Differential coupling of 5-HT₁ receptors to G proteins of the G_i family

Abstract

1. Since all 5-HT₁ receptors couple to G_i–type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5-HT₁ receptors has been unclear.

2. In previous studies we have used transfected cells to demonstrate that 5-HT_1B receptors can couple more efficiently than 5-HT_1A receptors to activation of extracellular signal-regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5-HT₁ receptors differentially couple to isoforms of G_iα.

3. In the present study we utilized a model system in which pertussis toxin resistant forms of human G_iα1, G_iα2, and G_iα3 were used to directly compare the coupling of human 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors to each G_iα in transfected human HeLa cells.

4. 5-HT_1A receptors displayed a preference for G_iα1 and G_iα2, relative to G_iα3. Pertussis toxin resistant forms of G_iα1, G_iα2, and G_iα3 rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5-HT in the absence of pertussis toxin.

5. In contrast, pertussis toxin resistant forms of G_iα1, G_iα2, and G_iα3 rescued 32%, 118%, and 35% of 5-HT_1B receptor-stimulated activity, respectively, indicating that 5-HT_1B receptors coupled primarily through G_iα2. A similar preference for G_iα2 was found in studies of the 5-HT_1D receptor, where toxin resistant G_iα1, G_iα2, and G_iα3 rescued 30%, 70%, and 40% of activity, respectively.

6. In conclusion, the observed differential coupling of 5-HT₁ receptors to isoforms of G_iα, provides additional evidence for our previous findings that the subtypes of 5-HT₁ receptors exhibit similar, but distinct, functions.

Introduction

At least 16 types of mammalian receptors for serotonin (5-HT) have been identified and classified within seven families (Hoyer et al., 1994; Scalzitti & Hensler, 1996). The physiological significance of such a large number of receptors is currently unclear. This is especially true regarding those receptors classified as 5-HT₁ receptors, which have been postulated to play a role in the treatment and pathophysiology of a number of disorders including depression, anxiety, and migraine headaches. All of the 5-HT₁ receptors, designated 5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, and 5-HT_1F, couple to G proteins of the G_i class and inhibit adenylyl cyclase. Although these receptors have clear distinctions in pharmacology and structure, there is little currently known about differences in coupling to cellular signals. However, we have previously demonstrated that 5-HT_1B receptors couple more effectively than 5-HT_1A receptors to activation of the mitogen-activated protein (MAP) kinase ERK and to inhibition of adenylyl cyclase in Chinese Hamster Ovary (CHO) cells (Mendez et al., 1999). In those studies we directly compared the intrinsic activity of each receptor subtype in stably transfected cells expressing receptors at the same densities. The observed differences in receptor function suggested the possibility that the receptors differentially couple to isoforms of G_iα.

However, binding studies utilizing membranes from infected Spodoptera frugiperda (Sf9) insect cells over-expressing receptors and G proteins, suggest that differential receptor/G protein coupling, in fact, might not occur. All three isoforms of G_iα have been reported to reconstitute high-affinity binding by 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors (Butkerait et al., 1995; Clawges et al., 1997; Brys et al., 2000). Nevertheless, it cannot be assumed that these findings translate into similar non-preferential utilization of G proteins in the coupling of receptors to cellular signals in mammalian cells. In fact, Garnovskaya et al. (1997) found that pertussis toxin resistant forms of G_iα1 were ineffective in rescuing coupling of 5-HT_1A receptors to Na⁺/H⁺ exchange in transfected CHO cells.

In the present studies we examined the coupling of human 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors to the MAP kinase ERK. The ERK pathway is known to enhance cell survival, and is required for normal neuronal functioning (Encinas et al., 1999; Erhardt et al., 1999). In particular, our studies were aimed at uncovering differences in the coupling of 5-HT₁ receptors to G_iα. Since G_iα is selectively ADP-ribosylated by pertussis toxin, we utilized toxin resistant mutants of human G_iα to ‘rescue' receptor/G protein-coupling from pertussis toxin-catalyzed inhibition of 5-HT₁ receptor/G protein coupling. In this way, the efficacy of receptor coupling to each subtype of G_iα could be studied in isolation. An advantage to this approach, over some other methods, is that such studies directly address functional specificity in a cell type-independent manner. Since pertussis toxin prevents the coupling of receptors to endogenous G_iα, the relative endogenous expression of G_iα isoforms in the particular cell type used does not alter the observed results.

In order to simulate the G protein-coupling of endogenous human 5-HT₁ receptors, as closely as possible, we utilized a model system in which the receptors, G proteins, and cell line were all human. Studies presented here demonstrate that 5-HT_1B and 5-HT_1D receptors couple more selectively than 5-HT_1A receptors to subtypes of G_iα. The expression of multiple receptors that display similar, but distinct, coupling to G proteins and cellular signals provides insight into the large number of receptors (at least 16) required to mediate the diverse and highly complex actions of 5-HT in the central nervous system.

Methods

Materials

5-HT, tranylcypromine, and pertussis toxin were purchased from Sigma (St. Louis, MO, U.S.A.).

Cell culture

HeLa cells were obtained from American Type Culture Collection (Rockville, MD, U.S.A.), and were routinely cultured in Eagle's minimum essential medium supplemented with non-essential amino acids and 10% dialyzed foetal bovine serum (dialyzed in membranes with 1000 Dalton molecular weight cut-offs against a 100-fold greater volume of 150 mM NaCl to remove endogenous 5-HT), 100 units penicillin-100 μg streptomycin/ml (95% air, 5% CO₂).

Transient transfections of cells

cDNAs for the human 5-HT_1B and 5-HT_1D receptors were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). cDNA for the human 5-HT_1A receptor has been previously described (Fargin et al., 1989; Cowen et al., 1996). cDNAs for pertussis toxin resistant mutants of human G_iα1 (C351I), G_iα2 (C352I) and G_iα3 (C351I) were obtained from the Guthrie cDNA Resource Center (Sayre, PA, U.S.A.). Expression of all sequences was under the control of the CMV promoter. Transient transfections were performed 48 hours prior to cellular studies using the Profectin calcium phosphate procedure (Promega, Madison, WI, U.S.A.). For studies of receptor coupling, cells were cultured in 60 mm plates and co-transfected with 6 μg of receptor plasmid DNA plus 6 μg of G protein plasmid DNA or empty vector. For studies of G_iα expression, cells were cultured in 100 mm plates and transfected with 20 μg of receptor plasmid DNA.

Immunoblots

Monoclonal anti-phospho-ERK1/ERK2 (Thr202/Tyr204) was obtained from Cell Signalling (Beverly, MA, U.S.A.). Goat polyclonal anti-G_iα recognizing all G_iα subtypes, rabbit polyclonal total ERK1/ERK2, and horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The day prior to use, cells were washed with phosphate-buffered saline and cultured overnight under serum- and geneticin free conditions. Cells were stimulated with the specified concentrations of agonists, and routinely lysed with a 26-gauge needle in (mM) HEPES 25 (pH 7.4), NaF 50, EDTA 5, sodium orthovanadate 1, 250 μM 4-(2-aminoethyl)-benzene-sulfonylfluoride hydrochloride, 0.1% aprotinin, and 10 μg ml⁻¹ leupeptin. In studies of G_iα expression, NaCl 150 mM, 1% Triton X-100, and β-glycerolphosphate 1 mM were included in the lysis buffer. Proteins were separated on 12% resolving gels (Bio-Rad Laboratories, Hercules, CA, U.S.A.) and transferred to 0.45 μM Immobolin-P polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, U.S.A.). Membranes were blocked overnight with 3% powdered milk before incubation with primary and secondary antibodies. Bound antibodies were visualized using Enhanced Luminol Chemiluminescence Reagent (NEN Life Sciences, Boston, MA, U.S.A.) and exposure to a Kodak Image Station 440CF with a cooled, full-frame-capture CCD camera (Kodak). Net intensity of bands was calculated directly from stored images using Kodak Digital Science 1D Image Analysis Software (version 3.5) on defined regions of interest.

Binding assays

Cells were transfected, as described above, with 6 μg of receptor plasmid DNA plus 6 μg of empty vector. The day prior to use, cells were washed with phosphate-buffered saline and cultured overnight under serum-free conditions. Receptor binding assays were performed using the radioligand [³H]-5-HT (Veldman & Bienkowski, 1992), obtained from Perkin-Elmer Life Sciences (Boston, MA, U.S.A.). Assays contained 10–20 μg of membrane protein, 15 nM [³H]-5-HT, and 1 μM tranylcypromine in a total volume of 100 μl. Displaceable binding of [³H]-5-HT was determined in the presence of 10 μM 5-HT.

Results

Activation of MAP kinase was assayed by measuring MAP kinase kinase (MEK)-dependent phosphorylation of ERK1 and ERK2 at threonine 202 and tyrosine 204 (Cobb & Goldsmith, 1995). When nontransfected HeLa cells were treated with 5-HT, no increase in the level of activated, phosphorylated ERK was detected (Figure 1A). Therefore, although HeLa cells were found in binding studies to apparently express endogenous receptors for 5-HT (Table 1), they did not express subtypes of 5-HT receptors that couple to activation of ERK. In contrast, 5-HT did stimulate phosphorylation of ERK in cells transfected with cDNA for 5-HT₁ receptors. When transfected cells expressing human 5-HT_1A receptors were treated with 5-HT, a 3.8-fold activation of ERK was observed. This activation represented primarily ERK2, as the observed band (Figure 1B) migrated at a relative weight equal to the lower band of a band doublet of p44 ERK1/p42 ERK2 seen from PC12 cell lysate run on the same gel (not shown). Activation of ERK2 by 5-HT was mediated by G_i, as pretreatment with pertussis toxin caused almost complete inhibition.

Figure 1.

5-HT_1A receptors couple to activation of ERK through pertussis toxin sensitive G proteins. (A) Nontransfected HeLa cells were treated for 5 min with 10 μM 5-HT (lane 2), and then lysed. (B) HeLa cells transfected with cDNA for the human 5-HT_1A receptor were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; ***P<0.001; n.s., statistically not significant vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Table 1.

Transfected 5-HT₁ receptors are all expressed at the same density

In contrast, when cells were co-transfected with cDNA for the 5-HT_1A receptor and pertussis toxin resistant forms of human G_iα1, G_iα2, or G_iα3, all three toxin-resistant subtypes were found to ‘rescue' receptor-mediated activation of ERK from inhibition by pertussis toxin (Figure 2). However, the receptor demonstrated a preference for G_iα1 and G_iα2, as transfection with toxin resistant G_iα3 caused the smallest activation of ERK. Pertussis toxin resistant forms of G_iα1, G_iα2, and G_iα3 rescued 73, 76 and 44%, respectively, of the increase in levels of activated ERK stimulated by 5-HT in the absence of pertussis toxin. While both G_iα1 and G_iα2 effectively coupled 5-HT_1A receptors to activation of ERK, concentration–response curves revealed somewhat more efficient coupling by G_iα2. The EC₅₀ (calculated by nonlinear regression analysis of the net intensities of bands) for 5-HT-stimulated activation was 12 nM for cells expressing toxin resistant G_iα2, but 40 nM for cells expressing G_iα1 (Figure 3A). The EC₅₀ for cells transfected with G_iα3 was similar to that for G_iα1, 32 nM, though the maximal effect was significantly reduced. Interestingly, the basal levels of activated ERK were higher in cells transfected with toxin resistant G_iα2 than in cells transfected with toxin resistant G_iα1 and G_iα3.

Figure 2.

5-HT_1A receptors couple to activation of ERK through multiple subtypes of G_iα. HeLa cells co-transfected with cDNA for the human 5-HT_1A receptor and pertussis toxin resistant forms of either (A) G_iα1, (B) G_iα2, or (C) G_iα3 were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). **P<0.01; ***P<0.001 vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Figure 3.

5-HT_1A receptors couple most efficiently to activation of ERK through G_iα2 despite equal expression of all transfected G_iα subtypes. (A) HeLa cells co-transfected with cDNA for the human 5-HT_1A receptor and pertussis toxin resistant forms of either G_iα1, G_iα2, or G_iα3 were treated overnight with 20 ng/ml pertussis toxin before treatment with the indicated concentrations of 5-HT for 5 min, and subsequent lyses. (B) HeLa cells were transfected with cDNA for pertussis toxin resistant forms of either G_iα1 (lane 2), G_iα2 (lane 3), or G_iα3 (lane 4) and the density of expression of G_iα subunits were compared to nontransfected (con) cells (lane 1). (A) Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK) or (B) 20 μg of total lysate was analysed by immunoblotting with antibody recognizing all forms of G_iα. Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³) **P<0.01; ***P<0.001 vs nontransfected (con) cells, ANOVA, Bonferroni analysis. A representative immunoblot from one of the three experiments is shown.

The observed preferential coupling of 5-HT_1A receptors to G_iα1 and G_iα2, relative to G_iα3, was not the result of differences in the levels of transfected G_iα subunits. Each subunit was expressed at an approximately 2.5-fold greater density than endogenously expressed G_iα subunits (Figure 3B).

As was seen with 5-HT_1A receptors, coupling of 5-HT_1B receptors to activation of ERK was almost completely inhibited by pertussis toxin (Figure 4A). However, the relative preference for coupling to subtypes of G_iα was different than that found for 5-HT_1A receptors. G_iα2 much more effectively rescued receptor-mediated activation of ERK than G_iα1 and G_iα3. In contrast to the complete (118%) rescue by G_iα2, pertussis toxin resistant forms of G_iα1 and G_iα3 rescued only 32 and 35%, respectively, of the activation of ERK stimulated by 5-HT in the absence of toxin (Figure 5). Significantly, the more selective G protein-coupling by 5-HT_1B receptors, relative to 5-HT_1A receptors, was not the result of expression of a lower density of transfected 5-HT_1B receptors. The level of displaceable binding of [³H]5-HT to membranes prepared from cells transfected with cDNA for the 5-HT_1B receptor was similar to that from membranes prepared from cells transfected with cDNA for the 5-HT_1A receptor (Table 1).

Figure 4.

5-HT_1B and 5-HT_1D receptors couple to activation of ERK through pertussis toxin sensitive G proteins. (A) HeLa cells transfected with cDNA for the human 5-HT_1B receptor or (B) human 5-HT_1D receptor were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; **P<0.01; n.s., statistically not significant vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Figure 5.

5-HT_1B receptors couple preferentially to G_iα2. HeLa cells co-transfected with cDNA for the human 5-HT_1B receptor and pertussis toxin resistant forms of either (A) G_iα1, (B) G_iα2, or (C) G_iα3 were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; **P<0.01; ***P<0.001 vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

5-HT_1D receptors were found to be similar to 5-HT_1B receptors in their coupling to G_iα. As was found with 5-HT_1B receptors, treatment with pertussis toxin almost completely uncoupled endogenous G_iα from 5-HT_1D receptors (Figure 4B). Pertussis toxin resistant G_iα2 rescued 70% of receptor-mediated activation of ERK. In contrast, toxin resistant forms of G_iα1 and G_iα3 rescued only 30 and 40%, respectively, of the activity stimulated by 5-HT in the absence of pertussis toxin (Figure 6).

Figure 6.

5-HT_1D receptors are similar to 5-HT_1B receptors in preferentially coupling to G_iα2. HeLa cells co-transfected with cDNA for the human 5-HT_1D receptor and pertussis toxin resistant forms of either (A) G_iα1, (B) G_iα2, or (C) G_iα3 were treated overnight in the presence (lanes 3 and 4) or absence (lanes 1 and 2) of 20 ng ml⁻¹ pertussis toxin before treatment with 10 μM 5-HT (lanes 2 and 4) for 5 min, and subsequent lyses. Total lysate was analysed by immunoblotting with antibody to phospho-ERK1/ERK2 (p-ERK). Membranes were then stripped and analysed with antibody to total ERK1/ERK2 (Total). Net intensities of bands were calculated from three separate experiments, performed in duplicate, and expressed as the means±s.e.mean (×10³). *P<0.05; **P<0.01; ***P<0.001 vs absence of 5-HT, two-sided paired Student t-test calculated separately for both the presence and absence of pertussis toxin. Representative immunoblots from one of the three experiments are shown to demonstrate that treatment with pertussis toxin does not alter the levels of total ERK.

Discussion

Our studies demonstrate that human 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors exhibit clear differences in coupling to subtypes of G_iα. 5-HT_1B and 5-HT_1D receptors were found to much more efficiently utilize G_iα2 relative to G_iα1 and G_iα3. In contrast, 5-HT_1A receptors demonstrated less selectivity, particularly with regards to G_iα1 vs G_iα2. These findings represent a progression of our earlier studies in which we used transfected CHO cells to demonstrate more efficient regulation of ERK and adenylyl cyclase by 5-HT_1B receptors relative to 5-HT_1A receptors (Mendez et al., 1999). Significantly, in each study the coupling by receptor subtypes was compared in an individual cell line, under identical conditions. Therefore, the difficulties inherent in comparing receptors expressed in different cell types was avoided. Together, these studies provide evidence that although all 5-HT₁ receptors, to some degree, negatively regulate adenylyl cyclase, they differentially couple to G proteins and consequently exhibit differences in coupling to cellular signals.

CHO cells were not utilized in the present study because they express, at low density, endogenous 5-HT_1B receptors that couple to activation of ERK (Mendez et al., 1999). That low level of expression was useful in our earlier studies in which the coupling of receptors to cellular signals was studied at various receptor densities. However, interpretation of results from studies of receptor coupling to G proteins is facilitated when all receptors are activated with the physiological agonist (i.e. 5-HT, in the present studies). In this manner, problems resulting from the use of different receptor-selective agonists are avoided. For example, it has been reported that the relative preference of 5-HT_1A receptors for subtypes of G_iα is modulated by the particular receptor agonist studied (Gettys et al., 1994). It is quite possible that the same also occurs with 5-HT_1B and 5-HT_1D receptors.

Significantly, our finding that 5-HT_1A receptors more effectively utilized G_iα1 than did 5-HT_1B and 5-HT_1D receptors, cannot be attributed to differences in expression of receptors or G proteins. Binding studies demonstrated that the density of 5-HT_1B and 5-HT_1D receptors was similar to the density of 5-HT_1A receptors. Similarly, the poor coupling by all 5-HT₁ receptor subtypes to G_iα3, was not the result of lower levels of expression of the G_α subunit relative to G_iα1 and G_iα2. Immunoblot analysis demonstrated similar levels of expression of all three G_iα subunits.

Our finding that all three subtypes of 5-HT₁ receptors effectively utilize G_iα2 to activate ERK is consistent with findings from studies of other G_i-coupled receptors. Winitz et al. (1994) demonstrated that a dominant-negative form of G_iα2 inhibited thrombin and ATP receptor-coupling to activation of ERK. Conversely, pertussis toxin resistant forms of G_iα2 have been reported to rescue activation of ERK by A1-adenosine receptors (Pace et al., 1995), and constitutively active forms independently stimulate activation of the MAP kinase (Edmastsu et al., 1998). However, our results additionally demonstrate that some G_i-coupled receptors can also, to varying degrees, utilize G_iα1 and G_iα3 to stimulate activation of ERK. Interestingly, in our studies of G protein coupling to 5-HT_1A receptors, we found that transfection with G_iα2 resulted in increased basal activity relative to that seen with transfection of G_iα1 and G_iα3. This may represent an enhancement of receptor constitutive activity by G_iα2.

The observed differences in 5-HT₁ receptor-coupling to isoforms of G_iα provide an explanation for our previous demonstration of differential coupling of receptors to cellular signals (Mendez et al., 1999). While all subtypes of G_iα, by definition, inhibit the activity of adenylyl cyclase in in vitro studies, there is increasing evidence that the particular isoforms differentially regulate cellular pathways in intact cells. This may result, in part, from their expression in different cellular locations. For example, in LLC–PK1 renal epithelial cells, G_iα2 is localized to the basolateral membrane where it negatively couples to adenylyl cyclase, while G_iα3 is expressed both in the apical membrane where it stimulates Na⁺ channel activity and in the Golgi (Ercolani et al., 1990). Interestingly, Garnovskaya et al. (1997) found that pertussis toxin resistant forms of G_iα2 and G_iα3, but not G_iα1, rescued coupling of 5-HT_1A receptors to activation of Na⁺/H⁺ exchange in CHO cells. Our findings suggest that this likely reflected a lack of coupling of G_iα1 to Na⁺/H⁺ exchange rather than a lack of coupling of G_iα1 to 5-HT_1A receptors. Perhaps, in CHO cells, the expression of G_iα1 is localized such that it cannot modulate Na⁺/H⁺ exchange. Our demonstration that 5-HT_1A, but not 5-HT_1B and 5-HT_1D, receptors efficiently utilize G_iα1 relative to G_iα2, is significant in that the differential coupling was observed in studies of the same cellular signal (ERK), in the same cells, under identical conditions.

Interestingly, our findings differ from those obtained in binding studies utilizing membranes from infected Sf9 cells. In those studies G_iα1, G_iα2, and G_iα3, each were found to reconstitute high-affinity binding by all three 5-HT₁ receptors (Butkerait et al., 1995; Clawges et al., 1997; Brys et al., 2000). However, our studies were different in that we attempted to more closely duplicate the coupling of endogenous receptor to endogenous G protein that would occur in human cells. While Sf9 cells are non-mammalian cells, our studies utilized human HeLa cells. Similarly, the previous studies utilized rat and mouse G proteins, while our studies examined coupling to human G proteins. These differences, as well as the much higher levels of protein expression achieved in Sf9 cells, relative to mammalian cells, could account for our different results.

Significantly, since our study utilized toxin resistant mutants of human G_iα to ‘rescue' receptor/G protein-coupling, we were able to study the efficacy of receptor coupling to each subtype of G_iα in isolation. In that pertussis toxin prevents the coupling of receptors to endogenous G_iα, the relative endogenous expression of G_iα isoforms in the particular cell type studied does not alter the observed results. Although HeLa cells express endogenous G_iα3 and G_iα1 in a 10 : 1 ratio, with little G_iα2 (Raymond et al., 1993), our results are relevant to any type of cell expressing 5-HT₁ receptors, regardless of the composition of expressed G_iα.

Our studies, in that they model the coupling of 5-HT₁ receptors to G proteins in human cells, may therefore be relevant to human central nervous system (CNS) neurons, and consequently to understanding the etiology of mood disorders. There is increasing evidence that patients with depression and bipolar disorder exhibit alterations in the expression of G proteins (Young et al., 1994; Avissar et al., 1997). Our findings suggest that changes in the expression of G_iα2 could have an effect on the activation of ERK stimulated by each of the three 5-HT₁ receptors. In contrast, alterations in the level of G_iα1 would impact primarily the activity elicited by 5-HT_1A receptors. Such changes in G protein expression could be postulated to contribute to the pathophysiology of mood disorders since the ERK pathway is required for normal neuronal functioning, and is known to enhance cell survival (Encinas et al., 1999; Erhardt et al., 1999).

Our findings that subtypes of 5-HT₁ receptors display similar, but distinct, patterns of coupling to G proteins and cellular signals (Mendez et al., 1999) is consistent with the hypothesis that a large number of receptors, with subtle differences, are required to mediate the actions of 5-HT. Serotonergic medications are known to have diverse and complex actions on the central nervous system. They are used to treat such complicated disorders as depression, anxiety, eating disorders, obsessive–compulsive disorder, and schizophrenia. As the functions of individual 5-HT receptors continue to be elucidated, it may become possible to design medications that act more selectively at the specific receptor/receptors relevant to particular disorders.

Abbreviations

5-HT

5-hydroxytryptamine or serotonin

ERK

extracellular signal-regulated kinase

MAP kinase

mitogen-activated protein kinase