More serotonin receptors?

RECEPTOR DIVERSITY

THERESA BRANCHIEK

More

serotonin

receptors?

Molecular cloning has identified 13 types of serotonin receptor. They differ in sequence, pharmacology, distribution and mechanism of action, but the reasons for so much diversity remain unclear. Serotonin 6hydroxytryptamine, 5-HT) is a neurotransminer that has profound and diverse effects in both the central and peripheral nervous systems [ 1 ] In the central nervous system (CNS)!, serotonin affects mood, sleep and arousal, satiety, vomiting, cardiovascular regulation, temperature control, pain, sedation, anxiety and depression. In the periphery, the primary actions of serotonin are on the gastrointestinal tract and cardiovascular system, but it also affects the respiratory tree and the genital-urinary system. Serotonin receptors have been found on neurons, glial cells, smooth muscle, endothelial and epithelial cells, blood cells and platelets. The actions of serotonin on cells, tissues or intact animals can be excitatory or inhibitory, slow or fast in onset, long or short in duration, rapidly or slowly desensitizing, and thoroughly confusing.

Subtypes

of serotonin

@HT,)

5-HT,,

(5-HT,,)

S-HT,,

&HT,c)

of adenylate

cyclase

Stimulation

of phospholipase

C

Stimulation

of adenylate

cyclase

Stimulation

of adenylate

cyclase

Stimulation

of adenylate

cyclase

Unknown

P-91. receptor

5-HT,,

receptor

One of the surprises of molecular cloning of serotonin receptors has been the extreme diversity of subtypes within the family. At present, we can tentatively assign seven subdivisions to the family, with multiple subtypes within many of the subdivisions (Table 1). With the exception of the 5-HT3 receptor, the others have been shown (or are expected) LO be m,embers of the G-protein coupled receptor superfamily, members of which are predicted to have seven CLhelices spanning the plasma membrane. The 5-HT, receptor family contains five members so far, each coupled to the inhibition of adenylate cyclase activity [2]. The 5-HT2 family has three members, each coupled to phosphoinositide hydrolysis [3]. The 5-HT3 receptor is a ligand-gated ion channel and possible subtypes are the subject of active investigation at the moment. Cloning of the 5-HT4 recezptor(s) has not yet been reported but as the pharmacologically identified 5-HT4 stimulates adenylate cyclase activity, it is also predicted to be a G-protein coupled receptor. Three new classes of serotonin receptor h,ave recently been reported, also with primary structures that predict G-protein coupling: the 5-HT, receptor subfamily [4,5], the 5-HTg receptor [63 and a seventh 5-HT receptor, provisionally named 5-HT,

5-HT,

Inhibition

diversity

One of the intriguing questions raised by the cloning of the five 5-HT, receptor genes is ‘why?‘. As they all respond to the same natural neurotransmitter, all couple to adenylate cyclase inhibition, and are all expressed in @ Current

Biology

?

adult human tissues, why have five separate genes been retained in the human genome? One factor contributing to this diversity is likely to be that related receptor subtypes couple to the same basic signalling pathway through different G proteins. Suggestive evidence in support of this speculation has come from observations of the different abilities of various mammalian cell lines to respond functionally to serotonin after transfection with different subtypes of 5-HT, receptor. For example, the 5-HTlDp and 5-HT1, receptors, but not the 5-HTlD, receptor, robustly inhibit adenylate cyclase activity when expressed in murine Y-l cells (unpublished observations). As G proteins are heterotrimeric, and there are known to be three a subunits for the inhibitory G protein G, two CCsubunits of Go, and four p and six y subunits in various cell types, there are quite enough combinatorial possibilities for cell-specific or tissue-specific pathways of signalling through five 5-HT, receptor subtypes. It should be noted that there are also four isoforms of adenylate cyclase, adding to the possible combinations of downstream response elements. The ability of different 1993, Vol

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Biology 1993, Vol 3 No 5

receptor subtypesto gateion channelsdirectly,which may be the first event of signalling from the receptor, has not yet been assessed. In addition to specificity of G-protein coupling, downstream branchpoints in ,the signalling pathwaystriggered by diierent 5-HT receptors m ight differ. The 5-HT1, receptor has been shown to elevate intracellular calcium [l]. What about the other 5-HT, subtypes?Studies of 5.HT,b, and 5-HT,,o receptors have indicated that activation of each subtype leads to calcium mobilization from intracellularstores through G proteins that are sensitiveto ADP-ribosylationby penussis toxin [lo], with a half-mtimal serotonin dose similar to that determined for adenylate cyclaseinhibition. F’arallelstudies are under way for the 5-HT,, and 5-HTlF receptors. Are the different subtypes of 5-HT, receptor diiferentially distributed in the brain and periphery?Distributions have been assessedby analysisof mRNA levelsand there seemsto be a substantialoverlap, particularly in several key structures in the brain, including the raphe nuclei [2]. However, 5HT, receptors can be differentially distributed on a cellular or subcellular level, for example between the somatode:ndriticportion and the synaptic terminals of neurons of the raphe nucleus [l]. In the case of the 5-HT,, receptor, the mKNA in the raphe nucleus has been shown to encode receptors on the soma and dendrites. The 5-HT,, receptor seems to be a terminal autoreceptor,present on serotonergicneurons and allowing serotonin to regulateits own release.A&ysis of the other subtypes must await the developmentof selectiveantibodies or specific radiolabelledligands. The distribution of receptors in the periphery may show greater variation. For example, the human 5-HT,, mRNA is most abundant in spleen and lymph nodes [ll J, whereas the human 5-I-IT,, receptor is most abundant in the uterus and mesentery [2]. In addition to these considerations, other potential diiferences between closely relatedsubtypesinclude the time coursesof activation,desensitizationor regulation, different ratios of high to low agonist atlinity states,developmentalregulation, or some other functional difference - there must presumablybe some justification for their preservationas separategenes in the genome.

Newly discovered

seraltonin receptor

subtypes

Three additional families of serotonin receptor have recently been described.Two membersof the 5-HTgfamily, 5-HT, and 5-HTsB(also known as 5-HT,, and 5-HT,p) have been cloned from the mouse [4] and rat [ 51 brain. Their pharmacologicalprofiles indicate some similarity to 5-HT, receptors, on the basis of their binding affinities for the drugs 5-carboxamidotryptamine(5CT) and methiothepin. It has become increasingly clear, however, that the use of restricted drug criteria for receptor classification will need to be revised. As was the case previously for the 5-HT,!, receptor [2], no correspondence is apparent between either of these two 5-HTs receptors

and any previously described 5-HT receptor. In addition, at the level of deduced amino-acid sequence,these m receptors form a subfamily with little sequencesimilarity to any of the cloned receptors for serotonin or other biogenic amines [4,5]. In neither study [4,5] was it possible to demonstratefunctional coupling of the 5-HT, receptors to either adenylate cyclaseactivity or phosphoinositide metabolism- their signaltransductionmechanismremainselusive,in spite of their expressionin a variety of cell lines containinga tide range of G proteins. Perhapstheir primary coupling is to ion channels,the Na+/K+ pump, or guanylatecyclase. The 5-HT, receptor has a widespreaddistribution in the CNS,including the dorsal raphe nucleus.By contrast, the 5-HT5n receptor has a much more lim ited distribution, but is also found in the dorsal raphe - the set of poten tial serotonin autoreceptorscontinues to expand. Neither transcript was detected in any peripheral organ studied, raising the possibility of CNS-specificdistribution for the 5HT, subfa&ily. The 5.HT6 receptor [6] is the first of the cloned 5-H? receptors that has been shown to couple to adenylate cyclase stimulation. The pharmacologicalprofile of this receptor is, however, distinctly different from that of the 5-HT,+receptor, which stimulates adenylate cyclasebut has so far been characterized only pharmacologically. The 5-HT6 receptor has some characteristicstypical of dopamine receptors, including its affinity for the drugs clozapine and pergolide, and its predominant distribution in the striatum, a brain region rich in dopamine receptors. Both ligand binding and functional responses of the 5HTg subtype are highly sensitiveto a number of tricyclic anti-depressantand anti-psychoticdrugs, indicating an expandedrole for serotonin receptorsin neuropsychiatric disorders.As with the 5HT5 receptors, no 5-HT6 transcriptswere detected in any peripheral organs. A second adenylatecyclase-stimulatory5-HT receptor has been cloned [7,8]. The sequence and pharmacological profile of this receptor are distinct from any of the other cloned mammalian5-HT receptors,including 5-HT6.It has high at&&y for 5-CT and low aflinity for sumatriptan,and thus resembles a component with high-a&ity 3H-5CT binding that was described recently [12]; it is not the 5-HT4 receptor. A receptor clone with similar properties was obtained from rat brain [9] but no functional COUpling was detected; its precise relationship to the newly cloned cyclase-stimulatory receptor is unclear.Using pharmacologicalcriteria, the newly cloned cyclase-stimulatory receptor could be consideredto be an additional member of the 5-HT, subfamily,as indicated by its high affinity for 5-CT and methiothepin. But as it stimulates rather than inhibits adenylate cyclase activity, and it has a primary sequencewith only 47-52% similarity to other human 5-HT, subtypes,it is potentially a ‘5.HT,’ receptor. Much progresshas been made towards understandingthe molecular basis of serotonin receptor variation. Nevertheless,severalknown subtypes, and undoubtedly some

DISPATCH

-

unknown subtypes,remain to be cloned. Why do we need so many serotonin receptor subtypes?As the 5-HT,, and 5-HT,, receptors both couple to adenylatecyclase inhibition, and both function as autoreceptors,what difference allows 5-HTIA to function as a somatodendritic autoreceptor whereas 5-HT,, functions as a terminal autoreceptor?Which members of the 5-HT, family can activateKf channels?Wlhy are the mRNAs for so many subtypes found in the raphe nuclei - are they colocalizedin the samecells?Why does serotonin also use a ligand-gatedion channelreceptor,whereasthesehavenot been found in dopamine or norepinephrine signalling? Understanding the differences between species in the pharmacologyof the clo:nedsubtypeswill help to relate thesesubtypesto in vivo and in vitro models of serotonin function. Determining the physiologicalrole of each subtype and its role in human diseasewill occupy at least the next decade.The availabilityof sets of cloned human receptorstransfectedinto cell lines will be instrumentalin the designof highly selective,high afhnity ligandsfor each receptor subtype.These studies should lead to the development of more potent tlherapeuticagentswith fewer side effects,as well as a better understandingof the molecular basisof the many roles of serotonin in the body. Although there has been much focus on the cloning and characterization of serotonin receiptorgenes,it is clear that the use of these cloned receptorsfor the investigationof normal and diseasedneural processesremainsa challengefor the future.

References 1. FOZARDJR, SAXENAPR: Serotonin: molecular biology, receptors, and functional effects I3asel: Birkhauser Verlag; 1991. 2. ADHAM N, KAO H-T, SCIUXHTERI, BARD J, OLSENM, URQUHART D, DURKINM, HARTIGP, WEINSHANKR, BRA~CHEKT: Cloning of another human serotonin receptor (5.HTrE): a fifth 5-HTr receptor subtype coupled to the inhibition of adenylate cyclase. Proc Natl Acad Sci USA 1993, 90:408412. 3. HUMPHEEYPPA, HAETIGPR, HOVERD: .A reappraisal of 5-m receptor classification. In Proceedings of the Second International

Sjvnposiam on Serotonin: From Cell Biology to Pharmacology and Expevimental Therapeutiw; Dordrecht: Kluwer Academic Publishers; 1993. 4. MATIES H, BOSCHERTU, AMUUKY N, GRABHF R, PIASSATJ-L, MUXATELLI F, MAT~EI M-G, HEN R Mouse 5-hydroxytryptamine, and 5-hydroxytryptamine5n receptors define a new family of serotonin receptors: cloning, functional expression, and chromosomal localization. Mol Pbarmacol 1993, 43:3&319. 5. EELANDERM, IOVENBERG T, BARRONB, DE Ixcw L, DANIEI~ON P, RACKE M, SLONEA, $&GAL B, FOVE P, CANNON K, ET AL.: Two members of a distinct subfamily of serotonin receptors differentially expressed in rat brain. Proc Nat1 Acud Sci USA 1993, in press. 6. MONSMAF, SHENY, WARD R, HAMEZN M, SIBLEYD: Cloning and expression of a novel serotonin receptor with high affinity for antipsychotic drugs. Mel Pharmacoll993, 43:32C-327. 7. BARD J, ZGoMiXCK J, ADHAM N, BRANCHEKT, WElNSHANKR Cloning of a novel human serotonin receptor functionally coupled to adenylate cyclase stimulation. Sot Neurosci Abstr 1993, in press. 8. HEN R, AMWKY N, PLAS~AL JL: Cloning and characterization of a novel mammalian serotonin receptor that activates adenylate cyclase. Sot Neurosci Abstr 1993, in press. D: 9. SHEN Y, MONSMAF, GERFENC, MAHAN L, JOSE P, SIF.$LEV Molecular cloning and characterization of a cDNA encoding a serotonin-related G-protein-linked receptor. Sot Neurosci Abs 1992, l&465. 10 ZGOMBICKJ, BORDENL, COCHRANT, Kuc HAREWICZS, WEINSHANK R, BRANCHEKT: Dual coupling of the cloned 5-HT,n, and 5-HTrop receptors to adenylate cyclase inhibition and elevation in intracellular calcium concentrations via pertussis toxin-sensitive guanine nucleotide bindiig proteins. SOC Neuvosci Abs 1993, in press. 11 KOBILKA BK, FR~ELLET, COLLINSS, YANG-FENGT, KOBILKA TS, FRANCEKU, LEFKOWITZRJ, CARON MG: Au iurrordess gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature 1987, 32975-79. 12. MAHLE CD, NOWAK HP, MATEON RJ, HURT SD, YoCo\ FD: [3H]5Carboxamidotryptamine labels multiple high atkity 5-HT&ilte sites in guinea pig brain. Eur J Phamzacol 1991, 205~323-324.

TheresaBranchek,Departmentof Pharmacology,Synaptic PharmaceuticalCorporation, 215 College Road, Paramus, New Jersey07652,USA.

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