucleotide transversion at positions 72 and 73 (adenine in ATG as position 1). All other G-protein  subunits have the transversion at these positions, arguing that the published G₁₄ sequence may be incorrect. The construct encoding G₁₅ was generously provided by Dr. Thierry Wurch (21), and the construct encoding G₁₆ was generously provided by Dr. Thomas Amatruda III (22).

Stress Fiber Assays-- NIH-3T3 cells stably expressing 5-HT_2CR isoforms were serum-starved overnight and incubated with various ligands or peptides for 30 min at 37 °C, fixed in 4% paraformaldehyde for 10 min, and permeabilized in 0.1% Triton X-100 for 5 min. Nonspecific background was reduced by incubating cells in 1% bovine serum albumin for 30 min. To visualize the cytoskeleton, cells were stained for polymerized actin by incubation with 1.65 µM Oregon Green-phalloidin (Molecular Probes, Inc., Eugene, OR) for 20 min. The glass slides were examined using an inverted microscope (Zeiss Axiovert 100). The ratio of stress fiber-positive cells (defined as the distinct presence of stress fibers overlying a nucleus) relative to the total number of counted cells in randomly chosen visual fields is reported as a percentage value. A minimum of 50 cells were included in each experiment, and experiments were repeated at least four times. Quantification was performed in a blind manner (i.e. the observer was not informed of the conditions or cells used in the experiment). Concentrations of compounds for INI treatments were 1 µM DOI, SB 206553, and LSD; 10 µM clozapine and lysophosphatidic acid (LPA); and 100 µM membrane-permeable sequence (MPS) Gq and G13 peptides. Treatments for VGV cells were identical, except 10 µM DOI and LSD were used.

Phosphoinositide Hydrolysis Assay-- NIH-3T3 cells stably expressing the INI receptor isoform were plated in 24-well plates and incubated for 16-20 h with 2 µCi/ml myo-[³H]inositol (20-25 Ci/mmol; PerkinElmer Life Sciences) in serum-free, inositol-free Dulbecco's modified Eagle's medium to label phospholipid pools. Labeling medium was aspirated, and the cells were washed twice with Hanks' balanced salt solution containing 1 mM Ca²⁺ and 1 mM Mg²⁺. Cells were treated with peptides solubilized in Hanks' balanced salt solution (+Ca²⁺/Mg²⁺) at 37 °C for 30 min. Subsequently, 10 mM lithium chloride was added to the cells for 10 min prior to agonist activation for 30 min at 37 °C. The reaction was stopped by aspirating the solution and fixing with 50 µl of methanol/well. [³H]Inositol monophosphates were isolated as previously described (23).

Peptide Design and Synthesis-- MPS peptide was based on a hydrophobic membrane-permeable sequence described previously (2). The G_q (Gq) and G₁₃ (G13) carboxyl-terminal peptides were designed based on the last 10 amino acids of the carboxyl terminus (Gq, amino acids 350-359; G13, amino acids 367-377), a region of other G subunits that has been identified as a site of interaction between G-proteins and receptors (24, 25). The MPS sequence is AAVALLPAVLLALLAK-S; the Gq-mimicking peptide sequence is CQLNLKEYNLV; the G13-mimicking peptide sequence is CLHDNLKQLME.

Cell Culture-- For the stress fiber assays, NIH-3T3 cells stably expressing human INI, VSV, and VGV 5-HT_2CRs were generated as described previously (5); receptor densities were 2047, 1292, and 5375 fmol/mg of protein, respectively. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units of penicillin/ml, and 100 µg of streptomycin/ml under 5% CO₂ at 37 °C.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Validation of R-SAT Assay-- Utilizing the cell-based assay, R-SAT, functional interactions between 5-HT_2CR isoforms (Fig. 1A) and the  subunits of various heterotrimeric G-proteins (G) were probed. Earlier studies of these edited isoforms have documented decreases in agonist potency to induce accumulation of inositol phosphates (3, 5, 6) and to release intracellular stores of calcium (7). As a validation of the assay, we initially determined if there were similar shifts in agonist potency in R-SAT. As illustrated in Fig. 1B, serotonin dose-response curves were shifted rightward for the VSV and VGV edited isoforms compared with the nonedited INI isoform (EC₅₀ values of 3, 14, and 104 nM for INI, VSV, and VGV isoform, respectively), similar to the results of earlier functional studies. In addition, the nonedited INI isoform exhibited dramatic constitutive activity (defined as the ability to promote productive G-protein coupling in the absence of an agonist) (26), such that there was very little stimulatory effect of 5-HT. The VSV isoform demonstrated a moderate level of constitutive activity. Ritanserin was a potent and highly efficacious inverse agonist, essentially eliminating the constitutive activity of both the INI and VSV isoforms (Fig. 1C). The fully edited isoform, VGV, had no measurable constitutive activity. Thus, the results obtained with the R-SAT assay reproduce and extend previous results obtained with more classical assays of 5-HT_2CR function.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Characterization of INI, VSV, and VGV isoforms of the 5-HT2CR in R-SAT. A, the positions of the editing sites within human 5-HT2CR RNA (top) and the predicted amino acid sequence (bottom) are shown for the nonedited INI isoform and for the edited VSV and VGV isoforms. B, serotonin responses observed with the R-SAT assay of cells transiently expressing the INI, VSV, or VGV receptor isoform (mean ± S.D.; n = 5). C, receptor isoforms exhibit dramatically different constitutive activities. Representative dose-response curves are shown for serotonin (filled symbols) and ritanserin (open symbols) for INI (left), VSV (middle), or VGV (right), with responses at the highest concentrations of serotonin and ritanserin set at 100 and 0%, respectively.

Co-expression of 5-HT_2CR and G-protein  Subunits-- To investigate potential differences in receptor/G-protein coupling, various G subunits (G_q, G₁₁, G₁₂, G₁₃, G₁₄, G₁₅, G₁₆) were co-transfected with a single receptor isoform, and the ability of each G subunit to shift agonist and inverse agonist dose-response curves was compared with receptor alone (i.e. signaling through endogenous G-proteins). Potency is defined as the amount of a drug needed to produce a half-maximal effect (EC₅₀). In the context of the extended ternary complex model of receptor activation (27), potency depends on a number of discrete partial reactions: 1) the affinity of the compound for the receptor; 2) the productive interaction of receptor and G-protein; and 3) activated G-protein interacting with downstream effector molecules. In this system, steps 1 and 3 are similar for each condition, so any shift in potency upon the addition of G subunits is a reflection of receptor/G-protein coupling. Previous R-SAT studies of the co-expression of muscarinic receptors with G_q/11 (17), G₁₂ (16), G₁₃ (18), and G₁₄ and G₁₅² provided evidence that 1) these G-proteins are expressed under these experimental conditions and 2) efficient receptor/G-protein coupling can be detected by leftward shifts in agonist dose-response curves.

Co-expression of the VGV receptor isoform with G_q or G₁₁ caused a significant shift in serotonin potency compared with receptor alone, whereas other G subunits were without effect (Fig. 2; Table I). Furthermore, no alterations in serotonin potency were observed with transfection of a 5-fold excess of G₁₂, G₁₃, G₁₄, G₁₅, and G₁₆ (data not shown), suggesting that the VGV receptor/G-protein interaction is specific to G_q and G₁₁. Serotonin had no effect in cells transfected with G subunits alone or in untransfected cells (data not shown), confirming that the responses result from interaction of heterologously expressed receptors and G-proteins. Similar experiments with the VSV isoform showed that co-transfection of G_q, but not other G subunits, caused a significant increase in 5-HT potency (Fig. 3; Table I). Analysis of dose-response curves for another 5-HT_2CR agonist DOI also revealed that only G_q increased potency when VSV was co-transfected with G subunits (data not shown). The potential physiological significance of the apparent difference in isoform coupling to G_q and G₁₁ is unclear, since these G-proteins typically mediate similar functional responses (28).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Expression of Gq and G11, but not other G subunits, shift 5-HT potency at VGV. Shown are representative agonist concentration-response curves generated by R-SAT assays of the VGV receptor isoform co-expressed with various G-protein  subunits. Plotted are the absorbance values of the -galactosidase substrate ONPG at 405 nm versus ligand concentration, normalized to the maximum response for each condition. Points are the mean of data from at least six independent experiments performed in duplicate. The asterisks indicate conditions significantly different from receptor alone (see Table I for summary).

View this table: [in this window] [in a new window]   Table I Effect of G subunits on serotonin and ritanserin potency at different 5-HT2CR isoforms Data represent potency values obtained in R-SAT assays utilizing receptor alone or in the presence of the indicated G subunit, cultured in the presence of either 5-HT or ritanserin. Data are mean EC50 values in nM ± S.E.; parentheses indicate n. * p < 0.05; ** p < 0.001; significantly different from receptor alone.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Expression of Gq shifts 5-HT potency at VSV, but other G subunits do not. Shown are representative agonist concentration-response curves generated by R-SAT assays of the VSV receptor isoform co-expressed with various G-protein  subunits. Plotted are the absorbance values of the -galactosidase substrate ONPG at 405 nm versus ligand concentration, normalized to the maximum response for each condition. Points are the mean of data from at least four independent experiments performed in duplicate. The asterisks indicate conditions significantly different from receptor alone (see Table I for summary).

The extended ternary complex model of receptor/G-protein coupling predicts that increasing the concentration of a constitutively activated receptor or its cognate G-protein should not only increase agonist potency but also decrease inverse agonist potency (27). We therefore hypothesized that co-transfection of G subunits that functionally couple to a particular receptor isoform would cause rightward shifts in inverse agonist dose-response curves, providing a method to determine receptor/G-protein coupling of the isoforms with significant constitutive activity (INI and VSV). To test this hypothesis, these isoforms were co-transfected with the various G subunits, and dose-responses curves were generated using the fully efficacious inverse agonist ritanserin. A significant rightward shift in the ritanserin dose-response curve was observed when G_q was co-transfected with VSV but not when other G subunits were co-transfected with VSV (Fig. 4; Table I). These results agree with previous data utilizing agonists and suggest that the VSV isoform couples principally to G_q.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Expression of Gq, but not other G, shifts ritanserin potency at VSV. Shown are representative inverse agonist concentration-response curves generated by R-SAT assay of VSV receptor isoform co-expressed with various G-protein  subunits. Plotted are the absorbance values of the -galactosidase substrate ONPG at 405 nm versus ligand concentration, normalized to the maximum response for each condition. Points are the mean of data from at least three independent experiments performed in duplicate. The asterisks indicate conditions significantly different from receptor alone (see Table I for summary).

Because of the high degree of constitutive activity of the nonedited INI receptor, we were unable to obtain reproducible dose-response curves for serotonin above agonist-independent activity when G subunits were co-transfected with the receptor. As predicted, co-transfection of G_q with INI caused a rightward shift in the ritanserin dose-response curve compared with receptor alone (Fig. 5; Table I). However, co-transfection of G₁₃ or G₁₅ with INI caused a leftward shift in dose-response curves for ritanserin (Fig. 5; Table I) as well as dose-response curves for another fully efficacious 5-HT_2CR inverse agonist, clozapine (data not shown). These data suggest that the INI receptor can productively interact with G_q, G₁₃, and G₁₅.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Expression of Gq, G13, and G15, but not other G, shift ritanserin potency at INI. Shown are representative inverse agonist concentration-response curves generated by R-SAT assay of INI receptor isoform co-expressed with various G-protein  subunits. Plotted are the absorbance values of the -galactosidase substrate ONPG at 405 nm versus ligand concentration, normalized to the maximum response for each condition. Points are the mean of data from at least seven independent experiments performed in duplicate. The asterisks indicate conditions significantly different from receptor alone (see Table I for summary).

Stress Fiber Formation-- To confirm the apparent alteration in G₁₃ coupling, we examined receptor-mediated rearrangements of the actin cytoskeleton, a G₁₃-mediated process important in cell shape and regulatory responses including chemotaxis, mitogenesis, and neurite retraction (29, 30). NIH-3T3 cells stably expressing the INI receptor isoform showed a high level of stress fibers in the absence of agonist (Fig. 6, A and C), which was decreased by treatment with the inverse agonists SB 206553 and clozapine (Fig. 6, B and C). Consistent with the R-SAT results, only a small DOI signal was detected, presumably due to the high degree of receptor constitutive activity. Importantly, the addition of a membrane-permeable peptide (G13) designed to mimic the receptor/G₁₃ interface and hence block coupling (2), markedly decreased actin stress fibers in INI-expressing cells (Fig. 6C), whereas the carrier peptide (MPS) alone had no effect. Although a similar peptide designed to block receptor/G_q coupling could attenuate 5-HT_2CR-mediated phosphoinositide hydrolysis, the G13 blocking peptide was not effective at blocking this signal (Fig. 6D), thus demonstrating the specificity of the G13 peptide. In contrast, cells stably transfected with the VGV isoform did not show actin rearrangement in response to the agonists DOI or LSD (Fig. 7, A and C), whereas LPA, an agonist at an endogenous G₁₃-coupled receptor (31), produced a 3-fold increase in the number of cells expressing stress fibers (Fig. 6, B and C). The addition of the G13 blocking peptide attenuated the LPA response, whereas the Gq peptide had no effect (Fig. 7D). These results confirm that G₁₃ protein-mediated rearrangements of the actin cytoskeleton occur as a downstream consequence of INI, but not VGV, receptor activation.

View larger version (96K): [in this window] [in a new window]   Fig. 6.   Stimulation of INI receptors initiates rearrangement of the actin cytoskeleton downstream of G13. A and B, stable cell lines expressing the INI isoform in the absence (basal) or presence of ligands were incubated with Oregon Green-conjugated phalloidin. A, INI cells in the absence of ligand illustrating presence of stress fibers (arrowhead); B, INI cells after treatment with inverse agonist, SB 206553, showing lack of organized stress fibers. C, quantification of stress fibers after various drug or peptide treatments (see "Experimental Procedures"). *, p < 0.05; **, p < 0.001, significantly different from basal; #, p < 0.01, significantly different from MPS; results are the mean of at least four independent experiments. D, stable INI cell lines were stimulated with 10 nM DOI and assayed for phosphoinositide hydrolysis in the absence or presence of blocking peptides. **, p < 0.001, significantly different from basal; , p < 0.001, significantly different from DOI; results are the mean of at least five independent experiments performed in triplicate.

View larger version (107K): [in this window] [in a new window]   Fig. 7.   Stimulation of VGV receptors does not cause rearrangement of the actin cytoskeleton via G13. A and B, stable cell lines expressing the VGV isoform in the absence (basal) or presence of ligands were incubated with Oregon Green-conjugated phalloidin. VGV cells showed no significant change in the expression of stress fibers in response to 5-HT2CR ligands (A) but did show an increase after LPA treatment (B, arrowhead). Quantification of stress fibers after various drug (C) or peptide treatments (D; see "Experimental Procedures"). **, p < 0.001, significantly different from basal; , p < 0.001, significantly different from LPA; results are the mean of at least four independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The original ternary complex model states that the ligand, the receptor, and the G-protein must interact to form a ternary complex in order to elicit second messenger production. However, recent revisions have occurred to accommodate the finding that some receptors have the ability to promote productive G-protein coupling in the absence of an agonist, termed constitutive activity (27, 32). The extended ternary complex model predicts that receptors have the capacity to spontaneously isomerize from an inactive conformation, termed R, to an active state, R*, with the R* version of the receptor having the ability to interact with and activate G-proteins. A relatively large proportion of the nonedited 5-HT_2CR exists in a G-protein-coupled state in the absence of agonist (23, 33). These results were confirmed in the current work using the R-SAT assay. Furthermore, this high level of constitutive activity is progressively eliminated by RNA editing, with the INI receptor being most efficacious at G-protein coupling, the VSV isoform being intermediate, and VGV receptors existing predominantly in the uncoupled state (present results; see Refs. 5, 34, and 35). The isoform-dependent constitutive activity could have important implications for the physiological effects of 5-HT by controlling basal tone and sensitivity at synaptic sites. In addition, region-specific generation of edited isoforms (3), coupled with reports of altered RNA editing in suicide (36) and schizophrenia patients (37), suggests that the repertoire of expressed edited 5-HT_2CRs may contribute to individual differences in brain serotonergic signaling and perhaps even responses to therapeutic agents such as atypical antipsychotic drugs, many of which have been shown to be inverse agonists at the 5-HT_2CR (34, 38).

Serotonergic agonists exhibit a decreased potency for eliciting inositol phosphate production and calcium release when interacting with the edited receptor isoforms stably expressed in NIH-3T3 fibroblasts (5-7). The current results obtained in the R-SAT assay are in agreement, demonstrating decreased agonist potencies for the edited isoforms (INI > VSV > VGV). The extended ternary complex model predicts that raising the concentration of a G subunit that productively interacts with the R* configuration (thereby increasing R*G) would increase agonist potency because agonists preferentially bind R*. Conversely, this model also predicts that the potencies of inverse agonists to reverse constitutive activity will decrease as the fraction of R*G increases (32). In agreement with these predictions, we observed that the addition of G_q and G₁₁ subunits to cells expressing the VGV isoform shifted 5-HT dose-response curves leftward (i.e. increased 5-HT potency), compared with receptor alone. Co-expression of G_q with the VSV isoform caused an increase in 5-HT potency, while co-expression of other G subunits (including G₁₁) did not cause a significant shift in the 5-HT concentration-response curve. Previous work has shown that co-expressing these G-proteins with other monoamine receptors in the R-SAT assay can shift agonist potency (16, 18),² suggesting that there would be a shift in 5-HT potency if the 5-HT_2CR could efficiently couple to these G subunits. Consistent with predictions of the effects of increasing [G] on inverse agonist potency, we observed a rightward shift in ritanserin dose-response curves (i.e. a decrease in inverse agonist potency) at the INI and VSV receptor with G_q co-transfection. These results are the first experimental evidence that increasing [G] can decrease inverse agonist potency. In contrast, ritanserin potency actually increased significantly when G₁₃ and G₁₅ were co-transfected with INI but not with the VSV isoform. This provided an initial suggestion of a functional interaction between G₁₃ and G₁₅ subunits and the INI isoform but not the VSV isoform. Taken together, these data support the hypothesis that the INI isoform has the ability to activate G_q, G₁₃, and G₁₅, whereas the edited VSV and VGV receptor isoforms can efficiently activate only the G_q family of G-proteins.

G_12/13 proteins, isolated as oncogenes and cloned by homology to other G-proteins, have been unique in their failure to regulate known G effectors such as adenylyl cyclase or phospholipases (reviewed in Ref. 39), although these G-proteins have been shown to mediate rearrangement of the actin cytoskeleton (31). Therefore, to confirm the apparent alteration in G₁₃ coupling exhibited by edited 5-HT_2CR isoforms, we examined the ability of the 5-HT_2CR isoforms to modulate the actin cytoskeleton. These studies demonstrate that the INI receptor has the ability to promote actin cytoskeleton rearrangement via activation of G₁₃, whereas the VGV receptor isoform does not. Importantly, these data confirm the R-SAT results suggesting an alteration in the ability of edited isoforms to activate G₁₃. This differential ability of the 5-HT_2CR edited isoforms to promote actin polymerization has important implications in the brain, where neurotransmission is dynamically regulated by cytoskeletal rearrangments. Furthermore, the finding that 5-HT_2CR isoforms with three residues altered within the putative second intracellular loop exhibit reduced functional coupling to G₁₃ provides the first evidence of a critical role for the second intracellular loop in coupling to G_12/13-type proteins.

A noteworthy perspective here is that although an acute response to G_12/13 activation results in the rapid formation of stress fiber and focal adhesion assemblies, a relatively longer response results in the activation of specific gene expression (40). The interactions between these different sets of signals may distinguish G_12/13 signaling from other signal transduction pathways. Thus, transient activation of G_12/13 may be involved in immediate housekeeping responses, such as the regulation of Na⁺/H⁺ exchange, stress fiber formation, ion channels, cell volume, and shape changes, while a more sustained level of activation, perhaps mediated by constitutively active receptors, may lead to cell division and differentiation. Detailed temporal and spatial analyses of 5-HT_2CR isoform profiles and G-protein signaling differences are needed. For example, the pleiotropic role of the 5-HT_2CR as a growth factor during development (41) and as a regulator of neuronal excitability in mature animals (42) may reflect temperospatial differences in RNA editing and subsequent G-protein coupling capacity.

Whereas RNA editing has been shown to have profound functional consequences in a number of proteins (43-45), this is the first report of a change in the pattern of receptor-mediated G-protein activation by RNA editing. As evidenced by both R-SAT data and stress fiber formation, extensively edited isoforms of the 5-HT_2CR, created by editing at four or five sites, have lost or greatly attenuated their ability to functionally couple to G₁₃-protein, which may produce functionally distinct signaling patterns in vivo. Thus, RNA editing is a novel mechanism for regulation of the pattern of activation of heterotrimeric G-proteins, molecular switches that control an enormous variety of biological processes.

	ACKNOWLEDGEMENTS

We thank Mattie Goodman and Kathleen Rutledge for expert technical assistance, Dr. Ethan Burstein for critical discussions, and Drs. Jon Backstrom, Erik Barton, Paul Gresch, Heidi Hamm, and Lee Limbird for critical reading of the manuscript. We also thank Dr. Thierry Wurch for generously providing the construct encoding G₁₅ and Dr. Thomas Amatruda III for generously providing the construct encoding G₁₆. Statistical analyses were performed by the Statistical Core of Vanderbilt University's John F. Kennedy Center for Research on Human Development.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants MH34007, NS35891, and GM07623.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: 459 Preston Research Bldg., Dept. of Pharmacology, Vanderbilt University, Nashville, TN 37232-6600. Tel.: 615-936-1685; Fax: 615-343-6532; E-mail: elaine.bush@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, September 25, 2001, DOI 10.1074/jbc.M106745200

² R. D. Price, D. M. Weiner, M. S. S. Chang, and E. Sanders-Bush, unpublished results.

	ABBREVIATIONS

The abbreviations used are: 5-HT, serotonin; G-protein, heterotrimeric guanine nucleotide-binding protein; 5-HT_2CR, serotonin 5-HT_2C receptor; R-SAT, Receptor Selection/Amplification Technology^TM; DOI, (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane; ONPG, o-nitrophenyl--D-galactopyranoside; PCR, polymerase chain reaction; LPA, lysophosphatidic acid; MPS, membrane-permeable sequence.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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