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Annu. Rev. Neurosci. 1991.14:335-360. Downloaded from www.annualreviews.org by University of California - Los Angeles - UCLA Digital Coll Services on 05/05/14. For personal use only.

1991. 14:335-60 Copyright © 1991 by Annual Reviews Inc. All rights reserved

MOLECULAR BIOLOGY OF SEROTONIN RECEPTORS David Julius Cell Biology and Neuroscience Programs, Department of Pharmacology, University of California, San Francisco, California 94143 KEY

WORDS:

mitogenesis, signal transduction, G protein-coupled receptors, cellular transformation, receptor subtype diversity

INTRODUCTION

Serotonin (S-hydroxytryptamine; SHT) is a biogenic amine that functions as both a neurotransmitter and a hormone in the mammalian central nervous system (CNS) and in the periphery. Within the brain, serotonergic neurons originate primarily in the raphe nuclei of the brainstem and project to most areas of the CNS, where they regulate a wide variety of sensory, motor, and cortical functions (Osborne 1982). In the periphery, serotonin is involved in such diverse functions as the regulation of enteric reflexes, the modulation of platelet shape change and aggregation, the modulation of vascular smooth muscle contraction, the initiation of activity in primary afferent nociceptors, and the regulation of lymphocyte cytotoxicity and phagocytosis (for review see Peroutka 1988, Richardson & Engel 1986). Pharmacological and physiological studies have shown that the multiple actions of serotonin are mediated by several distinct cell surface receptor subtypes, designated SHTl a, Ib, lc, Id, SHT2, SHT3, and SHT4 (Bradley et aI 1986). For example, the hallucinatory actions of lysergic acid diethyl­ amide (LSD) and other psychotropic serotonin analogues are probably elicited by activation of cortical 5HT2 (Rasmussen & Aghajanian 1988) or 5HTIc receptors. In contrast, the pain that is produced from applying serotonin to a blister base results from activation of 5HT3 receptors on primary sensory nerve endings (Richardson & Engel 1986). Individual serotonin receptor subtypes exhibit characteristic ligand­ binding profiles and couple to different intracellular signaling systems. 335 0147-006X/9l/030 1-0335$02.00

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5HTl a, 5HTl b, 5HTl d, and 5HT4 receptors activate or inhibit adenylate cyclase (AC) (DeVivo & Maayani 1986, Schoeffter & Hoyer 1989, Shenker et al 1987) to alter the levels of cyclic AMP (cAMP) in the cell. 5HTl c and 5HT2 receptors stimulate phospholipase C--catalyzed hydrolysis of phosphatidylinositol bisphosphate (PIP2) (Conn et al 1986, de Courcelles et al 1985) to yield the second messengers inositol trisphosphate (IP 3) and diacylglycerol (DAG); IP 3 promotes the release of Ca 2+ from intracellular stores and DAG serves as an endogenous activator of protein kinase C (PKC). A host of biochemical and genetic evidence (discussed below) indicates that 5HTl , 5HT2, and 5HT4 receptors belong to the class of neurotransmitter and hormone receptors that transduce extracellular sig­ nals by activating GTP-binding proteins (Stryer & Bourne 1986). In con­ trast to these G protein-coupled receptor subtypes, which mediate slow, modulatory responses via second-messenger signaling pathways, the 5HT3 receptor is a ligand-gated ion channel. Activation of this receptor leads to the opening of nonselective cation channels to promote fast, depolarizing responses in neurons (Derkach et al 1989, Yakel & Jackson 1988). Thus, serotonin, like several other transmitters (acetylcholine, GABA, and glu­ tamate) activates both G protein-coupled and ligand-gated ion channel receptor SUbtypes. The existence of multiple receptor subtypes that modu­ late a variety of ion channels and intracellular second messenger signaling pathways greatly extends the flexibility of neurotransmitter action and provides a mechanism whereby a single neurotransmitter can elicit a mul­ titude of cellular and physiological responses. A simplified compendium of 5HT receptor subtype characteristics is provided in the accompanying Table 1. This review focuses on the recent contributions of molecular biology to the characterization of vertebrate serotonin receptors. The isolation of cDNA and genomic clones encoding a variety of neurotransmitter recep­

tors has made expression of these proteins possible in new cellular environ­ ments devoid of other, closely related receptor subtypes in order to study their signal transduction and ligand-binding properties in greater detail. The structure, function, and tissue-specific expression of 5HTl a, 5HTl c, and 5HT2 receptors are discussed in this context.

RECEPTOR STRUCTURE Brief Cloning History The expression of functional receptors in Xenopus oocytes has provided a sensitive assay for the detection of messenger RNAs (mRNAs) encoding receptors that, like the 5HT l c receptor, couple to inositol phospholipid­ signaling systems (for review see Snutch 1988). High levels of 5HTIc

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Table 1

Abbreviated compendium of 5HT receptor subtype characteristics 5HT I a

5HTlb/ld

Primary effector

tAC

tAC

Labeled by

3[H]-DPAT

3[H]-5HT

5HTlc PI hydrolysis 25 I [I]-LSD

5HT2 PI hydrolysis 125[I]_LSD

3[H]-5HT

3[H]-LSD

3[H]-LSD

3[H]-LSD

3[H]-5HT

3[H]_Ketanserin

5HT3

5HT4

Na+ / K + channel J[H]-GR65360

JAC

2-Methyl-5HT

5-Methoxy-5HT

J[H]-ICS 205-930

3[H]-Spiperone Partially selective agonists

DPAT

BRL-24924 Partially selective antagonists

SDZ 21-009

Mesulergine

Ketanserin

ICS 205-930

Mianserin

Spiperone

MDL72222

Mesulergine

Cocaine

m � 0

Cl Z

Mianserin Location (examples)

Hippocampus

S. nigra

C. plexus

Raphe nuclei

B. ganglia

Hippocampus

Cerebral cortex

'"

Sensory nerves

Hippocampus

Enteric nerves

Enteric nerves

51

� m (j m "d --l 0 �

'"

...., ...., -..l

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receptors are expressed in the choroid plexus, a non-neuronal cell type in the brain responsible for the production of cerebral spinal fluid (CSF). In Xenopus oocytes that have been injected with choroid plexus mRNA, application of serotonin stimulates the hydrolysis of PIP 2 to liberate ino­ sitol phosphates that raise intracellular Ca 2+ levels. This promotes the opening of Ca 2+ -dependent chloride channels, resulting in a serotonin­ evoked inward current response. Lubbert and collaborators took advan­ tage of this functional assay, in combination with a hybrid depletion method, to isolate a partial cDNA clone encoding the carboxy-terminus of the mouse 5HTl c receptor (Lubbert et al 1987). Using the same func­ tional assay to screen a cDNA expression library, Julius et al ( l 988a) employed a direct in vitro transcription approach to isolate a full-length, functional clone encoding the rat 5HTl c receptor. Subsequently, a func­ tional cDNA clone encoding the 5HT2 receptor was isolated by screening a rat brain cDNA library, under reduced stringency hybridization con­ ditions, with synthetic oligonucleotide probes (Pritchett et al 1988) or coding region fragments (Julius et a1 1990) derived from the 5HTlc recep­ tor cDNA clone. A 5HTla receptor clone was obtained by screening a human genomic library under low stringency hybridization conditions and using a /32adrenergic receptor clone as a molecular probe (Kobilka et al 1987). This gene, originally named G-2l , was later shown by Fargin et al to encode a 5HTIa receptor based on its pharmacological profile when expressed in transfected mammalian cell lines (Fargin et al 1988). The rat 5HT l a receptor was isolated in a n analogous manner, using a hamster P2-adren­ ergic receptor clone to screen a rat genomic library (Albert et al 1990). Receptor Structure and Gene Organization The deduced amino acid sequences of the 5HTl a, 5HTlc, and 5HT2 receptors (see Figure 1) clearly indicate that they belong to the extended family of G protein-coupled neurotransmitter and hormone receptors that consist of a single polypeptide chain containing seven putative membrane­ spanning a-helices. Other members of this family include the visual opsins, muscarinic cholinergic receptors, adrenergic receptors, tackykinin recep­ tors, dopamine D2 receptor, and yeast pheromone receptors. On the basis of structural homologies with rhodopsin and the P2-adrenergic receptor, it is likely that the amino terminus of each of the serotonin receptors is located outside the cell, with the carboxy terminus located in the cytoplasm. Accordingly, the seven hydrophobic transmembrane domains would be linked together by three intracellular and three extracellular loops (reviewed by O'Dowd et aI1989). The regions of greatest homology among members of this family correspond to the seven hydrophobic domains. A

339

SEROTONIN RECEPTORS

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SHT2 MEILCEDNISLSSIPNSLMQLGDGPRLYHNDFNSRDANTSEASNWi]DAENRTNLSCEGYLPPT MVNLGNAVRSLLMHLIGLLVWQFDI�SPVAAIVTDTFNSSDGG SHT1C MDVLSPGQGNNTTSPPAPFETGGNTTGI SHT1A MGPPGNDSDFLLTTNGSHVPDHDV B2AR

II

I

em SILH LQE �

�FQFPDGVctNN

LTTlVlvlI:il rlTil SI�I��



SDVTVSYQVITSL

GTLIFCAVL­

TEERDEAWVVGMAI MSVIVLAIVF IV

III

V

�M LI L

r

F

ATLcvsDLSTRAKLASFSFLPQSSLSSEKLFQ----------------- --------------------­ TLMLLRGHTEEELANMSLNFLNCCCKKNGGEEENAPNPNP-----------------------------­ IRKTVKKVEKTGADTRHGASPAPQPKKSVNGESGSRNWRLGVESKAGGALCANGAVRQGDDGAALEVIEV LQKIDKSEGRFHSPNLGQVEQDGRSG-------------------------------------------VI

VII

TIPALAYKSSQLQVGQKKNSQEDAEQT�DCSMVTLGKQQ�ENCTDNIETVNEK�CV VAATALSGRELNVNIYRHTNERVARKA�PEPGIEMQVENtiELPVNPSNVVSERIS�V NSNGKTDYMGEASGCQLGQEKESERLC�PPGTESFVNCQGTVPSLSLDSQGRNCSTNDSPL Figure 1

Amino acid sequence comparison of the rat 5HT2 (Julius et al 1990, Pritchett et

al 1988) and 5HTIc (Julius et al 1988a) receptors, the human 5HTIa receptor (Kobilka et al 1987), and the hamster /32-adrenergic receptor (Dixon et al 1986). The boxed residues are identical in at least the 5HTIc and 5HT2 receptor sequences. Roman numerals and brackets identify the putative transmembrane domains (from Julius et al 1990).

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large body of biochemical and genetic evidence supports the notion that these membrane-spanning regions are intimately involved in ligand binding and G protein coupling (see O'Dowd et al 1989). This high degree of sequence conservation is therefore likely to reflect the fact that each of these receptors transduces signals by interacting with members of a highly conserved family of G proteins. Among the cloned serotonin receptors, the 5HT l c and 5HT2 subtypes are most closely related, exhibiting 50% amino acid sequence identity overall, and 80% identity within the putative transmembrane regions. This degree of sequence conservation may reflect a very recent gene duplication or stringent conservation to maintain a common set of functions. The nearly complete sequence divergence in the N- and C-terminal regions, as well as in the third cytoplasmic loop (between transmembrane domains V and VI), suggests that the two genes do not result from a recent duplication event, but rather, that stringent conservation is required to maintain similar ligand-binding and effector-coupling specificities. The 5HTla receptor is more closely related to the adrenergic receptors than it is to the 5HT l c and 5HT2 serotonin receptors. This is most apparent in the sixth membrane­ spanning domain, where the 5HTl a receptor is identical to the P2-adren­ ergic receptor at 90% of its amino acid residues (Albert et a1 1990, Fargin et al 1988). Pharmacological analysis of chimeric adrenergic receptors produced by gene fusion methods (reviewed by Lefkowitz et al 1988) suggests that the sixth and seventh transmembrane domains are major determinants of antagonist-binding specificity. The fact that thc 5HT l a receptor is particularly homologous to p-adrenergic receptors in this region may explain why various adrenergic antagonists, such as propranolol and cyanopindolol, exhibit moderate affinities for 5HT l a sites (Hoyer et al 1986, Middlemiss 1986). It is also interesting to note that within the transmembrane regions the aI-adrenergic receptor shares greater homo­ logy with the 5HTla receptor than with other members of the adrenergic receptor family (Cotecchia et al 1988). The notion that serotinin receptors derive from at least two gene families is supported by a comparison of their gene structures. The 5HT l c and 5HT2 receptor genes, like those of the visual opsins (Nathans 1987), contain multiple introns within the protein-coding region, and preliminary analysis suggests that at least some of the intron-exon boundaries are conserved between these two subtypes (K. Huang and D. Julius, unpub­ lished). In contrast, the 5HT l a receptor gene is intronless (Kobilka et al 1987), as are the genes encoding many of the adrenergic receptors (O'Dowd et aI1989). Thus, serotonin receptors derive from at least two gene families: the 5HTIc and 5HT2 receptors define one, intron-containing gene family; whereas the 5HT l a receptor has evolved from a distinct, intronless adren­ ergic receptor lineage. Mutation within a family may therefore generate

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SEROTONIN RECEPTORS

34 1

variant receptors that either bind different agonists or modulate different effectors. Since the 5HTlb receptor also binds adrenergic antagonists (Schoeffter & Hoyer 1989), it seems likely that this subtype will prove to be a member of the adrenergic receptor family. Outside of the putative transmembrane regions, no significant protein sequence homology exists among the different G protein-coupled recep­ tors. Even when the same receptor from different species is compared, the divergence within these regions is significantly greater than within the hydrophobic domains. For example, of the 48 amino acid differences that exist between rat and human 5HTl a receptors, 44 lie in cytoplasmic or extracellular domains. In spite of the apparent lack of sequence homology, the C-terminal tail and the third cytoplasmic loop regions do share some structural similarities. Most notably, they are rich in serine and threonine residues. Biochemical and genetic studies of rhodopsin, adrenergic recep­ tors, and yeast pheromone receptors (Blumer et al 1988, Lefkowitz et al 1988) have demonstrated that these residues are potential sites for phosphorylation by cAMP-dependent protein kinase, protein kinase C, or "private" receptor kinases such as rhodopsin kinase and p-adrenergic receptor kinase (Benovic et aI1989). Phosphorylation of these residues has been shown to attenuate receptor-G protein interactions and to account for heterologous and homologous mechanisms of desensitization (O'Dowd et al 1989), Although the 5HTl a receptor has a very short cytoplasmic tail, the large third cytoplasmic loop contains a number of potential phos­ phorylation sites. Whereas the rat 5HTlc and 5HT2 receptors share little or no primary sequence homology within the C-terminal tail, the sequence of a putative Xenopus 5HT2 receptor shows striking homology to the rat 5HT2 receptor in this region (D. Julius, unpublished). These comparisons suggest that the primary sequence of the. C-terminal tail may be func­ tionally important, and that divergence between 5HTlc and 5HT2 recep­ tors in this domain may permit the independent regulation of two receptor subtypes that bind the same ligand and activate the same effector system. Finally, limited regions of homology are seen at the ends of the third cytoplasmic loop and at the beginning of the C-terminal tail. From deletion and point mutation analysis of adrenergic receptors, these regions are thought to be important for receptor-G protein interactions (Dixon et al 1988).

FUNCTIONAL EXPRESSION 5HTla Receptor

As mentioned above, a human gene (G-21) encoding the 5HT l a receptor was originally isolated by virtue of its homology with a

PHARMACOLOGY

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fJz-adrenergic receptor clone (Kobilka et aI1987). Based on this structural similarity, membranes prepared from monkey kidney (COS-7) cells tran­ siently transfected with G-21 were examined for their ability to bind the fJ-adrenergic antagonists 125I-cyanopindolol (Fargin et aI1988). The mod­ erate, rather than high, affinity binding observed with this radioligand

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suggested, based on previous observations (Hoyer et al 1985), that G-21 might encode a 5HTla or 5HTlb receptor. Subsequently, the 5HTla­

selective agonist 3[H]-8-0H-2-(di-n-propylamino)-tetraline, or 3[H]-DPAT (Gozlan et al 1983), was shown to bind to these membranes with both high (KH 0.06 nM) and low (KL 14.5 nM) affinities (Fargin et aI1988). This ligand binding profile is consistent with the two agonist affinity states =

=

previously established for 5HTl a-binding sites in the brain (Hall et al 1985). Moreover, the potency with which various 5HT 1 a-selective agonists

and antagonists (e.g. ipsapirone and spiroxatrine) displaced 3[H]-DPAT binding to membranes from transfected cells is consistent with G-21's being a 5HTIa receptor; 5HT itself displayed a KH of 0.3 nM (Fargin et aI1988). Similar results have recently been reported for a rat 5HTl a receptor clone

stably expressed in mouse L cells (Albert et al 1990). In the presence of GTP or related analogues, receptor-G protein com­ plexes dissociate, leaving the receptor in a low-affinity state for agonists (Stryer & Bourne 1986). GTP can convert high-affinity 3[H]-DPAT binding sites to low-affinity sites in rat brain (Schlegel & Peroutka 1986) or trans­ fected cell (Albert et al 1990, Fargin et al 1988) membranes, thereby indicating that the 5HT l a receptor is functionally coupled to a G protein, both in vivo and in vitro. This is consistent with the observation that pretreatment of membranes with Bordetella pertussis toxin (PT), which modifies and inactivates some G protein species by ADP-ribosylation, also eliminates high affinity 3[H]-DPAT binding sites (Albert et aI 1990). In terms of effector coupling mechanisms, the 5HT l a receptor has been implicated in a number of different signaling pathways in the brain. In the rat or guinea pig hippocampus, 5HTIa receptors have been reported to activate (Mark stein et a1 1986, Shenker et a1 1985) or inhibit (Bockaert et al 1987, DeVivo & Maayani 1986) adenylatc cyclase, or to directly activate K+ channels through a G protein-dependent, second messenger-independent mechanism (Andrade & Nicoll 1987, Colino & Halliwell 1987). In general, the ability of a single receptor subtype to both inhibit and activate AC has been met with some skepticism. In mammalian cell lines transiently or stably expressing human (Fargin et al 1989) or rat (Albert et a1 1990) 5HTla receptors, serotonin inhibits adenyl­ ate cyclase (AC) activity in a dose-dependent manner (ECso 20 nM), as judged by its ability to antagonize the action of agents that stimulate AC SIGNAL TRANSDUCTION

=

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in these cells (such as isoproterenol, VIP, or forskolin). Serotonin by itself, however, does not stimulate cAMP synthesis. Through the work of Bockaert and colleagues, it now seems likely that the observed 5HT­ mediated activation of hippocampal adenylate cyclase involves a novel, pharmacologically distinct receptor subtype, predictably called 5HT4, which displays a relatively low ECso (100 nM) for serotonin (Dumuis et al 1988). This 5HT4 receptor may be related to another, previously "unclassifiable," subtype in the gut, which is known to promote aceyl­ choline release and smooth muscle contraction (for review, see Clarke et al 1989). In addition to inhibiting AC, the human 5HTla receptor can weakly stimulate phosphatidylinositol hydrolysis in transfected HeLa cells, but only if serotonin is present at fairly high concentrations (ECso 3.2 11M) (Fargin et al 1989). This observation is interesting because it implies that a neurotransmitter can elicit distinct cellular responses by activating a single receptor subtype, depending on the ambient concentration of the transmitter. Given the weak and ancillary nature of PLC activation, how­ ever, is this biochemical response physiologically relevent? To address this issue at the in vitro level, Raymond et al (1989) have shown that activation of 5HTl a receptors on transfected HeLa cells stimulates phosphate uptake via a sodium-dependent transport system. Because the EC50 for serotonin in this capacity is quite high (1 11M), stimulation of phosphate uptake is more likely to be mediated by PIPrPLC activation than by inhibition of AC. Additional evidence suggests that activation of protein kinase C (by the PIP 2-derived second messenger diacylglycerol) is a key step in this process (Raymond et al 1989). PT blocks all of these serotonin-evoked responses, including PI turnover, AC inhibition, and phosphate uptake, with equal potencies. In light of these findings, it is noteworthy that a serotonin-dependent Na+ pump (Na+/K+ ATPase) has been described in regions of the rat brain that contain a high density of serotonergic terminals (Hernandez & Condes-Lara1989). =

5HTlc and 5HT2 Receptors PHARMACOLOGY With a few minor discrepancies, the pharmacological profiles established for 5HTl c and 5HT2 receptors in the brain (see Schmidt & Peroutka 1989) are recapitulated in transfected mammalian cell lines. Thus, membranes prepared from NIH-3T3 mouse fibroblasts stably expressing rat 5HTIc or 5HT2 receptors have been shown to bind 12SI-Iysergic acid diethylamide C 2SI-LSD) with nanomolar affinity (Julius et a1 1989, 1990); human embryonic kidney 293 cells transiently expressing the rat 5HT2 receptor display high-affinity (KD 0.5 nM) binding sites for the neuroleptic 3H-spiperone (Pritchett et aI1988), a 5HT2- and dopa=

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mine D2-receptor antagonist that binds with moderate affinity to 5HT l a sites. The broad-spectrum antagonists mianserin and mesulergine block 5HT1c and 5HT2 receptors on transfccted cells with nanomolar potency, whereas the 5HT2-seleetive antagonist ketanserin is at least 100 times more efficacious at blocking 1 25I-LSD binding to 5HT2 receptors than to 5HT1c receptors (Julius et al 1988a, 1990). Ketanserin also competes for 3H_ spiperone binding to 5HT2 receptors on 293 cells in the nanomolar range (Pritchett et al 1988). Perhaps most significantly, 5HTlc receptors ex­ pressed in vitro bind serotonin in the nanomo1ar range, whereas 5HT2 receptors bind agonist in the micromolar range. As this difference in agonist affinity was one of the criteria originally used to distinguish 5HTl from 5HT2 binding sites in the brain (Peroutka & Snyder 1979), the 5HTIc receptor might still be considered a member of the 5HT1 receptor family in this respect. SIGNAL TRANSDUCTION In the choroid plexus, activation of native 5HTl c receptors promotes the hydrolysis of phosphatidylinositol (Conn et al 1986), thus generating the cytoplasmic second messengers IP3 and DAG. Activation of 5HT2 receptors in the cerebral cortex (Conn et al 1986), on platelets (de Courcelles et al 1985), or on aortic smooth musele cells (Kavanaugh et al 1988) has the same effect. When these receptors are expressed and activated on transfected NIH-3T3 fibroblasts, the IP 3 pro­ duced from PIP 2 hydrolysis serves to liberate Ca2+ from IP rsensitive intracellular stores. As such, receptor activation can be followed by using Ca 2+-sensitive dyes such as indo-l (Tsien 1989) to measure changes in intracellular free-Ca2+

Molecular biology of serotonin receptors.

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