Effector Coupling Mechanisms of the Cloned 5-HT1A Receptor*

The signal transduction pathways of the cloned hu- man 5-HT1A receptor have been examined in two mammalian cell lines transiently (COS-7) or perma- nently (HeLa) expressing this receptor gene. In both systems, 5-hydroxytryptamine (5-HT, serotonin) me- diated a marked inhibition of &adrenergic agonist-stimulated (80% inhibition in COS-7 cells) or forsko- lin-stimulated cAMP formation (up to 90% inhibition in HeLa cells). This serotonin effect (ECso = 20 nM) could be competitively antagonized by metitepine and spiperone (Ki = 81 and 31 nM, respectively) and could also be blocked by pretreatment of cells with pertussis toxin. In both cell types, 5-HT failed to stimulate adenylyl cyclase through the expressed receptors. In HeLa cells, 5-HT also stimulated phospholipase C (-40-75% stimulation of formation of inositol phosphates). Again, this effect was inhibited by metitepine. However, the EC60 of 5-HT was considerably higher (-3.2 NM) than that found for inhibition of adenylyl cyclase. Both pathways were demonstrated to be similarly affected by pertussis toxin. These findings indicate that like the M2 and M3 muscarinic cholinergic receptors, the 5- HTlA receptor can couple to multiple transduction pathways with varying efficiencies via pertussis toxin- sensitive G-proteins. The lack of stimulation of cAMP formation

Multiple types of serotonin receptors have been described in both the peripheral and central nervous systems . Bradley et al. (1) have proposed dividing the serotonin receptor family into three major groups, 5-HT1,' 5-HT2, and 5-HT3. The 5-HT1 receptors, which by definition bind serotonin with high affinity (in the nanomolar range), can be further subdivided into four subtypes: 5-HTlA, 5-HTlB, 5-HTlC, and 5-HT1D receptors. These distinctions are based on radioligand binding analyses (see Ref. 2 for review).
We recently reported the identification of a genomic clone for a 5-HT1A receptor (3). This clone, called G-21, was initially obtained from a human genomic library by crosshybridization with a full-length &adrenergic receptor probe (4). Subsequently, based on ligand binding studies in a tran-* This work was supported in part by Sandoz, Basel, Switzerland.
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Despite intense investigative effort in recent years, the effector coupling mechanisms of the various serotonin receptors remain highly controversial (7). Nowhere is this more evident than for the 5-HT1A receptor which has been reported to stimulate (8-10) or inhibit adenylyl cyclase (11-E), increase K' conductance (16, 17), and even regulate phosphatidylinositol turnover (18). The definitive assignment of the functional coupling of this receptor to various signalling pathways has been hampered by the marked heterogeneity of serotonin receptors present in the model systems studied, the unavailability of highly selective antagonist ligands for the 5 -HTlA receptor, and even the possible existence of other 5-HTlA related receptors. In the present studies we have characterized the biochemical actions of the cloned human 5-HTlA receptor by using both transient (COS-7 cell) and permanent (HeLa cell) expression systems.
[3H]8-OH-DPAT was used as the radioligand (110 Ci/mmol) and 10 FM serotonin (5-HT) was used to determine nonspecific binding. After incubation with ligands, samples were filtered through GF/F filters.
Expression Vector and Cell Transfection-The HindIII-BamHI fragment from the human 5-HT1A receptor genomic clone "G-21" (4), containing the complete coding sequence, was subcloned into the expression vector pBC12BI (19). This vector contains the SV40 origin of replication and the Rous sarcoma virus long terminal repeat which acts as the promoter for the expression of the 5-HT1A receptor gene. The same vector, in which the cDNA of &adrenergic receptor was inserted (20), was also used in transient expression experiments. For transient expression, COS-7 cells were transfected using the DEAEdextran method (19). Cells were allowed to grow 60 h before assays were performed. Constitutive gene expression was obtained by cotransfecting the 5-HT1A receptor construct with pRSVNeo into HeLa cells, using the calcium phosphate procedure (19). Both plasmids were linearized a t a unique PvuI restriction site prior to transfection. Transformed cells were selected for their resistance to the antibiotic G418 (0.8 mg/ml) and then pooled to check their ability to demonstrate specific [3H]8-OH-DPAT binding. Pooled cells were then subjected to single cell dilution cloning and each clone was tested for [3H]8-OH-DPAT specific binding activity.
Assessment of CAMP Formation-The effects of agonists and antagonists on cAMP formation were assessed in both COS-7 and HeLa cells. Briefly, adherent cells (-80% confluent) were incubated twice for 10 min with DMEM, 10 mM Hepes. Medium, containing 100 FM 3-isobutyl-1-methylxanthine and various concentrations of test drugs were then added to the cells. At the end of this treatment (10-min incubation), the reaction was stopped by aspiration of the media and addition of 2 ml of 100 mM HC1. Cells were scraped and centrifuged for 10 min at 1000 X g. Aliquots of the supernatant, corresponding to approximately 1000-2000 cells, were used to perform cAMP measurements by radioimmunoassay.
Assessment ojInositol Phosphate Formation-Cells grown in 6-well dishes (Falcon), were equilibrated for 24 h in regular medium containing 5 pCi/ml of my~[~H]inositol (14.6 Ci/mmol). After washing with phosphate-buffered saline (30 min, 37 "C), cells were incubated for 30 min in phosphate-buffered saline containing 20 mM LiC1. This medium was then replaced by fresh medium containing different concentrations of various drugs. Reactions were stopped by aspiration and addition of 1 ml of 0.4 M perchloric acid. Lysates were then used for the measurement of [3H]inositol phosphates as described by Martin (21), using ionic exchange chromatography with Dowex 1-X8 (0.8 ml, 100-200 mesh) in the formate phase.

Transient and Permanent Expression
For permanent expression of the 5-HT1A receptor, clonal HeLa cell lines were obtained as described under "Methods." As shown in Table I Stimulation of CAMP Levels-Wild type COS-7 cells contain a low level (50-100 fmol/mg protein) of &adrenergic receptors which can mediate a 5-&fold stimulation of CAMP formation by 2 pM isoproterenol. Transfection of COS-7 cells with the &adrenergic receptor expression vector, however, led to a further isoproterenol-induced increase in cellular cAMP levels (80-120%) (Fig. 2). Thus, this transient system can be used to investigate the positive coupling of an expressed receptor to adenylyl cyclase. sion vector. Treatment of these cells with 10 ~L M 5-HT failed to increase intracellular cAMP levels. Similarly, when stably expressed in HeLa cells, the 5-HT1A receptor was still unable to mediate a serotonin-induced increase in cAMP levels (data not shown).
Inhibition of cAMP Levels-To study the action of an inhibitory receptor on adenylyl cyclase, receptor-mediated inhibition of forskolin-stimulated cAMP levels is commonly used. However, in the transient COS-7 cell expression system, this paradigm cannot be effectively utilized. Forskolin stimulates the total cell population, whereas studies indicate that only 5-10% of COS-7 cells are competent and can be transfected (19). Thus, it would be difficult to demonstrate a significant inhibition occurring in only the transfected cells. Accordingly, we utilized a co-transfection protocol in which the vectors expressing both the 5-HT1A receptor and p2adrenergic receptors were co-transfected. Again, it was found that in the transfected COS-7 cells, isoproterenol was able to further stimulate cAMP production by 90 f 18% (n = 11) compared to nontransfected cells. As shown in Fig. 3, under optimal co-transfection conditions (see legend), 5-HT (10 PM) caused an 80% inhibition of the further isoproterenol-stimulated cAMP levels. In order to further document the ability of the 5-HT1A receptor to inhibit adenylyl cyclase, we selected four HeLa clonal cell lines which permanently express different levels of the 5-HT1A receptor ranging from 0.5 to 2.8 pmol/mg of protein (Table I). Since these cells are clonal, the ability of 5-HT to inhibit forskolin-stimulated cAMP levels can be assessed without the complications alluded to above for COS-7 cells. Forskolin in these cells increased cAMP from 2-to &fold over basal levels. 5-HT induced a decrease in forskolin-stimulated cAMP levels in all four cell lines, however, the extent of this reduction did not appear to correlate with the levels of expression of the 5-HT1A receptor (Table  I). Clone HA7, which expresses levels of receptors comparable to those reported in hippocampus, was chosen to pursue the characterization of this activity. As shown in Fig. 4, serotonin was able to elicit a reduction of forskolin-stimulated cAMP levels in a concentration-dependent fashion, with an ECs0 of 20 f 3 nM (n = 3). Maximal inhibition of -75% was routinely achieved. In this system, metitepine and spiperone were able to induce a parallel and comparable rightward shift of the serotonin dose-response curve without affecting the maximal effect (Fig. 4). This is the classical pattern of competitive inhibition. Dissociation constants of 81 and 31 nM (mean of two experiments) for metitepin and spiperone, were, respectively, derived from these shifts and are in good agreement with those reported in the literature (12-14).
5-HTIA Receptor Coupling to Phosphoinositol Hydrolysk-Because serotonin, seemingly through a pharmacologically characterized 5-HT1A response, has been reported to couple Effect of pertussis toxin on 5-HT-induced inhibition of cAMP formation and stimulation of PI hydrolysis. Cells were treated for 4.5 h with increasing concentrations of pertussis toxin (0-100 ng/ml) before being assayed for 5-HT-induced inhibition of forskolin-stimulated cAMP levels (e) and 5-HT-induced accumulation of inositol phosphates (0). For the latter assay, cells were prelabeled -20 h with 10 pCi of my~-[~H]inositol and pertussis toxin was directly added to the medium for the last 4.5 h of incubation. Each data point is the average of two independent experiments performed in triplicate.
to different second messenger systems (7-18), it was of interest to examine the ability of the cloned 5-HT1A receptor to affect hydrolysis of phosphatidylinositol. In the stable cell line HA7, a modest but significant increase of total inositol phosphates (IPS, 40-75% over basal) was observed upon stimulation of the cells with 10 FM 5-HT for 15 min, in the presence of 20 mM LiCl. It is interesting that a similar pattern of response has been reported for the M2 and M3 muscarinic receptor subtypes which preferentially couple to inhibition of adenylyl cyclase but weakly activate phosphatidylinositol hydrolysis (22-24). When the accumulation of individual inositol phosphates was measured (Fig. 5), a rapid stimulation of IP3 was observed within the first minute followed by a slower increase in IP2 and IP, probably resulting from the dephosphorylation of IP3 and IP2. This 5-HT-induced accumulation of IP3 could be antagonized by metitepine (not shown). A dose-response curve for the ability of 5-HT to stimulate inositol phosphate (total) production is shown in Fig. 6. The ECso (3.2 f 0.8 PM, n = 3) is considerably higher than that for the 5-HT1A receptor-mediated inhibition of adenylyl cyclase. In contrast, 5-HT1A receptors transiently expressed in COS-7 cells did not seem to couple efficiently to this pathway under conditions where al-adrenergic receptors can mediate a large increase in production of inositol phosphates (not shown;Ref. 25).
Effect of Pertussis Toxin on 5-HTIA Receptor-mediated Responses-HA7 cells, pretreated with pertussis toxin for 4.5 h, were used to assess the effect of 5-HT (10 p M ) on both forskolin-induced cAMP formation and accumulation of inositol phosphates. As shown in Fig. 7, both pathways appeared to be similarly sensitive to pertussis toxin, with 50% inhibition of activity observed at 20 ng/ml of toxin.

DISCUSSION
The cloning of various neurotransmitter receptors has opened new approaches to the study of their biology and regulation. In particular, expression of such receptor proteins in a variety of systems provides a powerful tool for assessing the biochemical mechanisms by which their signals are transduced. The situation for the serotonin receptor is particularly complex since at least 6 well-defined receptor subtypes have been described. Moreover, for the 5-HT1A receptor, seemingly contradictory reports of coupling to various effector systems, including both stimulation and inhibition of adenylyl cyclase in different systems have appeared in the literature (7-17). Accordingly, having cloned the gene for the human 5-HT1A receptor, we were particularly interested in exploring the biochemical mechanisms of its coupling in transfected cells expressing the receptor. In these studies we have utilized two different expression systems and have examined three different responses. Our results clearly indicate that, of the responses studied, the most important coupling of the 5-HT1A receptor is to adenylyl cyclase inhibition. This response was demonstrated both in transiently and permanently transfected cell lines and could be blocked by pertussis toxin, suggesting that it is mediated through a Gi protein.
In contrast, we have no evidence that the 5-HT1A receptor can couple through G, to stimulate adenylyl cyclase, as had been claimed based on reports using rat and guinea pig hippocampus, as well as rat brain cortex preparations (8-10). One possible explanation for the discrepancy is that the "5-HT1A" receptor binding studied in those preparations is in fact due to another very closely related sub-subtype of this receptor. This concept has recently been discussed by Dumuis et al. (12). Given currently emerging concepts relating the structure of G-protein coupled receptors to effector coupling specificity, it would be difficult indeed to postulate a mechanism for coupling a single receptor to both G, and Gi. In fact to our knowledge, no single receptor which both stimulates and inhibits adenylyl cyclase has been discovered.
Our data also indicate that in the HeLa cell clone HA7, the expressed 5-HT1A receptor (0.5 pmol/mg protein) is also able to couple to activation of phospholipase C. The serotonininduced stimulation can be blocked by the antagonist metitepine, which confirms that the effect is mediated by the expressed serotonin receptor. However, the ECS0 of 5-HT is about 100-fold higher than that calculated for the inhibition of adenylyl cyclase, suggesting that cyclase inhibition is the primary transduction pathway of the 5-HT1A receptor. A comparable response was obtained for the clone HB24, expressing a higher concentration of receptors (2.3 pmol/mg protein) suggesting that the extent of this effect does not increase at higher receptor levels (not shown). Furthermore, 5-HT1A receptor-induced phosphatidylinositol hydrolysis was not observed in COS-7 cells, which might indicate that the 5-HT1A receptor can elicit distinct biochemical responses in different cellular systems.
An interesting and important issue is the nature of the G protein(s) through which the various biological actions of the expressed 5-HT1A receptors are mediated. On the one hand, the identical pertussis toxin sensitivity of the two responses might be taken to suggest that the same, or at least very similar, G proteins mediate both effects. On the other hand, the very divergent dose-response relationships for 5-HT activation of the two pathways is more suggestive of distinct G proteins mediating the two effects. The pertussis toxin-sensitive G proteins thus far described include Gi1-3 and Go. In addition, "G," which is a candidate for coupling to phospholipase C seems to be composed of at least two types of G proteins, a pertussis toxin-sensitive and a pertussis toxininsensitive one (26). Further supporting the existence of distinct G,s is the differential pertussis toxin sensitivity of the cholecystokinin receptor-and M2 and M3 muscarinic receptor-mediated effects on PI turnover (24). It is thus possible that HeLa cells express a G, protein displaying a pertussis toxin sensitivity similar to that of Gi and to which 5-HT1A receptors can couple.
It is interesting to compare the characteristics of the responses mediated by the 5-HTlA, muscarinic cholinergic receptors (22-24), and the cy1-and a2-adrenergic receptors' which apparently fall into two groups. The M1 and M4 muscarinic receptors and al-adrenergic receptors are all primarily coupled to the phospholipase C system via a pertussis toxin-insensitive or poorly sensitive G-protein. These receptors mediate strong PI turnover responses. In contrast, M2 and M3 muscarinic receptors as well as &'-adrenergic receptors and the 5-HT1A receptor, are primarily linked to adenylyl cyclase inhibition but are also capable of mediating weak stimulation of the phospholipase C system via a pertussis toxin-sensitive G-protein. Whether the same G-protein mediates both the cyclase inhibition as well as PI stimulatory effects, whether the same phospholipase C mediates both the pertussis toxin-sensitive and -insensitive responses, and whether the weak PI responses mediated by 5-HT1A and similar receptors are physiologically relevant all remain subjects for future studies.