5-Hydroxytryptamine Receptor Subtypes: Molecular and Functional Diversity

Receptor Subtypes : Molecular and Functional Diversity

5-Hydroxytryp tamin e

Frederic Saudou and Rene Hen' De'partement de Neurobiologie Laboratoire de Ge'ne'tiqueMole'culaire des Eucaryotes du CNRS Unite' 184 de I'INSERM F-67085 Strasbourg, France

1. Introduction Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine that was first discovered in the gut (Vialli and Erspamer, 1933).5-HT was later shown to correspond to a vasotonic substance found in the serum and was therefore called serotonin (Rapport et al., 1948). Serotonin is also found in the brain (Twarog and Page, 1953) and is involved in a wide range of behaviors such as sleep, appetite, pain perception, locomotion, thermoregulation, and sexual activity. (For a review, see Wilkinson and Dourish, 1991.) Furthermore, serotonergic drugs are used in the treatment of a number of pathological states such as migraine, depression, and anxiety (Sleight et al., 1991). The multiple actions of serotonin are mediated by the specific interaction of this amine with several receptors. Pharmacological and physiological studies identified distinct receptors that were designated 5-HTIA,5-HTl,, 5-HTl,, 5-HTl,, 5-HT2, 5-HT3, and 5-HT4 (Bradley et al., 1986; Hartig, 1989; Peroutka, 1990,1991b; Bockaert r t al., 1992). However, some pharmacological studies suggested the existence of additional serotonin receptor subtypes. Recently, the molecular cloning of 13 different mammalian receptor subtypes revealed an unexpected heterogeneity within 5-HT re-

'

Present address: Center for Neurobiology and Behavior, Columbia University, College of Physicians and Surgeons, 722 West 168th Street, New York, NY 10032. Advances in PhormacoloRv. Volume 30 Copyright Q lW4 by Academic Press. Inc. All rights of reproductlon in any form reserved

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Frederic Saudou and Rene Hen

ceptors. The latest classification of 5-HT receptor subtypes takes into account not only their pharmacological profile and their coupling with second messengers but also their amino acid sequence (Hoyer er al., 1994). Except for the 5-HT3 receptors, which are ligand-gated ion channel receptors, all the other 5-HT receptors belong to the large family of receptors that interact with G proteins. Based on their amino acid sequence homology and coupling to second messenger, these receptors can be divided into distinct families (Table I, Fig. 1): The 5-HT, family contains receptors that are negatively coupled to D ~ 5-HT,,, , the 5-HT,,, adenylate cyclase: the 5-HT,,, the ~ - H T I B , Ithe and the S-HT,, (5-HTIEp)receptors as well as the Drosophila 5-HTdroZA and 5-HTdroZB receptors (see Section 11,A). The 5-HT2 family includes receptors that stimulate phospholipase C: the 5-HT2* receptor previously known as the 5-HT2 receptor and the 5HT,, and stomach fundus S-HT,-like receptors which were respectively renamed 5-HT,c and 5-HTZBreceptors because of their resemblance to the 5-HT2 receptor (for the new nomenclature, Hoyer er al., 1994). The adenylate cyclase stimulatory receptors are a heterogeneous group including the 5-HT4receptor which has not yet been cloned, the Drosophila 5-HTd,, receptor, and two mammalian receptors tentatively named 5-HT6 and 5-HT,. Despite their common coupling with second messengers, the 5-HT6 and 5-HT7 receptors display little homology to one another nor to other 5-HT receptors. In addition, the pharmacological profile of the 5-HT6 and 5-HT, receptors differs frrom the profiles of all other 5-HT receptors including the 5-HT4 receptor (see Section 11,C). The 5-HT5, and 5-HT5, receptors might constitute a new family of 5HT receptors. These receptors display little amino acid homology to the SHT,, 5-HT2, 5-HT,, and 5-HT, receptors. Furthermore, unlike all other G-protein-coupled 5-HT receptors, the 5-HT5 receptors do not modulate the activity of adenylate cyclase or phospholipase C. Their interaction with second messengers remains unknown (see Section 11,D). The goal of this chapter is to focus on the contribution of molecular biological techniques to the identification and characterization of serotonin receptors: 1. Molecular cloning studies identified not only receptors that had already been characterized pharmacologically but also novel subtypes such as the S-HT,,,, the S-HT,,, the ~-HT,A,the ~ - H T ~the B , 5-HT6, and the 5-HT7 receptors, which had not been predicted by classical techniques. 2. The availability of the genes encoding the ~ - H T , and B 5-HTlDreceptors allowed proof of the hypothesis that the ~ - H T I receptor B was the

Table I 5-HT Receptor Families Superfamily G-Protein-coupled receptors

Ligand-gated ion channels

Family

Transduction system

5-HTl

L AC

5-HTz

7 PLC

Adenylyl cyclase stimulatory 5-HT receptors 5-HT5

7 AC Ion channels?

Comments, previous names The 5-HTlEreceptor is the rodent homolog of the 5-HT,Doreceptor; 5-HTlFpreviously called 5-HTIEp 5-HTzApreviously called 5-HT2;5-HTIB called rat fundus receptor, SLR, and S-HT~F; 5-HT2cpreviously called 5-HTlc 5-HT4: No molecular data available; 5HT, also called 5-HTx S-HT~A.5-HT5,

S-HT~A

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Frederic Saudou and Rene Hen

Fig. 1 5-HT receptors and their effector systems. 5-HT,, 5-HT,, 5-HT3, and 5-HT5 correspond to the members of the 5-HT,, 5-HT2, 5-HT3, and 5-HT5 families. G, is a pertussis toxin-sensitive G protein; Gq is pertussis toxin-insensitive. Abbreviations: AC, adenylate cyclase; PLC, phospholipase C; CAMP, 3‘,5’-adenosine monophosphate; DAG, 1.2diacylglycerol; IP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; Kinase A, CAMP-dependent proteine kinase; Kinase C, calcium-lipid-dependent protein kinase; Cam Kinase, calcium-calmodulin-dependent protein kinase.

rodent homolog of the 5-HT,, receptor and that a single amino acid change was responsible for their different pharmacological profile. 3. The expression of the cloned receptors in “simple” environments such as cell lines allows the characterization of their pharmacological and functional properties in the absence of all other 5-HT receptors. Hopefully, these studies will enable the discovery of selective ligands for poorly characterized receptors such as the 5-HT,,, the 5-HT5,, the 5-HT6, and the 5-HT7 receptors. 4. Site-directed mutagenesis of the cloned receptors will permit a detailed study of the domains involved in ligand binding and coupling with G-proteins and should help in the design of more selective drugs. 5 . The availability of the nucleotide and amino acid sequences of the various serotonin receptor subtypes enables a precise determination of the pattern of mRNA expression using in situ hybridization and of protein expression using specific antibodies.

5-HT Receptor Subtypes

33 1

6. The recent development of the homologous recombination technique will allow the creation of mutant mice lacking specific receptor subtypes and should give insights into the possible functions of these receptors.

II. G-Protein-Coupled 5-HT Receptors These receptors interact with G-proteins and share a putative seven transmembrane structure. Sequence comparisons reveal striking amino acid conservations, particularly within transmembrane domains (Fig. 2).

A. The 5-HT, Family-5-HT Receptors Negatively Coupled to Adenylate Cyclase The members of the 5-HTI family are characterized by their amino acid homologies (Fig. 3) as well as by their common effect on second messengers, that is, inhibition of adenylate cyclase activity (Fig. I). Furthermore, and 5-HTd,,, receptors, with the exception of the Drosophila 5-HTdroZA these receptors do not contain any introns in their coding sequences. These receptors display distinct patterns of expression that may reflect distinct physiological functions.

1 . The SHT,, Receptor a. Molecular Structure The genomic DNA encoding the human 5-HTIA receptor was isolated by hybridization at low stringency with a human 02 adrenergic receptor probe (Kobilka et al., 1987). This genomic clone, named (321, contained an intronless gene that displayed 48% amino acid homology to the p2 adrenergic receptor. The G21 clone was later shown to encode a functional S-HT,, receptor (Fargin et al., 1988). The protein structure consists of a single polypeptide chain of 422 amino acids (Kobilka et al., 1987;Chanda et al., 1993). The rat 5-HT,Areceptor has been isolated by the strategy used for the human 5-HT,, receptor and displays similar characteristics (Albert et al., 1990). Amino acid comparison of the human and rat receptors revealed an 89% identity, which corresponds to the homology usually found between species homologs in this gene family. The 5-HTlAreceptor possesses all the characteristics of the G-proteincoupled receptors including seven hydrophobic domains suggested to be transmembrane domains (Dohlman et af., 1987) and putative N-linked glycosylation sites in the N-terminal tail. Furthermore, these receptors possess potential phosphorylation sites that have been suggested to be involved in heterologous and homologous mechanisms of desensitization (O’Dowd et al., 1989b).

5-HTlA human 5-HTIR mouse 5-HI'lDo hunian 5-HTIE human 5-HT1F mo"Se 5-HTdro:!A 5 -HTdro;!R 5-HTLym 5-HT5A m0"Se 5-HT5B mouse mouse 5-HT7 5 - HTdro:I rat 5-H% rat 5-H:ZA 5-HT228 r a t 5-HT2C r a t 5-HTIA human 5-HT18 mouse 5-HTlDahuman 5-HTIE human 5-HT1F mouse 5-HTdroZA 5-HTdro2B 5-HTLym 5-HT5A mouse 5-HT5B mouse 5-HT7 mouse 5 - HTdro 1 5-HT6 rat 5-HTZA r a t 5-HT28 r a t 5-HTZC I d t 5-HT1A human 5-HT18 mouse 5-HT1Da human 5-HT1E human 5-HT1F mouse 5-HTdroZA 5 -HTdroZB 5-H'ILym 5-HT5A mouse 5-HT50 mouse 5-HT7 rnou~r I,-HTdrol rat 5-HT6 5-HTZA rat 5-HT28 r a t 5-HT2C rat 5-HTlA human 5-HT1B mouse 5-HT1Da human 5-HTIE human 5-HT1F mouse 5-HTdroZA 5 - HTdra 28 5-HTLym 5-HT5A mouse 5-HT58 mouse 5-HTl mouse 5-HT3rol 5-HT6 rat 5-HTZA r a t 5-HT2B r a t 5-HT2C r a t

LPF-CESSCtI--MFTLLGAII LVMPI -CkDACW--FHMAIfDFF

.,,

LVLI'I-CRnSCW--IHPALFDFF

LIVCL-S-I-YT--VSSEVADFL LWNV-C-EKCK--1SEEMSNFL LTMPL-CAA-CO--1SU::VASLF LTM!:L-CRE - CE- ~IHTAVAS LF LIGI'F--VDPEG- - 1 FFFARLFV TELISF'L-CS-- -W-DVPAIWKSIF

TELISPL~CA---C-SLPPIWKSIF LSTARI'FICGTSCS-CIPLWERTC LRLIXPF---~ETM-HVPASLSSLF

AN-----IAQAVCD-CISPGLFDVL TN-IMAVICKESCNEWNIGALLNVF

TN-VTLALC-DSCNQTTLKTLLQ IF TN- ILSVLCGRACNPKLMEKLLNVF

Fig. 2 Amino acid similarity of 5-HT receptors. The amino acid sequences of human 5HTIA. mouse 5-HTIB,human 5-HTID,, human 5-HTIE,mouse 5-HTIF,Drosophila 5-HTdro,~ and 5-HTdro,*, Lymnea 5-HTLymr mouse 5-HT,, Drosophila 5-HTd,,, mouse S-HTJAand 5HTSB.rat 5-HT,, rat 5-HT,,, rat 5-HTZBand rat 5-HTZc receptors are aligned. Putative transmembrane domains are numbered I to VII. Black boxes show positions where more than 12 of the 16 sequences are identical.

5-HT Receptor Subtypes

-

333

5-HT 1B mouse 5-HT

human

5-HT 1~ humw 5-HT1

5-HTIF mouSe

Family

5-HT1A human

L A C

5-HT dro2A

5-H7- drozB

/rAc

7

P A C

[

ll

5-HT 5-HT7

5-HT 5A mouse

[ 5-HT 5B 5-HT

mouse

rat

'h

fig. 3 Dendrogram. The sequence of 5-HT receptors were compared and clustered with the programm CLUSTAL (Higgins and Sharp, 1988). The comparison was performed with the amino acid sequences presented in Fig. 2. The lengths of the horizontal lines are inversely proportional to the percentages of homology between receptors or groups of receptors.

Like all other 5-HT receptors negatively coupled to adenylate cyclase (Fig. 2), as well as other inhibitory receptors such as the dopamine D2 receptor (Bunzow et a / . , 1988), the 5-HTIAreceptor has a short C-terminal tail (18 amino acids) (Fig. 2). This region has been shown to be involved in the coupling of the receptor to G-proteins. The 5' flanking sequence of the human 5-HT,, receptor has been isolated (Parks et al., 1991). This DNA fragment exhibited promoter activity in HeLa cells. Interestingly, this fragment did not contain a TATA box but a GC-rich region characteristic of housekeeping genes. Whether this fragment contains all the promoter sequences required for specific expression of the 5-HT,, receptor gene is not currently known. The gene encoding the 5-HT,, receptor has been localized on the distal part of mouse chromosome 13 (Sundaresan et al., 1989; Oakey et al.,

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Frederic Saudou and Rene Hen

1991) and on human chromosome 5 at the locus 5q11.2-ql3 (Table 11; Kobilka et a f . , 1987). b. Functional Expression To study the pharmacological properties of the human 5-HT,, receptor, the G21 DNA clone was inserted into a eukaryotic expression vector and transfected into monkey kidney cells (cos-7 cells). The receptor displayed a moderate affinity for the P-adrenergic antagonist radioligand [ '2SI]iodocyanopindolol( Fargin et af., 1988). The cloned 5-HT,, receptor also bound to the 5-HTI,-selective agonist radioligand [3H]8-hydroxy-2-(di-n-propylamino)tetraln ([3H]S-OHDPAT) with two affinity constants: a high-affinity constant of 0.06 nM and a low-affinity constant 14.5 nM. Competition displacement curves of ['H]8-OH-DPAT binding identified the binding site as that of the 5-HT,, receptor (5-carboxyamidotryptamine (5-CT > 8-OH-DPAT > 1(2-methoxy-phenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN190) = ipsaspirone = 5-HT > buspirone > spiperone > mesulergine > ketanserin}. Similarly the rat 5-HT,, receptor, when expressed in the mouse Lmtk- fibroblast cell line, displayed the pharmacological profile characteristic of the 5-HT,, receptor (Albert er al., 1990). Treatment of the cells expressing the 5-HT,, receptor with pertussis toxin, which ADP-ribosylates the mi and a, subunits of G proteins caused the loss of the high affinity state of the receptor. These data suggested that the high-affinity state for agonists of the receptor is due to its interaction with pertussis toxin-sensitive G-protein. Similarly, incubation of membranes with guanosine nucleotides decreased the high-affinity binding of [3H]8-OH-DPAT(Fargin et al., 1988; Albert et al., 1990). Mutagenesis experiments performed on the human 5-HTI, receptor revealed that Asn 385 in the seventh transmembrane domain (Fig. 2) is critically involved in the high-affinity binding interaction between the receptor and P-adrenergic antagonists such as pindolol and other aryloxyalkylamines but not in binding of other 5-HT,, ligands such as 8-OHDPAT (Guan et al., 1992). In contrast, Asn 396 and Ser 393 as well as Asp 82 in the second transmembrane domain are necessary for the specific binding of ['H]8-OH-DPAT to the human S-HT,, (Chanda et al., 1993). Several reports demonstrated that the 5-HT,, receptor was linked in vduo to the inhibition of adenylate cyclase (De Vivo and Maayani, 1986; Dumuis et a f . , 1988b; Schoeffter and Hoyer, 1988). However, other authors (Shenker et al., 1985; Markstein et al., 1986; Fayolle et al., 1988) reported a positive coupling to adenylate cyclase for the 5-HTIAreceptor. Fargin er al. (1989) and Albert et al. (1990) showed that the cloned 5HT,, receptor, when activated, inhibits adenylate cyclase activity in various cell lines (cos-7 cells, HeLa cells, and GH4Cl pituitary cells) but they did not detect any stimulation of adenylate cyclase in those cell

Table II 5-HTl Receptors Receptor

Species

Amino acids

Locus

5-HT,,

Human Rat

422 422

5q11.2-qI3 13 (mouse)

5-HTIB

Rat Mouse Human

386 386 390

9E 6q13

Human Dog Rat

377

lp34.3-p36.3

377 374

4 (mouse)

5-HTlE

Human

365

5-HT,,

Human Rat Mouse

366 366 367

S-HT,,@

5-HT,,

Introns in coding sequence

mRNA size (kb)

mRNA regional distribution (main sites)

Binding site distribution (main sites)

No

6.0 3.9 (3.6; 3.3)

Hippocampus (CAI -CA3DG), raphe nuclei, amygdala, septum Striatum, hippocampus (CAI ), ganglion cells (retina), subthalamic nuclei, entorhinal and cingulate cortex, Purkinje cells of cerebellum, spinal cord, raphe nuclei Striatum, nucleus accumbens, dorsal raphe nuclei, hippocampus

Identical to mRNA distribution

No

No No No

6.0

No No No

Not detected

-

No

-

3pll

No No No

Not detected

16C2-16 C4

5-6, 5.5

(monkey)

Caudate putamen, parietal cortex

Hippocampus (CAI-CA3). spinal cord, uterus, mesentery

Substantia nigra, globus pallidus, superior colliculus, deep cerebellar nuclei

Substantia nigra, globus pallidus, caudate putamen, subthalamic nuclei Amygdala, caudate, putamen, frontoparietal motor cortex, olfactory tubercle

-

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Frederic Saudou and Rene Hen

lines. These data suggest the existence of an additional receptor having a pharmacological profile similar to that of the S-HTIAreceptor and coupled positively to adenylate cyclase. Alternatively, increases in CAMP levels observed in hippocampus in response to S-HT,, agonists might be indirect consequences of the activation of the 5-HTlA receptor. Note that the recently cloned 5-HT, receptor displays a relatively high affinity for 8OH-DPAT (Ki = 35 n M ) and stimulates adenylate cyclase (see Section II,C,3). This receptor might therefore correspond to the stimulatory 5HTIA-likereceptor. In transfected cells, the 5-HTlAreceptor has also been shown to interact with phospholipase C. Interestingly, in HeLa cells the EC,, of 5-HT for phospholipase C activation is about 10- to 100-fold higher than that for the inhibition of adenylate cyclase (Fargin et al., 1989). In contrast, in Lmtk- cells the EC,, of 5-HT for phospholipase C and adenylate cyclase is about the same (Liu and Albert, 1991). In other cell lines such as Cos7 cells and GH4C1 cells, no activation of phospholipase C was observed (Fargin et al., 1989; Liu and Albert, 1991). This result indicates that the biochemical response elicited by receptor activation may depend on the cell type in which the receptor is expressed and on the nature of the Gproteins present in the cell type. Interestingly, the native ~ - H T , receptor A in brain tissues is apparently not functioning via the modulation of phospholipase C (Hamon et al., 1990). Other functional coupling of the 5-HTlAreceptor has also been reported; for example, in HeLa cells the activation of phospholipase C is followed by a stimulation of sodium-dependent phosphate uptake via protein kinase C, a mechanism sensitive to pertussis toxin (Raymond et al., 1989,1992), and by an activation of Na+/K+-ATPase(Middleton et al., 1990). In CHO cells, the human 5-HTlAreceptor, in addition to inhibiting adenylate cyclase and stimulating phospholipase C, potentiates the effect of Ca2+ ionophore A23187 on [3H]arachidonicacid release (Raymond et al., 1992), and in GH4Cl cells an inhibition of Ca2+ influx has been reported (Liu and Albert, 1991). The 5-HT,, receptor has also been shown to open K+ channels in hippocampal neurons (Andrade et al., 1986)via a pertussis toxin-sensitive G-protein without soluble cytoplasmic intermediates. Activation of K + channels leads to the hyperpolarization of the membrane and to a decrease of neuronal firing. In good agreement, Karschin et al. (1991) showed that the human 5-HTIAreceptor, when heterologously expressed in cardiac atrial cells, can open K+ channels that are normally activated by the endogenous muscarinic acetylcholine receptors. The ligand-binding properties and the interaction of the human 5-HTlA receptor with recombinant G-protein a subunits have been studied in

5-H T Receptor Subtypes

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Escherichia coli (Bertin et al., 1992). Among various a subunits, ai.)had the highest affinity for the human 5-HTIAreceptor and shifted the receptor to its high-affinity state. In contrast, a, was unable to interact with the receptor. These results are in good agreement with the work of Fargin et al. (1991), which demonstrated with antibodies directed against ai.2, and ai.3subunits of G-proteins that the 5-HTlAreceptor interacts preferentially with the ai-3subunit. A mitogenic effect for the 5-HTlAreceptor has been reported in NIH3T3 cells, in which activation of the 5-HTlAreceptor induces focus formation and potentiates the effect of epidermal growth factor (EGF) on DNA synthesis in a pertussis toxin-sensitive manner (Abdel-Baset et al., 1992; Varrault et al., 1992). In conclusion, the 5-HTIAreceptor is capable of interacting with multiple pathways depending on the cell type in which it is expressed. Although the inhibition of adenylate cyclase and the activation of K + channels by the 5-HT1, receptor have been well documented in uivo, further studies are needed to determine whether the other coupling mechanisms are of physiological relevance. c. Regional Distribution and SubcellularLocalization The localization of the mRNA encoding the 5-HT,, receptor has been studied in rat brain by Northern analysis and in situ hybridization (Table 11). The highest levels of 5-HTlAmRNA were detected in the .hippocampal formation [CAl-CA3, dentate gyrus (DG)], the entorhinal cortex, the raphe nuclei, the amygdala, and the lateral septum. Lower levels of mRNA were found in the olfactory bulb, the cerebral cortex, and some thalamic and hypothalamic nuclei (Albert et al., 1990; Chalmers and Watson, 1991; Miquel et al., 1991; Pompeiano et al., 1992). Autoradiographic studies using [3H]8-OH-DPATor '251-labeledBolton-Hunter-8-MeO[N-propyl-Npropylamino]tetralin (['251]BH-8-MeO-N-PAT)detected the protein in the same regions of the rat and human brain (Table 11; Marcinkiewicz et al., 1984; Pazos and Palacios, 1985; Hoyer et al., 1986; Verge et al., 1986; El Mestikawy et al., 1990; Chalmers and Watson, 1991; Miquel et al., 1991; Riad et al., 1991; Pompeiano et al., 1992). In addition, antibodies raised against the receptor allowed its visualization in the same brain regions (El Mestikawy et al., 1990; Miquel et al., 1991; Riad et al., 1991; Azmitia et al., 1992). The colocalization of mRNA and protein suggests that the 5HTIA receptor is expressed at the somatodendritic level rather than an axon terminals. This somatodendritic localization has been confirmed in the case of the raphe nuclei by Sotelo et al. (1990), who demonstrated that the 5-HT,, receptors are located on the perikarya and the dendrites of the raphe neurons. This result is in agreement with electrophysiological studies demonstrating that the 5-HTlAreceptors localized in the raphe

338

Frederic Saudou a n d Rene H e n

nuclei are involved in the inhibition of 5-HT neuronal firing (Vandermaelen et al., 1986). A developmental polymerase chain reaction (PCR) analysis revealed the presence of ~ - H T , transcripts A as soon as embryonic day 12 in the rat (Hillion et al., 1993). The highest levels were detected at embryonic days 14 and 15, which correspond to the time of differentiation of the target cells of serotonergic neurons, suggesting that the 5-HT,, receptor might be involved in the development of serotonergic neurons. Activation of 5-HT,, receptors located on astroglial cells induces the release of the protein S-100, which promotes the growth of serotonergic neurons in culture (Whitaker-Azmitia and Azmitia, 1989; Whitaker-Azmitia et al., 1990). Kobilka et ai. (1987) detected transcripts of the human receptor in the lymphoid tissues, suggesting a potential role for this serotonin receptor in the immune response. In good agreement, researchers have shown that ~-HT,A receptors might be involved in the regulation of cell contactmediated interaction between natural killer cells and monocytes (Hellstrand and Hermodsson, 1993).

2. The 5-HTIBand 5-HT1, Receptors The 5-HTIBreceptors are found in rat, mouse, hamster, and oppossum but are absent from all other mammalian species (Hoyer et al., 1985; Waeber et a/., I989a), whereas the 5-HTIDreceptors are absent from rodents but detected in dog, guinea pig, and humans with a distribution similar to that of 5-HT,, receptors. Researchers therefore postulated that 5-HTIBreceptors are the rodent homologs of 5-HTIDreceptors. This hypothesis has been confirmed by molecular cloning studies showing that the human counterpart of the rat 5-HT,, receptor is the ~ - H T , receptor D~ (for a review, see Hartig et a / . , 1992). Furthermore, investigators have shown (Metcalf et al., 1992; Oksenberg et al., 1992; Parker et al., 1993) that a single amino acid is responsible for the pharmacological difference between these two receptors. Therefore, the 5-HTlBand 5-HTlDoreceptors, which also have the same distribution and presumably the same function, will be presented together. a. Molecular Structure The SHT,, receptor has been cloned in the rat (Voigt et al., 1991; Adham et al., 1992) and in the mouse (Maroteaux et al., 1992) and the 5-HTIDo.receptorhas been cloned in humans (Demchyshyn et al., 1992; Hamblin et al., 1992b; Jin et al., 1992; Weinshank et a/., 1992). These receptors have been isolated by using oligonucleotides derived from consensus sequences of certain transmembrane domains. The receptor consists of a polypeptide chain of 390 amino acids in human (Demchyshyn et al., 1992; Hamblin et al., 1992b;Jin et al., 1992; Veldman

5-HT Receptor Subtypes

339

and Bienkowski, 1992; Weinshank et al., 1992) and of 386 amino acids in the rat and the mouse (Voigt et al., 1991; Adham et al., 1992; Maroteaux et al., 1992). Like the other receptors coupled to G-proteins, the receptor contains even putative transmembrane domains. There are no introns in the coding sequence, as in the 5-HTIAreceptor and the other members of the 5-HT, family (Table II), but there is at least one intron in the 5' noncoding sequence (S. Ramboz and R. Hen, unpublished observation). The amino acid sequence similarity between the human 5-HT,, receptor and the rodent ~ - H T Ireceptor B is high (96%) and is consistent with the fact that the 5-HTIBand 5-HT,Dpreceptors are species homologs. Only 32 amino acids are different in the human and rat receptors, and only 8 are located in the transmembrane domains. These domains are believed to constitute a ligand-binding pocket (Findlay and Eliopoulos, 1990;Hibert et al., 1991; Trump-Kallmeyer et al., 1992). In particular, the seventh transmembrane domain of the rodent 5-HT,, receptor contains an asparagine residue that has been suggested to be important for the binding of P-adrenergic antagonists such as pindolol derivatives (Guan et al., 1992). The 5-HT,, receptor, which has a low affinity for these p-antagonists, does not contain this asparagine residue but contains a threonine instead (Figs. 2 and 4). Replacement of Thr 355 of the human 5-HTlDpreceptor by an asparagine shows that this mutated receptor displays a high affinity forp-blockers, a characteristic of the 5-HTIBreceptor (Metcalf et al., 1992; Oksenberg et al., 1992;Parker et al., 1993). These studies demonstrate that the marked difference in pharmacological profiles of the 5-HTIBand 5HT,,, receptors is due to only one amino acid. The gene encoding the 5-HTlBlIDp receptor is localized on mouse chromosome 9 (position 9E) and on human chromosome 6 at the locus 6q13 (Table 11; Demchyshyn et al., 1992; Jin et al., 1992; Simon-Chazottes et af., 1993; Ramboz et al., 1994). b. Functional Expression The cDNAs encoding the rat and mouse 5HT1, receptors have been expressed in various cell lines (Voigt et al., 1991; Adham et al., 1992; Hamblin et al., 1992a; Maroteaux et af., 1992). Membranes prepared from these transfected cells display saturable binding of [3H]5-HTwith two affinity states, the high-affinity state corresponding to the receptor coupled to G-proteins since it is displaced by guanine nucleotides analogs. The pharmacological profile of the transfected receptor corresponds well with that of the rat ~ - H T , Breceptor (cyanopindolol > 5-CT = RU24969 > 5-HT > sumatriptan; Schoeffter and Hoyer, 1989). Ketanserin, mianserin, yohimbine, spiperone, and 8OH-DPAT have a low affinity for this receptor (Voigt et af.,1991; Harnblin et al., 1992a; Maroteaux et al., 1992). Similar results were obtained using the P-adrenergic antagonist [ '2SI]iodo-cyanopindololas a radioligand (Adham et d., 1992).

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Frederic Saudou a n d Rene H e n

Fig. 4 Seven-transmembrane-domain model of the mouse 5-HTIBreceptor. Solid circles indicate amino acids that are identical to corresponding positions in all 5-HT receptors. Squares indicate amino acids that are different from those in corresponding positions in the 5-HTID0receptor. Y indicates potential sites of N-linked glycosylation. The arrow indicates the asparagine involved in the binding of pindolol derivatives.

The 5-HT,, receptor (human homolog of the rat ~ - H T , Bhas ) also been expressed in mammalian cells (Demchyshyn et al., 1992; Hamblin et al., 1992b; Jin et al., 1992; Weinshank et a/., 1992). This receptor displays a pharmacological profile characteristic of the 5-HT1, subtype [5-CT > 5HT > sumatriptan > yohimbine > 5-methoxy-3-( 1,2,5,dtetrahydropyrid4-yl)-1H-indole (RU24969) > cyanopindolol > 8-OH-DPAT; (Schoeffter and Hoyer, 1989). Compounds such as pindolol derivatives and RU24969 have a lower affinity for the 5-HT,,, receptor than for the 5-HTIBreceptor. In contrast, the antimigraine drug sumatriptan exhibits a higher affinity for the 5-HT,,, receptor. When activated by agonists, the 5-HT,, and ~ - H T , Dreceptors , have been shown to inhibit adenylate cyclase (Adham et al., 1992; Hamblin et a / . , 1992a,b; Maroteaux et al., 1992; Weinshank et al., 1992). This inhibition is blocked by 5-HT,,,,, antagonists such as methiothepin. Moreover, the inhibition of adenylate cyclase is blocked by pertussis toxin, indicating that the 5-HT1, receptor is coupled to a pertussis toxin-sensitive G-protein (Maroteaux et al., 1992). These results are in good agreement with studies

5-HT Receptor Subtypes

34 1

reporting that the ~ - H T , and B ~-HT,D receptors are negatively coupled to adenylate cyclase in the rat substantia nigra (Boulehal et al., 1988; Schoeffter and Hoyer, 1989). There is no evidence for coupling of the 5-HTIBI receptor to phospholipase C (Adham et al., 1992). However, in uiuo the coupling of the receptor could be different since inhibition of 5-HT release resulting from the stimulation of 5-HTIBautoreceptors located on serotonergic terminals is not affected by CAMP (Blier, 1991). In CCL39 cells, the 5-HTl, receptor potentiates the effect of fibroblast growth factor (FGF) on DNA synthesis via a pertussis toxin-sensitive Gprotein (Seuwen et al., 1988). c. Regional Distribution and Subcellular Localization Northern analysis revealed a single 5-HTlBIlDp receptor mRNA of 5-6 kb in the brain, particularly in the striatum (caudate-putamen), the frontal cortex, the cerebellum, the hippocampus, the amygdala, and the medulla (Demchyshyn et al., 1992; Jin et at., 1992; Maroteaux et al., 1992). In situ hybridization confirmed this distribution within the brain. Hybridization signals are detected over medium spiny neurons in the striatum, and are restricted to the Purkinje cell layer in the cerebellum and to CA1 pyramidal neurons in the hippocampus (Voigt et al., 1991; Jin et al., 1992; Maroteaux et al., 1992). The S-HTIB mRNA is also found in layer IV of the entorhinal and cingulate cortices, subthalamic nucleus, nucleus of stria terminalis, nucleus accumbens, spinal cord, and retinal ganglion cells (Table 11; Voigt et al., 1991; Maroteaux etal., 1992; Boschert et al., 1993). During development, expression was detected in E l 7 embryos and in later stages with the same distribution as in the adult (Voigt et al., 1991). 5-HTlBand 5-HTl, binding sites have been localized in the basal ganglia (substantia nigra and globus pallidus), in the deep cerebellar nuclei, in the subiculum, and in the superior colliculi (Table 11) using the 5-HT,~-SpeCifiC radioligand ['2SI]iodo-cyanopindolol (Hoyer et a / ., 1985) or the 5-HTIBIlDspecific radioligand serotonin-O-carboxymethylglycyl[~2sI]tyrosinamide (S-CM-G[I2'I]-TNH2) (Boulenguez et al., 1991; Bruinvels et al., 1991; Segu et a!., 1991; Palacios et al., 1992; Boschert et al., 1993). The localizations of the mRNA and the protein are different, and the protein is localized in the projection zones of the neurons expressing the 5-HT1, mRNA. Researchers have therefore concluded that the 5-HT, receptor is localized predominantly on axon terminals (Hen, 1992; Boschert et al., 1993). This localization is in good agreement with lesion studies performed in the D striato-nigral loop of guinea pigs, in which a reduction of ~ - H T , binding sites in the substantia nigra was reported following quinolinic acid-induced lesions in the caudate-putamen (Waeber et al., 1990b). In postmortem brains of patients with Huntington's disease, which exhibit a degeneration of striatal neurons, the density of 5-HT1, receptors was significantly re-

342

Frederic Saudou a n d Rene H e n

duced in the substantia nigra (Waeber and Palacios, 1989). In keeping with their localization, 5-HTlBreceptors are involved in the inhibition of neurotransmitter release from nerve terminals. For example, stimulation of 5-HTlBautoreceptors triggers an inhibition of 5-HT release in rat cortex (Engel et al., 1986; Hoyer and Middlemiss, 1989; Limberger et al., 1991). In the rat hippocampus, ~ - H T ,receptors B located on cholinergic terminals inhibit acetylcholine release (Maura and Raiteri, 1986). Similarly, S-HTIB receptors have been suggested to inhibit the release of the vasoactive neuropeptide CGRP (calcitonin gene-related peptide) from trigeminal nerve endings in the rat dura mater (Buzzi et al., 1991). This result is supported by the finding that S-HT,B mRNA was found in trigeminal ganglion neurons (Bruinvels et a/., 1992). 5-HTID/,receptor localized in the dura mater has been proposed to be important in the pathology of migraine since the antimigraine drug sumatriptan is an agonist of 5-HT,B, receptors. In particular, stimulation of these receptors by sumatriptan might lead to the inhibition of CGRP release from trigeminal nerve endings (Buzzi and Moskowitz, 1991). 5-HT,,, mRNA has also been detected in the blood vessels themselves (Bruinvels et al., 1994).

3. The 5-HTlD, Receptor Researchers first reported that 5-HTl, binding sites were not present in rodents. However, the existence of “5-HTl,-like” sites has been suggested in several species including rats (Herrick-Davis and Titeler, 1988; Limberger et al., 1991; Mahle et al., 1991; Peroutka, 1991a; Beer et al., 1992). The molecular cloning of the 5-HT,,, receptor that displays a 5HTI, pharmacological profile in several species including the rat and the mouse demonstrated the existence of 5-HTlDsites in rodents. a. Molecular Structure In 1989, Libert et al. reported the cloning of four new members of the G-protein-coupled receptor family from a canine thyroid cDNA library. One of these receptors, called RDC4, exhibited 43% sequence identity to the 5-HTIAreceptor; researchers therefore suggested that this receptor was a serotonin receptor. The RDC4 receptor was later shown to encode a 5-HT receptor with a 5-HTl, pharmacological profile (Maenhaut et al., 1991; Zgombick et al., 1991). RDC4 is an intronless gene encoding a protein of 377 amino acids. The species homologs of RDC4 have also been cloned in human (Hamblin and Metcalf, 1991; Weinshank et al., 1992), in rat (Voigt et al., 1991; Hamblin et al., 1992a; Bach et al., 1993), and in mouse (Maroteaux et al., 1992). This receptor displays a high homology (74-77% in the transmembrane domains) to the 5-HTIBIIDP receptor (Fig. 3; Hartig et a / . , 1992) and was therefore named 5-HTlD,. Despite the high homology, the genes encoding these two receptors are not located on the same chromosome since the ~-HT,D,subtype

5-HT Receptor Subtypes

343

has been mapped in humans to the locus 1~34.3-36.3(Libert et al., 1991). The mouse homolog of S-HTID,has been mapped onto chromosome 4 (Weydert et al., 1992), which is syntenic to the region of human chromosome 1 carrying the 5-HTlD, receptor gene (Table 11). b. Functional Expression When expressed in mammalian cells, the human and canine 5-HTlD, receptors as well as the rat homolog display a pharmacological profile characteristic of the 5-HTlDtype receptor (Hamblin and Metcalf, 1991; Maenhaut et al., 1991; Zgombick et al., 1991; Hamblin et al., 1992a; Weinshank et al., 1992; Bach et al., 1993). Furthermore, this profile matches closely that of the 5-HTI, receptor when expressed under the same conditions (5-CT > 5-HT > sumatriptan > yohimbine > RU24969 > 8-OH-DPAT > spiperone; Weinshank et al., 1992). Interestingly, ketanserin and ritanserin, two 5-HT2 antagonists, displayed a high affinity for the rat 5-HTlD, receptor in one report (Bach et al., 1993). Stimulation of the ~ - H T , Dreceptor, , like that Of S-HT,, receptor, yields inhibition of adenylate cyclase in mammalian cells transfected with the corresponding genes (Hamblin and Metcalf, 1991; Zgombick et al., 1991; Hamblin et al., 1992a; Weinshank et al., 1992). This inhibition is blocked by the nonselective antagonist methiothepin. Treatment of the cells by pertussis toxin abolished the inhibition, indicating that the ~ - H T , Drecep, tor is coupled to a pertussis toxin-sensitive G-protein (Hamblin and Metcalf, 1991). Like 5-HTl,, the 5-HTlD,receptor does not seem to couple to phospholipase C in Lmtk- cells since activation of the receptor does not produce any modifications in phosphatidylinositol metabolism (Weinshank et al., 1992). c. Regional Distribution Libert et al. (1989)performed Northern analysis using the canine 5-HTlD, receptor gene as a probe, but they could not detect any mRNAs among the various tissues tested, leading to the suggestion that the 5-HTlD,receptor mRNA might be present at very low levels. In situ hybridization experiments performed on rat brain revealed that the ~ - H T , DmRNA , is expressed in the pyramidal cell layer of the olfactory tubercle, the striatum, the nucleus accumbens, the dorsal raphe nuclei, the lateral mamillary bodies, and the pyramidal and granule cells of the hippocampus (Hamblin et al., 1992a; Bach et al., 1993) whereas no signal was detected in the globus pallidus or substantia nigra. Autoradiographic studies performed on mouse and rat brains using the ~-HT,,,,Dspecific radioligand S-CM-G['251]-TNH2(Boulenguez et al., 1991; Segu et al., 1991)in the presence of 100 nM 34 1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP93129) to block the 5-HTl, binding sites revealed that 5-HTlD-likebinding sites are present in the globus pallidus, ventral pallidum, caudate-putamen, substantia nigra, and subthalamic

344

Frederic Saudou and Rene Hen

nuclei (Boschert et al., 1993; Bruinvels et af., 1993b). This distribution is similar to that of the S-HT,, binding sites (Boulenguez et al., 1991; Bruinvels et al., 1991,1993b; Segu et al., 1991; Palacios et al., 1992; Boschert et al., 1993) except for certain regions such as the cerebellum from which 5-HT,, sites appear to be absent (Table 11). However, the S-HT,, binding sites have a much lower density than the S-HTIBbinding sites (Boschert et al., 1993; Bruinvels etal., 1993b),and represent only a minor component of the S-CM-G['251]-TNH2binding in rodent brains. As in the case of the ~ - H T I B Ireceptor, ID~ the comparison between the pattern of expression of the mRNA and the pattern of expression of the protein suggests that the 5-HTlD,receptor might also be localized predominantly on axon terminals (Boschert et af., 1993).

4. The 5-HTlEReceptor The 5-HT,, receptors correspond to 5-HTl-like receptors with a low affinity for 5-CT that have been reported in the brains of various mammalian species (Leonhardt et af., 1989; Weisberg and Teitler, 1992). a. Molecular Cloning The 5-HT,, receptor has been isolated from a human genomic DNA library employing a low stringency screening approach with oligonucleotides derived from the human 5-HT,, receptor and the rat 5-HTIc receptor (Levy et al., 1992). This receptor, which was E first named S3 1 (Levy et al., 1992),was later identified as a ~ - H T Ireceptor (McAllister et al., 1992; Zgombick et al., 1992; Gudermann et al., 1993). The human 5-HTI, receptor gene contains an open reading frame of 1095 nucleotides that encodes a protein of 365 amino acids. Like the other members of the 5-HTl family, this receptor does not contain any introns in the coding sequence (Table 11). The S-HT,, receptor displays a higher degree of homology to the 5HTID,and 5-HT,,p receptor subtypes (64%) than to other 5-HT receptors (Fig. 3). b. Functional Expression The human ~ - H T , receptor E was expressed stably in Lmtk- cells. Membranes prepared from these murine fibroblasts reveal a high-affinity binding site for [3H]5-HT (Kd = 9.7 a). Addition of Gpp(NH)p, a nonhydrolyzable analog of GTP, shifts the receptor into a low-affinity state, suggesting that this receptor is coupled to G-proteins. The pharmacological profile of this receptor (5-HT > methysergide > ergotamine > sumatriptan > spiperone) corresponds to that of S-HT~E binding sites (Leonhardt et al., 1989; McAllister et al., 1992; Weisberg and Teitler, 1992; Zgombick et al., 1992; Gudermann ef ul., 1993). In particular, sumatriptan and 5-CT have a lower affinity for 5-HTI, receptors than for S-HT,, receptors.

5-HT Receptor Subtypes

345

The ~ - H T , E receptor is negatively coupled to adenylate cyclase in transfected Lmtk- cells (Levy et al., 1992; Gudermann et al., 1993), Y1 cells (Zgombick et at., 1992), and HEK 293 cells (McAllister ef al., 1992). In all the cell lines used, adenylate cyclase inhibition produced by 5-HT,, receptor stimulation is weak (30-35%) compared with that resulting from the stimulation of other "5-HT," receptors such as the human and rat 5-HT,, receptor (Fargin et al., 1989; Albert et af., 1990) or the mouse 5-HT,, receptor (Maroteaux et af., 1992) (50-60% of inhibition). This result may reflect a less efficient coupling of the SHT,, receptor to G-proteins in these cells and may indicate that this receptor interacts with different G-proteins in vivo. In cells expressing the S-HT,, receptor, 5HT did not stimulate phospholipase C and did not affect intracellular Ca2' concentration (Gudermann et al., 1993). c. Regional Distribution In situ hybridization experiments performed on human and monkey brains revealed that 5-HTIEreceptor transcripts are detected in cortical areas, caudate, and putamen (Bruinvels et al., 1994). Furthermore, autoradiographic studies based on the pharmacological properties of the 5-HT,, receptor led Bruinvels and collaborators (1993a) to examine the distribution in the rat brain of ['HIS-HT binding sites insensitive to 5-CT, sumatriptan, and S-CM-G['2SI]-TNH2.These sites were found in amygdala, caudate-putamen, frontoparietal motor cortex, and olfactory tubercle, all of which are regions in which the 5HTIE receptor mRNAs are detected and might therefore correspond to the 5-HTIEreceptor (Table 11).

5. The 5-HTlFReceptor, Also Called 5-HTIE, a. Molecular Structure Amlaiky et al. (1992) isolated a mouse 5-HT1,like receptor by screening a mouse brain cDNA library at low stringency using the mouse S-HT,, receptor gene (Maroteaux et af., 1992). Sequence analysis of the cDNA revealed on long open reading frame (367 amino acids) and a poly(A) tail. Amino acid sequence comparisons revealed that the highest percentages of homology were to the 5-HTIEreceptor (61%) and the SHT,, and 5-HTID receptors (54%). Pharmacological studies demonstrated that this receptor had a low affinity for 5-CT and resembled the 5-HTIEreceptor (Leonhardt et a / . , 1989; Weisberg and Teitler, 1992). The human and rat homologs of the mouse 5-HT,, receptor have been cloned (Adham et al., 1993; Lovenberg et al., 1993b) and consist of a polypeptide chain of 366 amino acids in both species (Table 11). The 5-HT,, gene does not contain any introns in the coding sequence but contains at least one intron in the 5' noncoding sequence (Lovenberg et al., 1993b). The gene is located on mouse chromosome 16 (position

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Frederic Saudou and Rene Hen

16C2-16C4) and on human chromosome 3 (locus 3pll) (Ramboz et al., 1994). b. Functional Expression Expression of the rat, mouse, and human receptor in various cell lines has allowed the establishment of its pharmacological profile. [1251]-Labeledlysergic and diethylamide (LSD) and [3H15HT bound to a single high-affinity site (Amlaiky et al., 1992; Adham et al., 1993; Lovenberg et al., 1993b). The rank order of potencies of various serotonergic drugs in displacing these ligands was 5HT > sumatriptan > methysergide > yohimbine > ergotamine. Ketanserin, cyanopindolol, and 5-CT displayed a low affinity (Amlaiky et al., 1992;Adham et al., 1993; Lovenberg et al., 1993b). This profile resembles that of 5-HTIEsites that have previously been found in human and rat brain (Leonhardt et al., 1989; Weisberg and Teitler, 1992). However, yohimbine and sumatriptan have a higher affinity for the 5-HTl, receptor than for the ~ - H T , receptor E (Amlaiky et al., 1992; McAllister et al., 1992; Zgombick et al., 1992; Adham et al., 1993; Lovenberg et al., 1993b). The fact that sumatriptan has a high affinity for the 5-HTlFreceptor suggests that this receptor might also be a site of action for this drug (Amlaiky et al., 1992; Adham et al., 1993; Lovenberg et al., 1993b). In NIH-3T3 and HeLa cells expressing the 5-HTlFreceptor, serotonin induced a decrease in forskolin-stimulated adenylate cyclase activity that was dose dependent and saturable (EC, = 7 nM), indicating that this receptor is negatively coupled to adenylate cyclase (Amlaiky et al., 1992; Adham et al., 1993). There is no coupling to phospholipase C in NIH3T3 cells (Adham et al., 1993). c. Regional Distribution Quantitative PCR allowed the analysis 5HTIF mRNA levels in various tissues. Specific PCR fragments could be amplified from spinal cord and brain RNAs (Amlaiky et al., 1992; Lovenberg et al., 1993b). Using the same method, Adham et al. (1993) detected transcripts in the human brain but also in uterus (endometrium and myometrium) and mesentery. By in situ hybridization experiments, 5-HTlFtranscripts were detected in CAI-CA3 layers of the hippocampus. Expression was also found in lamina V of frontal cortex and in the dorsal raphe nuclei (Table 11; Amlaiky et al., 1992; Adham et al., 1993).

B. The 5-HT2 Family-5-HT Phospholipase C

Receptors Coupled to

This family contains three receptors with striking amino acid sequence homology (Fig. 3) and the same coupling to second messengers, that is, activation of phospholipase C (Fig. 1). According to the new nomenclature (Hoyer et al., 1994),these receptors are referred to as 5-HT,,, correspond-

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347

ing to the classical 5-HT2receptor; 5-HT2,, corresponding to the stomach fundus 5-HT2-likereceptor; and 5-HT2,, corresponding to the previous 5HT,, receptor.

1 . The 5-HT2, Receptor a. Molecular Structure The 5-HT,, receptor was first cloned in the rat (Pritchett er al., 1988; Julius et al., 1990) by homology to the rat 5HT,, receptor (Julius er al., 1988). The receptor encodes a protein of 471 amino acids in the rat (Julius et al., 1990), the mouse (Foguet et al., 1992b), the hamster (Chambard et al., 1990), and human (Saltzman et al., 1991) and belongs to the G-protein-coupled receptor family. In contrast to receptors negatively coupled to adenylate cyclase, the 5-HT,, receptor, like the other receptors positively coupled to phospholipase C or adenylate cyclase, possesses a long C-terminal tail (Fig. 2). Analysis of the 5-HT2, receptor gene revealed the presence of two introns (Chen et al., 1992;Foguet et al., 1992b; Yang et al., 1992).Interestingly, the position of these two introns is conserved among the 5-HT2,, 5-HT2,, and 5-HT,c receptors (Foguet et al., 1992b). The S-HT~A gene has been mapped onto human chromosome 13 at position 13q14-q21 and onto mouse chromosome 14 (Table 111; Hsieh et al., 1990; Liu et al., 1991; Sparkes et al., 1991). b. Functional Expression Expression of the rat 5-HT2, receptor in different mammalian cell lines such as HEK 293 (Pritchett et al., 1988) and NIH-3T3 (Julius et al., 1990) revealed that it encodes a functional 5-HT receptor with binding properties similar to those of the SHT, subtype. The 5-HT2, receptor displays a high affinity for [12’I]LSD (Kd = 1.6nM)and [’Hlspiperone (Kd = 0.5 nM). [12’I]LSDbinding was displaced efficiently by ketanserin and mianserin, which are 5HT, antagonists; the pharmacological profile was ketanserin = DO1 > mesulergine > 5-HT. Expression of the cloned 5-HT2, receptor in NIH-3T3, Lmtk-, and cos-7 cells revealed the presence of [3H]ketanserin binding sites as well as [’HI( +)-1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane hydrobromide ([’HIDOB) and 12’I-labeled(~)-l-(2,5-dimethoxy-4-iodophenyl)-2aminopropane hydrochloride ([I2’I]DOI)binding sites. The DO1 and DOB binding sites represented only 10% of the ketanserin binding sites and were GTP sensitive (Branchek et al., 1990; Teitler et al., 1990). The fact that the introduction into a cell line of the cloned receptor generated both types of sites suggests that they correspond to two affinity states of the same receptor rather than two distinct receptors, as had been proposed by Pierce and Peroutka (1989). Activation of the cloned 5-HT2, receptor in various cell lines leads to accumulation of inositol phosphates (Pritchett et al., 1988; Van

Table 111 5-HTz Receptors Receptor

Species

Amino acids

Locus

Introns in coding sequence

mRNA size (kb)

mRNA regional distribution (main sites)

Binding site distribution (main sites)

S-HTzA

Human Rat Mouse Hamster Rat Mouse

47 1 47 1 47 1 471 479 504

13q14-q21

2

4.5 5.2-6.5 5-6 6.4 2.3

Identical to mRNA distribution

Human Rat Mouse

458 460 459

Cerebral cortex, hippocampus, striatum, spinal cord, olfactory bulb Stomach fundus, intestine, heart, kidney, lung. brain Choroid plexus, medulla pons, striatum, hippocampus, (CAICA3), hypothalamus, spinal cord

5-HT2, 5-HTzc

14

Xq24

5.2 X D-X F4

Stomach fundus Identical to mRNA distribution

349

5-HT Receptor Subtypes

Obberghen-Schilling et al., 1991; Yang et al., 1992) and subsequently to Ca2+ release from inositol triphosphate-sensitive intracellular stores (Pritchett et al., 1988; Julius et al., 1990; Van Obberghen-Schilling et al., 1991). When expressed in Xenopus oocytes, activation of the 5-HT2, receptor leads to opening of Ca2 -sensitive chloride channels subsequent to the release of Ca2+from intracellular stores (Pritchett e t al., 1988). Deletion of the N-terminal tail of the rat ~-HT,Areceptor did not modify the activity of the receptor in Xenopus oocytes, suggesting that this region and/or the N-glycosylated residues present in this region are not necessary for the binding and the coupling of the receptor to second messengers (Buck et al., 1991). Similarly, deletions of the C-terminal part had little effect on receptor activity. However, replacement of Cys 397 produced a complete loss of activity (Buck et al., 1991). This result is in agreement with the fact that, in the case of the P2-adrenergic receptor (O'Dowd et al., 1989a), this cysteine (which is highly conserved among G-proteincoupled receptors) is palmitoylated and its replacement by mutagenesis resulted in a partial loss of coupling activity. Within transmembrane domain V, replacement of Ser 242 found in the human 5-HT2, receptor by an alanine which is found in the rat receptor (Fig. 2) resulted in an increased affinity for mesulergine (Kao et al., 1992), which is characteristic of the rat receptor. Within the sixth transmembrane domain, replacement of Phe 340 by a leucine has been shown to decrease the affinity of [3H]mesulergine, ['251]DOI,and [3H]5-HT for the 5-HT2, receptor whereas replacement of Phen 339 by a leucine decreased only the affinity for [3H]ketanserine (Choudhary et al., 1993). This finding is in good agreement with the model proposed by Hibert et al. (1991), suggesting that Phe 340 but not 339 is involved in anchoring the aromatic ring of 5-HT to the receptor. Like the 5-HT2, receptor, the 5-HT2, receptor when expressed in NIH3T3 cells leads to cellular transformation (Julius et al., 1990), indicating a tight link between phospholipase C signaling pathways and cellular transformation in these fibroblasts. Interestingly, when expressed in CCL39 cells, the 5-HT2, receptor had no such properties (Van ObberghenSchilling et al., 1991). In contrast, in these cells, a5-HT-inducible mitogenesis is mediated by 5-HT,, receptors via a pertussis toxin-sensitive Gprotein (Seuwen et al., 1988). In rat aortic smooth muscle cells, 5-HT has a weak mitogenic effect by activating 5-HT, receptors (Corson et al., 1992). c. Regional Distribution Northern analysis and in situ hybridization experiments revealed expression of the 5-HT2, receptor mRNA in the brain. The 5-HT2, receptor mRNA is detected in the frontal cortex, lamina V of the neocortex, hippocampus, striatum, nucleus accumbens, olfactory bulb, and spinal cord (Julius el al., 1990; Mengod et al., 1990b). The +

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Frederic Saudou and Rene Hen

distribution of 5-HT2, binding sites determined using [ 1251]DOI, [3H]ketanserin, [3H]mesulergine, [3H]LSD, and [3H]spiperoneas radioligands is comparable to the distribution of 5-HT,, receptor mRNA (Pazos et al., 1985; Mengod et al., 1990b), suggesting a somatodendritic localization of the protein (Table 111). 5-HTzAreceptor mRNA and binding sites are also found in the periphery (Bradley et al., 1986). For example, S-HT~A mRNA is found in aorta and in cultured rat aortic and uterine smooth muscle cells (Corson et al., 1992; Rydelek-Fitzgerald et al., 1993). In uterine smooth muscle cells, 5-HT has been shown to increase 5-HT2, mRNA levels (Rydelek-Fitzgerald et al., 1993). In rats, 5-HT2, mRNAs were found at low levels in late embryonic stages (embryonic day 17) and increased postnatally until day 13 (Roth et al., 1991).

2. The 5-HTZBReceptor The 5-HTzBreceptor was first described in the rat stomach fundus where serotonin mediates a contractile activity that was used as a bioassay for serotonin (Vane, 1957). a. Molecular Structure The 5-HTzBreceptor has been isolated in the rat (Foguet et al., 1992a; Kursar et al., 1992) and the mouse (Foguet et al., 1992b; Loric et al., 1992). This receptor consists of a polypeptidechain of 479 amino acids in the rat (Foguet et al., 1992a; Kursar et al., 1992) and 504 amino acids in the mouse (Loric et al., 1992). Although the difference in length between the mouse and the rat receptors is important, these receptors seem to correspond to species homologs since the difference is localized in the C-terminal part where a point mutation appears to be responsible for an increase in the size of the open reading frame of the mouse receptor (Table 111). The ~ - H T z receptor B amino acid sequence exhibits a high degree of homology to the 5-HT,, and 5-HTzcreceptors (45 and 5 1%, respectively). Analysis of the genomic structure reveals that the 5-HT2, receptor gene possesses the same intron-exon boundaries as the genes encoding the 5HT,, and 5-HT2, receptors. b. Functional Expression The 5-HTZBreceptor, when expressed in mammalian cells, displays a high-affinity binding for [3H]5-HT(Kd = 7.9 nM) (Foguet et al., 1992a; Kursar et al., 1992; Wainscott et al., 1993) and ['251]DOI(Kd = 25.8 nM) (Loric et al., 1992). Its pharmacological profile [ritanserin > 5-HT > 5-HT > 1-(3-chlorophenyl)piperazine hydrochloride (m-CPP) > ketanserin > mianserin] was consistant with its being a member of the 5-HT2family. However this receptor can be distinguished from the 5-HT,, and 5-HT,c receptors since ketanserin and spiper-

5-HT Receptor Subtypes

35 1

one have a higher affinity for the 5-HT,, receptor than for the 5-HT2, receptor. Mianserin, which has a high affinity for the 5-HT2, and 5-HTzc receptors, has a low affinity for the 5-HT2, receptor. Yohimbine has a higher affinity for the ~-HT,Breceptor than for the two other members of the 5-HT2 family. This pharmacological profile correlates well with the functional characteristics of the contractile 5-HT receptor of the rat stomach fundus (Kalkman and Fozard, 1991). Activation of the 5-HT2, receptor leads to the accumulation of inositol phosphates in transfected cells (Kursar et al., 1992; Wainscott et al., 1993). Whereas 5-HT is a full agonist, rn-trifluoromethylphenylpiperazine hydrochloride (TFMPP) and quipazine are only partial agonists. Phospholipase C stimulation can be antagonized by mianserin and methysergide. In Xenopus oocytes, activation causes the opening of Ca2+-sensitivechloride channels and is blocked by yohimbine (Foguet et al., 1992a). c. Regional Distribution A single mRNA of 2.3 kb encoding the 5HT,, receptor is detected in rat stomach fundus (Kursar e f a f . , 1992). By quantitative PCR experiments, the 5-HT2, receptor transcripts are also detected in heart, intestine, and brain (Table 111; Foguet et af., 1992a; Loric et al., 1992). During development, the 5-HT2, mRNAs are present in rat and mouse embryos at embryonic day 9 (Foguet et al., 1992a; Loric et a / . , 1992).

3. The 5-HT2, Receptor The 5-HT2c receptor was first described in the choroid plexus using 5HT, receptor radioligands (Pazos et al., 1984). Because of the high affinity of this receptor for serotonin, it was initially classified in the 5-HT, family and named 5-HT,,. However, analysis of its pharmacological profile and coupling with seconds messengers, that is, activation of phospholipase C, and later of its amino acid sequence, revealed that this receptor belongs to the 5-HT2 family. It has therefore been renamed 5-HT,, (Hoyer et al., 1994). a. Molecular Structure The 5-HT2creceptor was first isolated by functional expression in Xenopus oocytes of RNAs prepared from rat choroid plexus (Lubbert et al., 1987; Julius et al., 1988). The 5-HT2, receptor consists of a polypeptide chain of 458 amino acids in human (Saltzman et a / . , 1991), 460 amino acids in rat (Julius et al., 1988), and 459 amino acids in mouse (Table 111; Yu et al., 1991). In contrast to the other G-protein-coupled receptors, the 5-HTzc receptor appears to possess an eighth hydrophobic domain in the N-terminal part of the protein. This unusual feature has also been observed in Drosophila receptors (see Section II,E, I).

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Frederic Saudou and Rene Hen

The 5-HTzc receptor possesses three introns in its coding sequence. The second and third exons are located at the same positions as they are in the S-HT,, and 5-HTZBreceptors (Foguet et ai., 1992b). The gene encoding the 5-HTZc receptor is located in the mouse on chromosome X at position XD-F4 and in human on chromosome X at position Xq24 (Yu et al., 1991; Milatovich et al., 1992). Promoter sequences of the mouse 5-HTZc receptor gene have been identified (Bloem et at., 1993). The sequence located upstream of the transcription initiation site contains a potential TATA box found in most eukaryotic promoters and binding sites for the transcription factors AP 1 and AP2. b. Functional Expression In mammalian cells such as NIH-3T3 cells (Julius et al., 1988),the cloned 5-HT2creceptor displays a pharmacological profile characteristic of the 5-HT,, binding sites characterized in choroid plexus (ritanserin = mianserin = mesulergine > 5-HT = ketanserin >> spiperone; Conn ef al., 1986). When activated, the 5-HTzc receptor stimulates phospholipase C, leading to the liberation of Ca2+from intracellular stores (Julius et al., 1988). In Xenopus oocytes, this increase in intracellular CaZ+levels triggers the opening of Ca2+-sensitivechloride channels, resulting in an inward current. This 5-HT-evoked current can be blocked by mianserin (Julius er al., 1988; Yu et al., 1991). When the 5-HTZc receptor mRNA was coinjected with mRNAs isolated from rat cerebral cortex, activation of the expressed 5-HTZc receptor resulted in the closing of K + channels in a Ca2+-independent manner (Panicker et al., 1991). The 5-HTzC-evoked increase of intracellular Ca2+ can cause the activation of the a isoform of protein kinase C in cos-7 cells (Lutz et al., 1993), and the activation of Ca2+-sensitivepotassium channels in mouse fibroblasts A9 cells (Boddeke e? al., 1993). As in the case of the 5-HT2, receptor, activation of the 5-HTZcreceptor induces mitogenesis and cellular transformation at high frequency in NIH3T3 cells. When injected into nude mice, transformed cells expressing the 5-HT,, receptor generate tumors. Like the ~ - H T receptor, ~A the 5-HT,, receptor has no mitogenic properties in a different cellular environment such as Chinese hamster lung CCL39 cells (Kahan et al., 1992), where serotonin has been shown to have mitogenic properties via 5-HT,, receptors (Seuwen et al., 1988). c. Regional Distribution A single 5-HTZcreceptor mRNA of 5.2 kb is detected in rat brain (Julius et al., 1988; Mengod et al., 1990a; Roth et al., 19911. Northern analysis and in situ hybridization experiments indicate that 5-HTZc mRNA is found at high levels in the choroid plexus and at lower intensities in the anterior olfactory nucleus, lateral habenula,

5-HT Receptor Subtypes

353

hippocampus, amygdala, cingulate cortex, striatum, subthalamic nuclei, substantia nigra, suprachiasmatic nuclei, and spinal cord (Julius et al., 1988; Hoffman and Mezey, 1989; Molineaux et al., 1989; Mengod et al., 1990a; Roth et al., 1991; Roca et al., 1993). Autoradiographic studies detect 5-HT,, binding sites in the same regions (Pazos and Palacios, 1985; Mengod et al., 1990a), the region expressing the highest levels of binding sites being the choroid plexus (Table 111). 5-HT,c mRNA is present in small amounts at embryonic days 17 and 19 and increases in the immediate postnatal period (Roth et al., 1991).

C. 5-HTReceptors Positively Coupled to Adenylate Cyclase 1 . 5-HT4 Receptor There are no molecular data available on the 5-HT4 receptor since it has not yet been cloned. However, the identity of this receptor is well established now. (For a review, see Bockaert et al., 1992.) Stimulation of the 5-HT, receptor has been found to activate adenylate cyclase in cultured rat colliculus neurons (Dumuis et al., 1988a), in the guinea pig hippocampus (Bockaert et al., 1990), in the guinea pig ileum (Craig and Clarke, 1990), and in the tunica muscularis mucosae preparation of the rat esophagus (Baxter et al., 1991; Reeves et al., 1991). The resulting increase in CAMP production leads to protein kinase A activation (Kaumann et al., 1991; Fagni et al., 1992; Ouadid et al., 1992). In colliculus neurons researchers have proposed that, as in the case of Aplysia 5-HT receptors, the activation of the 5-HT4receptor closes K+ channels causing a prolonged depolarization, an increased opening of voltage-sensitive Ca” channels, and an increase in neurotransmitter release (Dumuis et al., 1988a,1991; Fagni et al., 1992). In cardiac muscle, voltage-sensitive Ca2+ channels are activated, resulting in an increase of muscle contraction (Kaumann, 1990; Kaumann et al., 1991; Ouadid et al., 1992), whereas a relaxation of smooth muscle is observed in esophagus in response to 5HT, ligands (Baxter et al., 1991; Reeves et al., 1991; Ford et al., 1992). The pharmacological profile of the 5-HT4 receptor is GR113808 > 2-methoxy-4-amino-5-chlorobenzoicacid 2-(diethylamino)ethyl ester (SDZ205557) > (endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro3-isoprop yl-2-0x0- I H-benzimidazol- 1-carboxamide hydrochlo ride (BIMU8) > cisapride > 5-HT > 5-methoxytryptamine (5-MeOT) (Bockaert et al., 1992). Agonists belong to the benzamide family (e.g., cisapride, renzapride, zacopride, metoclopramide) and to benzimidazolones (BIMU8). The synthesis of tritiated GR113808, a potent and selective 5-HT4antagonist, allowed autoradiographic studies in guinea pig, rat, and

354

Frederic Saudou and Rene Hen

human brain and revealed the presence of 5-HT4binding sites in striatum, globus pallidus, substantia nigra, olfactory tubercle, and hippocampus (Grossman et al., 1993; Waeber et af., 1993). Molecular cloning of the 5HT, receptor should give more information about its pharmacological profile, coupling mechanisms, and tissue distribution.

2. The 5-HT, Receptor a. Molecular Structure The 5-HT, receptor (Table IV) was first isolated by PCR amplification from rat striatal mRNA. This receptor consists of a polypeptide chain of 437 (Monsma et af., 1993) or 436 (Ruat et al., 1993a) amino acids. The two published amino acid sequences differ in their C-terminal tail. However, the nucleotide sequences are identical except for one nucleotide, which is absent from one of the sequences resulting in a frameshift. The 5-HT6 receptor contains seven hydrophobic regions and is distant from all other 5-HT receptors, as seen in the dendrogram (Fig. 3). The third cytoplasmic loop of the 5-HT6 receptor is short compared with those of the other serotonin receptors, whereas the Cterminal tail is long (Fig. 2). These characteristics are also observed in receptors such as the 5-HTdrolor the 5-HT2receptor that stimulate adenylate cyclase or phospholipase C activity. Both groups reported the presence of at least one intron in the coding sequence of the receptor (Monsma et al., 1993; Ruat et al., 1993a). All these molecular characteristics suggest that the 5-HT6receptor corresponds to a new subclass of serotonin receptor. b. Functional Expression The 5-HT, receptor was expressed in Cos7 cells and displayed a high affinity for the serotonergic ligand [1251]LSD (& = 1.26 nM). The pharmacological profile of the receptor (methiothepin > clozapine > 2-bromo-LSD > ritanserin > 5-HT > 5CT) did not correspond to those of any of the previously described serotonin receptors except a serotonin receptor positively coupled to adenylate cyclase in the NCB-20 neuroblastoma cell line (Conner and Mansour, 1990). Ergoline derivatives such as LSD and lisuride displayed high affinity for the 5-HT6 receptor. Interestingly, atypical and typical antipsychotic drugs such as clozapine and loxapine as well as tricyclic antidepressant drugs (amoxapine and clomipramine) exhibited relatively high affinities for the 5-HT6 receptor. This results suggests that this receptor could be a target for these psychotropic drugs. Activation of the receptor in HEK-293 cells or cos-7 cells resulted in a stimulation of adenylate cyclase (Monsma et al., 1993; Ruat et al., 1993a). In this functional assay, lisuride and dihydroergocriptine were

5-HT Receptor Subtypes

355

partial agonists whereas amoxapine, methiothepin, and clozapine were antagonists. c. Regional Distribution Northern analysis of poly(A + ) RNA from various tissues revealed that a 4.2-kb transcript corresponding to the receptor is found predominantly in brain. 5-HT6 mRNA is detected in corpus striatum, olfactory tubercle, nucleus accumbens, cerebral cortex, and hippocampus (CA1-CA3, dentate gyrus) (Table IV; Monsma et al., 1993; Ruat et al., 1993a).

3. The 5-HT7 Receptor a. Molecular Structure This 5-HT receptor (Table IV) positively coupled to adenylate cyclase has been cloned in human (Bard et al., 1993, in rat (Lovenberg et al., 1993a; Meyerhof et al., 1993; Ruat et al., 1993b; Shen et al., 1993), and in mouse (Plassat et al., 1993), and consists of a polypeptide chain of 448 amino acids. The 5-HT, receptor is most homologous to the 5-HTdro,receptor that also activates adenylate cyclase (see Fig. 3) but is a distant relative of all the other 5-HT receptors. Like the 5-HTdr0,and 5-HT6 receptors, the 5-HT7 receptor possesses a long Cterminal tail (Fig. 2; Meyerhof et al., 1993; Plassat et al., 1993; Ruat er al., 1993b; Shen et al., 1993) and contains at least one intron in its coding sequence (Ruat et al., 1993b; Shen et al., 1993). b. Functional Expression When expressed in mammalian cells, the 5HT, receptor displays a high affinity for [3H]5-HT (& = 3.6 nM) and [‘*’I]LSD (Kd = 1.2 nM) with the following unique pharmacological profile: 5-CT > methiothepin > 5-HT > clozapine > 8-OH-DPAT (Lovenberg et al., 1993a; Meyerhof et al., 1993; Plassat et al., 1993; Ruat et al., 1993b; Shen et al., 1993). This pharmacological profile might correspond to that of some of the 5-CT-sensitive sites reported in mammalian brain (Mahle et al., 1991) and to those of “5-HTI-like” receptors positively coupled to adenylate cyclase in the cardiovascular and gastrointestinal systems (Saxena et al., 1985; Connor et al., 1986). Furthermore, because of the affinity of the 5-HT, receptor for 8-OH-DPAT, this receptor might correspond to 5-HT,,-like receptor positively coupled to adenylate cyclase (Shenker et al., 1985; Markstein et al., 1986; Fayolle et al., 1988). Such receptors have been suggested to play a role in circadian rhythms (Lovenberg et al., 1993a; Prosser et al., 1993). The relatively high affinity of the 5-HT, receptor for neuroleptics such as ( + )-butaclamol and clozapine suggests that this receptor might also play a role in certain neuropsychiatric disorders. When the 5-HT, receptor is transiently expressed in cos-7 cells or stably expressed in CHO, HEK-293, or HeLa cells, its activation leads to an

Table IV 5-HT5, 5-HT6, and 5-HT, Receptors Introns in coding sequence

mRNA size (kb)

mRNA regional distribution (main sites)

Yes (one)

5.8 ( 5 ; 4.5) 3.8-4.5

Yes (one)

Not detected

437 436

Yes

4.2

448 448

Yes

4.0 3.9

Hippocampus (CAICA3-DG), cerebral cortex, granular layer of cerebellum, olfactory bulb, habenula, spinal cord Hippocampus (CAI), dorsal raphe nuclei, habenula Corpus striatum, amygdala, cerebral cortex, hippocampus (CAI-CA3-DG) Hypothalamus, thalamus. hippocampus (CA2CA3). amygdala, intestine, heart

Receptor

Species

Amino acids

Locus

5-HT5A

Mouse Rat

357 357

5B

5-HT5B

Mouse Rat

370 370

I E4-1 EG 2ql I-q 13 (human)

5-HT6

Rat

5-HT7

Mouse Rat

~

7q36 (human)

~

357

5-HT Receptor Subtypes

increase in adenylate cyclase activity (Lovenberg et al., 1993a; Plassat et al., 1993; Ruat et al., 1993b; Shen et al., 1993).This effect can be blocked by nonspecific 5-HT receptor antagonists such as methiothepin, methysergide, and ergotamine but also by the neuroleptics (+)-butaclamol and clozapine. LSD is a partial agonist (Ruat et al., 1993b). c. Regional Distribution Quantitative PCR experiments reveal that the 5-HT, receptor is expressed in the central nervous system (forebrain, brainstem, cerebellum, and embryonal colliculus neurons) but also at the periphery (intestine and heart) (Table IV). In situ hybridization experiments detect the 5-HT7in hippocampus (CA2-CA3), hypothalamus, thalamus, amygdaloid complex, retrospenial cortex, tenia tecta, indosium griseum, superior colliculus, and dorsal and paramedian raphe nuclei (Lovenberg et al., 1993a; Meyerhof et al., 1993; Plassat et al., 1993; Ruat et al., 1993b).

D. The 5-HT5 Family-5-HT5,

and 5-HT5, Receptors

The 5-HT5family contains two receptors, 5-HT5, and 5-HT5,, that define a new family of serotonin receptors (Table IV). These receptors do not resemble receptors of the 5-HT, and 5-HT2 families in terms of amino acid sequence (Fig. 3), pharmacological profile, or transduction system. They display a high affinity for 5-CT and a low affinity for sumatriptan. They might therefore correspond to the nonclassical 5-CT-sensitive sites reported in the brain of various mammalian species (Mahle et al., 1991).

1. The 5-HT5, Receptor a. Molecular Structure Using degenerate oligonucleotides derived from transmembrane domains I11 and VI of G-protein-coupled serotonin receptors, Plassat er al., 1992 isolated a new mouse serotonin receptor. Hydropathy analysis of the corresponding protein of 357 amino acids revealed seven hydrophobic domains. Amino acid sequence comparisons indicated that this receptor was a distant relative of all previously identified 5-HT receptors and was therefore named 5-HT5receptor. The percentages of homology to known receptors are low, the best score being 37% with the Drosophila serotonin receptor 5-HTd,,, (Saudou eta!., 1992). Analysis of the genomic fragment of the 5-HT5, receptor gene indicates the presence of one intron about 8- kb long located in the middle of the third cytoplasmic loop (Matthes et al., 1993). The rat homolog of the mouse 5-HT5, receptor has been cloned (Erlander et al., 1993). The gene encoding the 5-HT5, receptor is located in human at locus 7q36 and in mouse on chromosome 5 at position 5B (Table IV; Matthes et al., 1993).

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Frederic Saudou and Rene Hen

b. Functional Expression When expressed in Cos-7 cells, the S-HT,, receptor displays a high affinity for ['2SI]LSD(& = 340 pM) and the following profile: LSD > ergotamine > 5-CT > methysergide > 5HT = RU24969 > bufotenine = yohimbine (Plassat et al., 1992). This profile does not correspond to the profile of any of the previously characterized serotonin receptors. Binding experiments using ['HIS-CT reveal that the 5-HTSAreceptor displays a high affinity (Kd = 0.84 nM)and a low affinity (Kd = 13 nM) for this radiolabeled compound (Amlaiky et al., 1993) and has a pharmacological profile that can correspond to the 5CT-sensitive sites reported by Mahle et at. (1991). When expressed in CosM6 cells, the rat 5-HT5, receptor displays a similar pharmacological profile (Erlander et al., 1993). In cells expressing the mouse or the rat receptor, Plassat et al. (1992) and Erlander et al. (1993) could not detect any change in adenylate cyclase or phospholipase C activity. The 5-HTs, receptor might therefore interact with a different signaling system, for example, an ion channel. c. Regional Distribution The 5-HTSAreceptor is expressed in the central nervous system (Table IV). In the mouse, Northern analysis reveals three transcripts in brain and cerebellum (5.8, 5.0, and 4.5 kb) (Plassat et al., 1992) whereas in rat two transcripts (3.8 and 4.5 kb) are detected (Erlander et al., 1993). Quantitative PCR demonstrates the presence of specific fragments only in the brain and the spinal cord among the various tissues tested (Plassat et al., 1992). Within the brain, 5-HT,, transcripts are detected in the cerebral cortex, the hippocampus (pyramidal cells of CA1-CA3 layers and granule cells of the dentate gyrus), the granule cells of the cerebellum, the habenula, and the tufted cells of the olfactory bulb (Plassat et al., 1992; Erlander et al., 1993).

2. The 5-HTSBReceptor a. Molecular Structure The 5-HT5, receptor gene has been isolated by PCR experiments on mouse brain RNA using degenerate oligonucleotides derived from transmembrane domains I11 and VI of G-protein-coupled 5HT receptors. The 5-HT,, receptor consists of a polypeptide chain of 370 amino acids both in rat and in mouse (Erlander et al., 1993; Matthes et al., 1993). The 5-HTSBreceptor is highly homologous to the 5-HT,, receptor (77%) (Plassat et al., 1992), whereas the percentages of homology to other known receptors are low (Fig. 3). The genomic fragment containing the ~-HT,Bgene has been isolated. Partial sequence analysis reveals that the ~-HT,B gene contains one intron located in the middle of the third cytoplasmic loop. Interestingly, the intron is located at exactly the same position in the 5-HT,, and ~-HT,B genes (Matthes et al., 1993).

5-HT Receptor Subtypes

359

The mouse 5-HT5Bgene is localized on chromosome 1 (position 1E41EG) whereas its human homolog is on chromosome 2 (position 2qllq13) (Table IV; Matthes et al., 1993). b. Functional Expression The 5-HT5, receptor expressed in Cos-7 cells exhibits a high affinity for ["'I]LSD (& = 470 pM) (Matthes et af., 1993) and two affinities for ['"IS-CT, a high affinity (& = 0.6 nM) and a low affinity (& = 14 nM). The fraction of the sites that exhibit a high affinity for [3H]5-CTmight correspond to receptors coupled to G-proteins (Amlaiky et al., 1993). Displacement of bound ['*'I]LSD by various serotonergic drugs gives the following rank order of potencies: LSD > ergotamine > methiothepin > 5-CT > methysergide > 5HT = RU24969 > bufotenine. Similar results were obtained by Erlander et al. (1993) in CosM6 cells transfected with the rat homolog. Like the 5-HT5, receptor, the 5-HT5, receptor does not interact with adenylate cyclase or phospholipase C (Erlander et al., 1993; Matthes er al., 1993). c. Regional Distribution Expression of the receptor is restricted to limited regions in the brain (Table IV). Zn situ hybridization experiments mRNA performed on mouse brain sections reveal the presence of ~ - H T , B only in the CAI field of the hippocampus, the medial and lateral habenula, and the dorsal raphe nucleus (Erlander et al., 1993; Matthes et al., 1993).

E. Serotonin Receptors in Invertebrates 1. Drosophila

a. Molecular Structure Using two degenerate oligonucleotides corresponding to consensus sequences found in the transmembrane domain VI of G-protein-coupled receptors, three Drosophila serotonin receptors have been isolated: the 5-HTd,, (Witz et al., 1990), the 5-HTdro2,,and 5-HTdr02B receptors (Table V; Saudou et al., 1992). The protein of 564 amino acids corresponding to the 5-HTdrolreceptor exhibits some homology to G-protein-coupled receptors. Hydropathy analysis of the 5-HTdroIreceptor sequence reveals the existence of eight hydrophobic domains in contrast to seven domains for all the other members of the family. The eighth domain, which is located near the N-terminal tail of the protein, might be an additional transmembrane domain or an unusually long cleavable signal sequence. Another feature of the 5-HTdrolreceptor is the presence of the terminal tail of a Ser-Gly motif that is repeated 10 times. This kind of motif is a putative attachment site for glycosaminoglycans such as chondroitin sulfate or keratan sulfate. Similar motifs are found in the Drosophila clock gene period and in the Neurospora clock gene frequency. This observation, in conjunction with the known role of serotonin in the modulation

Table V Invertebrate 5-HT Receptors Receptor

Species

Amino acids

Locus

Introns in coding sequence

mRNA size (kb)

mRNA regional distribution (main sites) Brain, ventral cord Brain, ventral cord (ventral unpaired median, neurons) Brain, ventral cord Central nervous system (growth-controlling light green cells), heart

5-HTdr0, 5-HTdro2~

Drosophila Dsosophila

564 a34

3 R lOOA 2 R 56A-B

No Yes

5.5 6.2

5-HTdm2~ 5-HT,,m

Drosophila Lymnea sragnalis

645

2 R 56A-B

Yes

4.9 2.3-3.2

509

-

-

5-HT Receptor Subtypes

36 1

of circadian rhythms, suggests that the 5-HTdro,receptor might modulate biological rhythms. The gene encoding the 5-HTdro,receptor does not contain any intron in the coding sequence and is located on the right arm of the third chromosome at position lOOA (Saudou et al., 1992). The .5-HTdro2,(834 amino acids) and 5-HTdrO2,(645 amino acids) receptors are highly homologous (84.3% within the putative transmembrane domains) (Fig. 3). The 5-HTdro2, receptor, like the 5-HTdrolreceptor, contains an additional hydrophobic domain located in the N-terminal tail. Interestingly, the N-terminal tail of the Drosophila serotonin receptors, as well as of other Drosophila G-protein-coupled receptors, is long compared with the mammalian receptors and might be characteristic of Drosophila G-protein-coupled receptors (Fig. 2). Although the 5-HTdrolreceptor appears to be a relative of the 5-HT7 receptor, the 5-HTdrO2,and 5-HTdro2,receptors belong to the 5-HTl family, as seen in Fig. 3. Unlike all the mammalian receptors of the 5-HT, family, the 5-HTdro2, gene contains at least four introns in the coding sequence. The 5-HTdro2,and 5-HTdro2B genes are located at the same position on the right arm of the second chromosome in the region 56A-B. This common location, as well as the high sequence homology found between these two genes, suggests that they result from a recent duplication event (Table V). b. Functional Expression When expressed in cos-7 cells, the Drosophila serotonin receptors display a high affinity for [12SI]LSD.The Kd is similar for the three receptors (ranging from 0.2 to 0.4 nM). Dihydroergotamine has a high affinity for the three receptors, which might therefore correspond to the ['Hldihydroergotamine binding sites reported in membranes prepared from Drosophila heads (Dudai and Zvi, 1982). Prazosin, an antagonist of the al- and a2-adrenergic receptors, has about 50-fold higher affinity for the 5-HTdr02, and 5-HTdro2~ receptors than for 5-HTdrol (Saudou et al., 1992). In NIH-3T3 cells stably expressing the receptors, the 5-HTdr0lreceptor was shown to be positively coupled to adenylate cyclase whereas 5-HTdrO2, and 5-HTdro2,were negatively coupled to this enzyme (Fig. 1). The adenylate cyclase inhibition induced by the activation of the 5-HTdroZA and 5HTdro2~receptors could be blocked by pertussis toxin, suggesting that these receptors interact with a pertussis toxin-sensitive G-protein such as Gi. The 5-HTdro2,and 5-HTdroZB receptors were also responsible for a 1.5- to 2-fold increase in the level of inositol phosphates in response to serotonin. The fact that Drosophila receptors are able to couple to mammalian G-proteins suggests that the mechanisms of transduction have been well conserved during evolution. Indeed, Drosophila serotonin re-

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Frederic Saudou and Rene Hen

ceptors and DrosophifaG-proteins are highly homologous to their mammalian counterparts. and 5-HTdrOzB Deletion of the N-terminal part of the S-HTd,,, , 5-HTdro2~, receptors does not alter their affinity for various ligands nor their coupling with second messengers (Saudou et al., 1992),indicating that the additional hydrophobic sequences is not necessary for receptor activity, at least in mammalian cells. c. Pattern of Expression In adult Drosophila, the mRNA of the 5HTdrol,,5-HTdrO2,,and 5-HTdroZB receptors is detected only in heads, with sizes of 5.5,6.2,and 4.9 kb, respectively (Table V). Analysis of expression during development reveals that the receptor mRNAs are expressed late in embryogenesis (stage 16 embryos) and their expression culminates when the larval nervous system is essentially formed (stage 17 embryos). This expression coincides with the appearance of serotonin immunoreactivity , which supports a role for serotonin as a neuromodulator when the nervous system is functional. In situ hybridization on stage 17 embryos reveals that the receptor mRNAs are present in distinct cell populations in the ventral cord. In particular, the 5-HTdroZA receptor mRNA is localized in ventral cord motoneurons (VUM neurons) that innervate larval muscles, suggesting a role for 5-HTdro2~ in the control of larval movements (Saudou et al., 1992).

2 . Lymnea stagnalis In the molluscan nervous system, the role of 5-HT in various behaviors (e.g., learning and memory in Apfysia) has been well studied and the existence of several 5-HT receptors has been demonstrated (Gerschenfeld and Paupardin-Tritsch, 1974). To gain insight into the pharmacology and the diversity of the serotonergic system in molluscs, Sugamori et al. (1993) isolated a 5-HT receptor from Lymnea stagnafis. The corresponding protein (509 amino acids) showed the highest degree of homology to the Drosophifa serotonin receptors and the receptors of the 5-HT, family (5-HT,, receptor) (Fig. 3), the best score being with the 5-HTdrO2,(61% within the transmembrane domains). This receptor, called 5-HTL,,, possesses a long third cytoplasmic loop and a short C-terminal tail; these are characteristics of receptors coupled negatively to adenylate cyclase, suggesting an equivalent coupling for 5-HTL,, (Fig. 2). However, the functional coupling of the receptor has not yet been analyzed. When introduced into COS-7 cells, the 5-HT,,, receptor displays a high-affinity binding site for [3H]LSD (& = 0.9 nM). Addition of the nonhydrolyzable guanine nucleotide Gpp(NH)p shifts the receptor into a low-affinity state, suggesting that 5HTLymis able to interact with G-proteins in Cos-7 cells.

363

5-HT Receptor Subtypes

The 5-HTLymreceptor is expressed in the central nervous system of L. stagnalis. Two mRNA species are detected (2.3 and 3.2 kb). By quantitative PCR, Sugamori et a/. (1993) detected 5-HTLymreceptor mRNAs in heart and showed that the transcripts are present within the central nervous system in specific neurons such as the growth-controlling Light Green cells (Table V).

111. 5-HT-Gated Ion Channels-5-HT3

Receptors

A. Molecular Structure In contrast to most of the 5-HT receptors, which are coupled to G-proteins, the 5-HT3 receptor is a ligand-gated ion channel. This receptor, when activated, causes a rapid excitatory response by depolarizing neurons, a property shared with the nicotinic acetylcholine receptor (Fig. 1). The SHT, receptor has been isolated by expression in Xenopus oocytes. By injecting size-fractionated poly(A + ) RNA from NCB20 cells into Xenopus oocytes, Maricq et a / . (1991) tested for the presence of serotoningated currents characteristic of the 5-HT, receptor. These investigators constructed a cDNA library from the positive mRNA fractions, and serial dilutions of the positive pools yielded the isolation of a cDNA encoding a functional 5-HT3 receptor. The predicted protein of 487 amino acids showed a high sequence homology to members of the ligand-gated ion channel superfamily. The receptor exhibits the characteristics of this superfamily including four hydrophobic transmembrane regions (M 1 to M4) and a large N-terminal extracellular domain containing a Cys-Cys loop, which has been proposed to be involved in the formation of a disulfide bond. A splice variant of the 5-HT, receptor has been cloned from another cell line, the NlE115 neuroblastoma cell line (Hope et al., 1993). This variant differs from the 5-HT, receptor described by Maricq et al. (1991) by a deletion of 6 amino acids in the large cytoplasmic loop between the putative M3 and M4 transmembrane regions. The two forms are present in the N l E l l 5 and NCB20 cell lines (Table VI).

B. Functional Expression Expression of the 5-HT3 receptor in Xenopus oocytes and in Cos-1 cells has allowed its pharmacological and electrophysiological characterization (Maricq et al,, 1991). The SHT, receptor is a cation-specific ion channel but, among cations, this receptor is poorly specific, allowing the passage of large cations such as Na+ and K + which have approximately the same

Table VI 5-HT3Receptors ~~

Receptor

5-HT3

~~

Species NCBZO cells Mouse1 hamster hybrid

Amino acids

487 48 1 (splice variant)

Locus

-

Introns in coding sequence

mRNA size (kb) 2.2

~

mRNA regional distribution (main sites)

Binding site distribution (main sites)

Cerebral cortex, hippocampus, amygdala, spinal cord, olfactory bulb, dorsal root ganglia

Identical to mRNA distribution with addition of medullary dorsal vagal complex and intestine

5-HT Recepfor Subfypes

365

permeability through the channel. Binding studies performed in Cos-1 cells expressing the receptor gave the following rank order of potencies: tropisetron > curare > l-aH-3a-5aH-tropane-3-yl-3,5-dichlorobenzoate (MDL72222) > 5-HT > methysergide. The fact that a single clone is able to generate currents with all the characteristics of the native 5-HT3 receptor (Maricq et al., 1991;Yakel et al., 1993)suggests that the 5-HT3receptor functions as an homopolymer. This result is surprising since the other members of the ligand-gated ion channel superfamily are heteropentameric proteins composed of two to four different homologous subunits. Although single subunits from some members of this superfamily can form functional homomeric receptors, these receptors generally lack some properties of the native multisubunit receptors. The electrophysiological and pharmacological properties of the short-splice variant are similar to those of the 5-HT3 receptor except for 2-methyl-5-HT, which behaves as a partial agonist in the case of the short variant and as a full agonist in the other case (Maricq et al., 1991;Yakel et al., 1993). The 5-HT3 receptor, when expressed in oocytes, displays desensitization in the continued presence of the agonist (Maricq el al., 1991). This property is common to the ligand-gated ion channels. As in the case of the nicotinic acetylcholine receptor, the second hydrophobic domain is involved in the mechanism of desensitization (Revah et al., 1991).In particular, substitution of Leu 286 with phenylalanine, tyrosine, or alanine results in a faster desensitization of the receptor whereas a replacement with threonine results in a slower desensitization (Yakel et af., 1993).These modifications are similar to those reported for the nicotinic acetylcholine receptor (Revah et al., 1991),suggesting that the conformational change that underlies desensitization might be common to ligandgated ion channels.

C. Regional Expression Northern analysis reveals the presence of a 2.2-kb transcript encoding the

5-HT3 receptor in neuroblastoma cells. By quantitative PCR experiments,

transcripts of the 5-HT3receptor can be detected in mouse cortex, brainstem, midbrain, spinal cord, and heart. In siru hybridization experiments reveal that the 5-HT3 receptor mRNA is detected in the hippocampal formation (interneurons); the piriform, cingulate and entorhinal cortices; the amygdaloid complex; the olfactory bulb; the trochlear nerve nucleus; the dorsal tegmental region; the facial nerve nucleus; the nucleus of the spinal tract of the trigeminal nerve; the dorsal horn of the spinal cord; and dorsal root ganglia (Table VI; Tecott et al., 1993). Autoradiographic studies using selective 5-HT3 radioligands such as [3H]tropisetron, r3H1-

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zacopride, '2sI-labeled zacopride, and l-methyl-N-(8-methyl-8-azabicyclo[3.2.l]oct-3-yl)-H-indazole-3-carboxamide ([3H]LY278584) has allowed the mapping of the 5-HT3 receptor binding sites in the central nervous system and the periphery of various species (Hamon et al., 1989; Hoyer et al., 1989; Waeber et al., 1989b,1990a; Wong et al., 1989; Anzini et af., 1990; Gehlert et at., 1991; Champaneria et af., 1992; Laporte el al., 1992). 5-HT3receptor binding sites are detected in the hippocampus, cortex, amygdala, facial nerve nucleus, dorsal horn of the spinal cord, gastrointestinal tract, and nucleus of the solitary tract (Table VI). The distribution of 5-HT3binding sites matches the distribution of 5-HT3receptor mRNA (Maricq et al., 1991; Tecott et al., 1993). However, no mRNA was detected in the medullary dorsal vagal complex, which comprises the aera postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus nerve. These regions have been suggested to be involved in the anti-emetic properties of 5-HT3 antagonists. Similarly, transcripts of the 5-HT3receptor A subunit (Maricq et al., 1991)are absent from intestine. This result suggests that the 5-HT3 receptor expressed in intestine could correspond to another distinct receptor. Indeed, preliminary reports revealed the existence of two pharmacologically distinct 5HT, binding sites in mouse cortex and ileum (Wong et a/., 1992).

IV. Conclusion Why are there so many 5-HT receptors? To try to answer such a question, it is worth considering what parameters distinguish the various receptor subtypes. The receptor families differ in their effector systems. Whereas the 5-HT3receptors are ion channels, the 5-HTI receptors inhibit adenylate cyclase, the 5-HT4, 5-HT6, and 5-HT, receptors stimulate adenylate cyclase, the 5-HT2receptors stimulate phospholipase C, and the 5-HT5receptors are probably coupled to a different effector system. Why then are there so many 5-HTI receptors (5-HTIA,5-HT,,, 5-HTID,,5-HTIE,and 5HT,,)? First, these receptors might not always share the same effector systems. The ~ - H T , A receptor, for example, can couple with adenylate cyclase, phospholipase C, or ion channels, depending on the cell type in which it is expressed. The other 5-HT, receptors can also inhibit adenylate cyclase in fibroblasts but their neuronal effectors are not known and might be different from those of the 5-HTIAreceptor. Second, the S H T , receptors differ markedly in their patterns of expression. Whereas the 5HTIA receptors are expressed in the raphe nuclei and in the hippocampus, the 5-HT,, receptors are found predominantly in the basal ganglia. In addition, even when two receptors are expressed by the same neurons,

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they are not necessarily found in the same subcellular compartment. The 5-HT,, receptors, for example, are localized in the somatodendritic compartment of raphe neurons whereas the 5-HT,, receptors are localized on the axon terminals of these neurons. The existence of a large number of receptors with distinct signaling properties and expression patterns might enable a single substance such as 5-HT to generate simultaneously a large panel of effects in many brain structures. Most complex behaviors require the synchronized modulation of several physiological functions. In a flight situation, for example, locomotor activity and fear will increase while sexual activity and digestive functions might decrease. The fact that several 5-HT receptors have similar pharmacological properties renders the study of their function by classical techniques exceedingly difficult. However, the availability of the genes encoding these receptors makes it possible to create mouse mutants lacking these receptors by homologous recombination or to block their expression with specific oligonucleotides. These techniques will hopefully allow us to understand why there are so many 5-HT receptors and what their functions are.

Acknowledgments We wish to thank U. Boschert, N. Amlaiky, S. Ramboz, R. Grailhe, A. Ghavami, H. Matthes, and J. L. Plassat for helpful comments and discussions and S. Metz for artwork. We are grateful to M. Hamon and D. Hoyer for their critical reading of the manuscript.

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