The EMBO Journal vol. 1 0 no. 1 3 pp.4017 - 4023, 1991

Molecular cloning and characterization of a rat brain cDNA encoding a 5-hydroxytryptaminelB receptor

Mark M.Voigt', David J.Laurie, Peter H.Seeburg and Alfred Bach2 Laboratory of Molecular Neuroendocrinology, Center for Molecular Biology, University of Heidelberg, Heidelberg, D-6900, FRG and 2BASF, AG. Abt. Biotechnologie, Carl-Bosch-Str.38, Ludwigshafen, D-6700, FRG 'Present address: Dept. of Pharmacology and The Biotechnology Centre, University College Dublin, Belfield Campus, Dublin 4. Ireland Communicated by P.Seeburg

To date, there have been at least eight different receptors for the neurotransmitter serotonin (5-HT) identified in the central nervous system. These receptors fall into four pharmacological classes: 5-HTI, 5-HT2, 5-HT3 and 5-HT4. The 5-HT, class has been shown to contain at least four pharmacologically distinct subtypes,

5-HTIA_D. Of these, cDNAs encoding the 5-HTlA and

5-HT,c

receptors have been previously characterized. We now report the cloning and expression of a rat brain cDNA encoding another member of the 5-HT, receptor family. Transient expression of this clone demonstrated high-affinity binding of [3H]5-HT with a pharmacological profile corresponding to that of the 5-HTIB subtype: 5-CT, 5-HT > propanolol > methysergide > rauwolscine > 8-OH-DPAT. In situ hybridization revealed expression of cognate mRNA within cells of the dorsal and median raphe nuclei, consistent with previous reports that the 5-HTlB receptor acts as an autoreceptor on 5-HT terminals in this species. mRNA expression was also detected in cells within the CAl region of hippocampus, striatum, layer 4 of cortex and in the cerebellum, suggesting a previously unrecognized post-synaptic role for the 5-HTIB receptor. Key words: 5-HTIB receptor/brain cDNA/G-proteincoupled receptor/serotonin/in situ hybridization

Introduction Serotonin (5-hydroxytryptamine, 5-HT), historically one of the first clearly identified neuroactive substances, is postulated to play important roles in a multitude of cognitive and behavioural functions and dysfunctions involving feeding, depression and sex (Glennon, 1990; GonzalezHeydrich and Peroutka, 1990). This is in keeping with its diffuse distribution throughout the neuroaxis, which arises entirely from a relatively small population of neurons located in the midbrain and brainstem (Steinbusch, 1985). Unfortunately, 5-HT has proven to be the most recalcitrant of all the classical neurotransmitters in yielding information concerning the mechanisms by which it influences neuronal function, due in large part to the number of different receptor (C) Oxford University Press

proteins to which it can bind. This pharmacological complexity of 5-HT is reflected in the current identification of eight different receptor subtypes, which can be segregated into four classes: 5-HTI, 5-HT2, 5-HT3 and 5-HT4. These categories are delineated by both the binding and effectorcoupling properties of the respective prototype receptors (for an overview, see Frazer et al., 1990). To date, the 5-HT1 receptor subclass has been examined in most detail. Pharmacological studies have identified at least four unique receptors within the central nervous system belonging to this receptor family, labelled 5-HTIA-5-HTID (Hoyer et al., 1986; Peroutka, 1986; Heuring and Peroutka, 1987). More recently, a putative 5-HTIE subtype has been found in human brain homogenates (Leonhardt et al., 1989). The characteristic signature of members of the 5-HT1 receptor subclass is high-affinity binding of 5-HT (K, < 100 nM) and transduction of biological actions via an interaction with guanine-nucleotide binding proteins (G-proteins). Distinction between the receptors in this group has been made on the basis of the affinities for a series of drugs showing some subtype selectively, e.g. 8-OH-DPAT for the 5-HTIA, mesulergine for the 5-HTic and rauwolscine for the 5-HTID receptors (Frazer et al., 1990). Unfortunately, there are no antagonists available that are selective for the 5-HT, receptor family; all known antagonists also bind with high affinity to at least one other neurotransmitter receptor group. Additionally, many drug classes that act as antagonists at one subtype have been reported to be agonists at another [e.g. (-)propranolol is an antagonist at the 5-HTlA, but acts as an agonist at the 5-HTIB and 5-HTID (Schoeffter and Hoyer, 1989; Murphy and Bylund, 1989)]. This, together with the paucity of subtype-selective compounds, especially for the 5-HTIB and 5-HTID, has hampered efforts to assign specific functional and behavioural correlates to each member of this family. The availability of recombinantly expressed receptors for each of the 5-HT1 subtypes, expressed as a pure population, would enhance such characterizations and aid in the development of selective drugs. Therefore, we have undertaken the cloning and recombinant expression of 5-HT, receptors from rat brain for use as a tool in the elucidation of the specific pharmacophores and functional coupling properties for each subtype. Recent molecular cloning experiments have demonstrated that some of the proteins belonging to the 5-HT, and 5-HT2 receptor subclasses, i.e. the 5-HTIA, 5-HTIc and the 5-HT2 are encoded by members of the seven-transmembrane domain containing gene superfamily (Julius et al., 1988; Kobilka et al., 1987; Pritchett et al., 1988; Albert et al., 1990). We have taken advantage of the close sequence similarities between related members of this superfamily to aid in the cloning of new members of the 5-HT, receptor class. In this report, we describe the cloning and characterization of a rat brain cDNA encoding a 5-HTlB receptor. 401 7

M.M.Voigt et al.

Results Isolation of a putative 5-HT receptor cDNA We used the polymerase chain reaction (PCR) as a first step in our cloning of cDNAs encoding members of the 5-HT1 receptor class. Degenerate oligonucleotides directed against highly conserved sequences present in the third and seventh putative transmembrane domains of several cloned G-protein coupled biogenic amine receptors were used to amplify cDNA segments from rat forebrain cDNA. Several products, ranging in size from 400 to 800 bp, were obtained and their sequence determined. Among these products a novel sequence (RB-10) was identified that was most similar to that of the rat 5-HTlA receptor (Albert et al., 1990), suggesting that it might be a fragment derived from a cDNA encoding a 5-HT receptor. This PCR fragment was then used to screen a rat hippocampal XgtlO cDNA library (Werner et al., 1991) to obtain a full-length cognate cDNA. Several clones were isolated and characterized and the longest (2.3 kb) was chosen for further analysis.

1 36 CAAGAGCTGCGCTCCGCAGCCAGGACGAGGAGAGCTATGGAGGAGCAGGGTATTCAGTGC M E E Q G I Q C

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The nucleotide and predicted amino acid sequences of clone RB-10 are shown in Figure 1. This cDNA contains a 1158 bp open reading frame encoding a predicted protein of 43 162 mol. wt. As has been reported for most G-protein coupled receptors, RB-10 appears to be a glycosylated protein as indicated by two potential glycosylation sites which reside within the putative N-terminal extracellular domain at amino acid residues 24 and 28. Other consensus sequences for post-translation modifications include three target sites for protein kinases within the putative third intracellular loop; two for protein kinase A at threonine residues 248 and 309, and one for protein kinase C at threonine residue 243. The presence of such sites suggests that this receptor may undergo phosphorylation-mediated desensitization similar to that which has been reported for the 02-adrenergic receptor (Hausdorff et al., 1989). When compared with other cloned G-protein coupled biogenic amine receptors, RB-10 exhibits its highest degree of identity (45%) with the rat 5-HTlA receptor (Figure 2) This figure is even higher when only transmembrane domains are considered (60%). In contrast, it has only 28% identities with both the rat 5-HTic and 5-HT2 receptors (Figure 2). Based on this sequence similarity, we postulated that this clone encodes a member of the 5-HT1 receptor family. This possibility was examined in fuirther detail through the following expression studies. Pharmacological characterization of RB- 10 The pharmacological characteristics of RB-10 were examined using a transient expression system in human embryonic kidney cells (HEK-293) (Gorman et al., 1990). Steady-state binding assays with [3H]5-HT demonstrated that this ligand bound in a saturable fashion with high affinity (Figure 3A), thus supporting the postulate that RB-10 is a 5-HT receptor. The resulting saturation data, when converted to a Rosenthal plot, exhibited a best-fit to a two-site mode (Figure 3B) that yielded apparent Kd of 3 0.5 and 60 + 3.4 nM (n = 3). The observed Bmax varied between 9 and 22 pmol/mg protein for the different experimental trials. The nucleotides GTP-ys and GTP inhibited [3H]5-HT binding in a concentration-dependent fashion (results not shown),

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Fig. 1. Nucleotide and deduced amino acid sequence for clone RB-10. Numbering of nucleotides and amino acids is shown on the right. Squared amino acid residues are potential glycosylation sites, ovals marking potential protein kinase A target residues and an underline delineating a potential site for protein kinase C.

suggesting that the two affinity states observed were due to RB-10 interaction with G-proteins. No consistent effect of 5-HT on either basal or stimulated cAMP levels was observed, however. Competitive inhibition studies were then performed in order to determine the specific 5-HT subclass to which RB-10 belongs. As seen in Figure 4 and Table I, the 5-HT agonist 5-carboxyamidotryptamine (5-CT) exhibited high affinity for the expressed clone. This confirmed that RB-10 is a member of the 5-HT, receptor family (Frazer et al., 1990) while at the same time excluding the possibility that it encodes a 5-HTlE receptor, at which 5-CT has 100-fold lower affinity (Leonhardt et al., 1989). Two putative rat hippocampal 5-HTIA receptor subtypes have been described that act to alter adenylate cyclase activity in opposite fashions (Markstein et al., 1986; Dumuis et al., 1987), with the inhibitory receptor already cloned (Albert et al., 1990). The possibility that RB-10 is the missing stimulatory 5-HTIA receptor was, however, excluded by the low affinity of the 5-HTIA selective agonist 8-OH-DPAT (Hoyer et al., 1986; Peroutka, 1986; Heuring and Peroutka, 1987) for RB-10

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Fig. 3. Radioligand binding analysis of RB-10 trans fected cells. (A) Saturation analysis of [3H]5-HT binding to men: RB-10-transfected HEK cells. Shown are the amoun ts of specifically bound [3H]5-HT deterrnined at various concentratiol ns, which were calculated by subtracting the total amount of bindinl g at those concentrations in the presence of excess unlabelled '5-HT (non-specific binding) from values obtained in the absence of unl abelled ligand (total binding). The results shown are the means of dupliccate determinations and are representative of three independent experim ents. (B) Rosenthal plot of data from (A). The data presented in (A) weere transformed, using the GraphPad program, into bound/free (B/F) values, which were then plotted against their corresponding bound (B) values. Values for this experiment, calculated using the GraphPad program, were KDI = 4.2 nM, KD2 = 67 nM.

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4020

for a selected group of drugs at the expressed RB-1O receptor to be 5-CT, 5-HT > propranolol > methysergide > rauwolscine > 8-OH-DPAT. This rank order as well as the apparent Ki values for these compounds correlate very well with those found for the rat 5-HTIB receptor, thus allowing us to assign RB-l0 as a 5-HTIB receptor. The effects of guanine nucleotides on binding strongly suggest that RB-1O interacts with a G-protein in HEK 293 membranes. Therefore, our inability to measure reliably 5-HT-induced changes in either basal or stimulated cAMP levels most probably reflects a limitation of this assay using cells transiently expressing RB-10, although the absence of an appropriate G-protein or activation of an effector pathway other than adenylate cyclase could be alternative explanations. Neurons which utilize 5-HT as a transmitter are relatively few in number, and are grouped in discrete nuclei in the midbrain and brainstem (Steinbusch, 1985). The RB-10 mRNA was detected in cell bodies lying within many of these groups (e.g. dorsal raphe, dorsal interpeduncular nucleus and central grey), suggesting that this clone is expressed by serotonergic neurons and encodes a 5-HT autoreceptor. This is in agreement with studies in the rat that have demonstrated the regulation of 5-HT release from forebrain terminals by a 5-HTIB receptor subtype (Engel et al., 1986). Of particular interest was the finding that neurons in many forebrain regions also express RB-10 InRNA, especially the CAI pyramidal neurons. This, together with results from receptor autoradiographic studies showing high levels of 5-HTlB receptor binding in subiculum but very low levels in CAI (Pazos and Palacios, 1985; Waeber et al., 1989; Radja et al., 1991), suggest that these receptors are located on CAl efferents in the subiculum, and not on pyramidal cell dendrites or somas within the CAl. Projections from cells within the caudate-putamen and raphe nuclei could also account for the contrasting dense 5-HTIB receptor binding and undetectable mRNA levels within the globus pallidus and substantia nigra. The expression of RB-10 mRNA by non-serotonergic neurons in hippocampus and caudate-putamen implicates 5-HTIB receptors as previously unrecognized post-synaptic mediators of some 5-HT effects on limbic and motor functions. Additionally, a functional role for 5-HTIB during the formation and consolidation of striatal projections is suggested by the higher expression levels for RB-10 mRNA in the young rat caudate-putamen when compared with the adult animal. Interestingly, RB-1O exhibits a higher identity to the canine orphan receptor RCD-4 (Libert et al., 1989) (63%) than it

Molecular cloning of a brain 5HTlB receptor

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Fig. 6. Emulsion autoradiographs showing the location of RB-10 mRNA in rat cerebellum and hippocampus. (A) Cerebellum, dark field microscopy. Arrowheads indicate Purkinje neurons. Abbreviations: 100 um. m, molecular layer; g, granule cell layer. Scale bar Scattered silver grains in the molecular and granule cell layers are non-specific signals. (B) and (C) Hippocampus, bright field and dark field microscopy, respectively. Arrowheads indicate the CAI -CA3 border, as observed by Nissl stain. Arrows indicate interneurons hybridizing with probe. Abbreviations: or, oriens layer; py, pyramidal 100 jtm. cell layer; rad, radiatum layer. Scale bar =

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Fig. 5. Film autoradiographs showing the distribution of RB-10 mnRNA in rat brain sections. (A) Sagittal section. Scale bar = 2 mm. (B) and (C) Coronal sections cut at the level of the nucleus accumbens and substantia nigra, respectively. Scale bar = 2 mm. (D) Coronal section cut at the level of the dorsal raphe. Scale bar = 1 mm. Abbreviations: CAl, field of hippocampus; CB, cerebellum; CG, central grey; CPu, caudate -putamen; Ctx, neocortex; DG, dentate gyrus; DR, dorsal raphe; GP, globus pallidus; IP, interpeduncular nucleus; MGM, medial geniculate, medial part; NA, nucleus accumbens; OT, olfactory tubercle; PaS, parasubiculum; R, red nucleus; S, septum; Sb, subiculum; SN, substantia nigra; SuG, superficial layer of superior colliculus; T, thalamus: IV, layer IV of neocortex.

does to the rat 5-HTIA gene (43%), suggesting that the RDC4 product may also encode a 5-HT, receptor subtype. We have recently cloned and characterized the rat RDC4 homologue and found that it encodes a receptor with a

5-HTID-like pharmacological profile (Bach,A., Unger,S.,

Palacios,J., Sprengl ,R., Seeburg,P. and Voigt,M., submitted), and another group has also reported similar findings with the human homologue (Hamblin and Metcalf, 1991). The presence of this receptor in the rat brain serves to confirm that the rat expresses genes encoding the 5-HTIB and the 5-HTlD receptors. The overriding difficulty with investigation into 5-HT function in the central nervous system has been the pharmacological complexities presented by the large number 4021

M.M.Voigt et al.

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of receptors for this substance. The availability of recombinant receptors expressed as pure populations is a first step in the elucidation of the roles that these receptor subtypes play in various 5-HT-mediated behaviours and dysfunctions. Additionally, the availability of stable cell lines expressing these receptors can play an important role in the design of subtype-selective drugs. The availability of such selective compounds is especially crucial for future studies investigating the functional roles in vivo of the 5-HTIB receptor subtype which, due to both its pre- and post-synaptic location, may play an important role in the aetiology and treatment of neuropsychiatric disorders (Glennon, 1989; Gonzalez-Heydrich and Peroutka, 1989).

Materials and methods Materials All basic chemicals along with 5-hydroxytryptamine HCI pargyline, and (-)propranolol were obtained from Sigma Chemical Co. and 5-carboxyamidotryptamine, rauwolscine, methysergide, ICS205,930 and 8-OH-DPAT were obtained from Knoll Pharmaceuticals (Ludwigshafen, FRG).

Polymerase chain reaction Approximately 10 ng of rat forebrain cDNA was used as template and two degenerate oligonucleotides, GCR-6 (ATATAAGCTTCTGTG(C/

T)GCCAT(C/T)T(T/G)CCCT(G/T)GACCGCTAC) and GCR- 14 (TATGAATTCG(T/C)(G/A)TAGA(T/C)GATGGG(G/A)TTG), corresponding to amino acid residues 136-144 and 362 -367 respectively, were present at a final concentration of 1 MM in a standard PCR mixture. PCR was carried out using a 1.5 min denaturation at 94°C, 2.5 min annealing at 55'C and 4 min extension at 72°C, for a total of 30 cycles. After electrophoresis through 1.5% low melting point agarose (BRL-GIBCO), product bands of 0.4-1 kbp were excised. The DNA was isolated, digested with Hindml and EcoRI and the fragments subcloned into M13mpl8 vectors for sequencing using the dideoxy chain termination method. cDNA library screening The RB-10 PCR fragment was 32P-labelled using a random primer kit (Boehringer-Mannheim) and used to screen - 1 x 106 recombinant phage from a rat hippocampal cDNA library in XgtlO using previously described conditions (Werner et al., 1991). Several positives were isolated after further rounds of screening and subcloned into M13 for sequencing. An insert containing a full-length coding sequence was then subcloned into a previously described mammalian expression vector (Gorman et al., 1990) for further studies. Cell culture and transfections for transient expression Human embryonic kidney cells 293 (HEK 293) (ATCC CRL 1573) at 50-70% confluency were transfected as described (Chen and Okayama, 1987), with growth medium replaced 18 h post-transfection. Both the binding and cAMP assays were carried out 48 h post-transfection. cAMP assays were carried out as previously described (Braun et al., 1991), with the exception that cells were co-transfected with the rat luteinizing hormone receptor construct pCLHR (Braun et al., 1991) together with the RB-10 construct, using 10 Mig'10 cm plate of an equal mix of the two plasmids.

Binding

Fig. 7. Film autoradiographs showing the developmental expression profile of RB-10 mRNA in the rat brain. (Ai) rat embryo E17, (ii) rat embryo E19, both sagittal sections. (Aiii)-(D), horizontal sections of rat brain at ages (Aiii), P0; (B), P6; (C), P12 and (D), adult. Abbreviations: AON, accessory olfactory nucleus; CA3, field of hippocampus; Ctx IV, neocortex, layer IV; Di, diencephalon; ER, entorhinal cortex; Hi, hippocampal formation; L, liver; Me, mesencephalon; Mt, metencephalon; My, myelencephalon; OB, olfactory bulb; SC, spinal cord; SCo, superior colliculus. Punctate labelling in the embryonic liver is non-specific. Other abbreviations as in Figure 6. Scale bar 4.8 mm. =

4022

Transfected cells were washed twice with warmed PBS and then rinsed off the plate with ice-cold PBS. Plates were pooled and the cells pelleted. After exchanging the PBS with the standard binding assay buffer (50 mM Tris-HCI, pH 7.7, 4 mM CaC12, 10 MM pargyline and 0.1% ascorbic acid) (Peroutka, 1986), the cells were resuspended and membranes prepared as previously described (Werner et al., 1991). The final pellet was then resuspended in binding buffer at a concentration of 0.04 mg protein/ml, and 0.4 ml of this homogenate used per assay point. Binding of [3H]5-HT (New England Nuclear, 23.8 Ci/mmol) was performed at 22°C for 20 min in a final volume of 0.5 ml, after which the reaction was quenched by addition of 5 ml ice-cold buffer and filtered through GF/C filters (Schleicher and Schuell Number 34). Filters were washed twice with 5 ml ice-cold buffer and the counts per minute (c.p.m.) bound determined by liquid scintillation counting. For saturation analysis, [3H]5-HT was used at concentrations ranging from 0.01 nM to 600 nM, with non-specific binding defined in the presence of 10 MM unlabelled 5-HT. Competition assays for [3H]5-HT binding were performed using drug concentrations ranging from

Molecular cloning of a brain 5HTlB receptor 0.1 to 50 mM and 1 nM [3H]5-HT, again with non-specific binding determined using 10 AM unlabelled 5-HT. No detectable specific binding could be detected in non-transfected HEK cells at any concentration of [3H]5-HT tested. In situ hybridization These experiments were carried out as previously described (Wisden et al., 1991). Hybridization was performed on 14 zm thick sections of whole embryos at 17 (E17) and 19 (E19) days of gestation, and on brains of post-natal rats of ages 0 (P0), 6 (P6) and 12 (P12) days, in addition to adult animals. Oligonucleotides used for hybridization were SX-1 and SX-2, which were complementary to codons encoding amino acid residues 149 - 164 and 249-264, respectively. Both oligonucleotide probes gave identical patterns of hybridization. As an additional check on signal specificity, competition experiments in which radiolabelled probes were hybridized to sections in the presence of excess (50-fold) unlabelled probe were performed.

Radja.F., Laporte.A.-M.. Daval.G.. Verge.D., Gozlan,H. and Hamon.M. (1991) Neurochem Int., 18, 1-15. Schoeffter,P. and Hoyer.D. (1989) Naunyn-Schmieclebergs Arch. Pharmacol., 340, 285-292. Steinbusch,H.W.M. (1985) In Bjorklund,A., Hokfelt,T. and Kuhar,M.J. (eds), Hamtdbook of C7iemical Neuroanatomv. Vol. 3. Elsevier, Amsterdam, pp. 68-125. Waeber,C., Dietl,M.M., Hoyer.D. and Palacios,J.M. (1989) NaunvnSchmiedeberg.s Arch. Phanracol., 340, 486-494. Werner,P., Voigt,M., Keinanen,K., Wisden,W. and Seeburg,P.H. (1991) Nature, 351, 742-744. Wisden,W., Morris,B.J. and Hunt,S.P. (1991) In Chad,J. and Wheal,H. (eds), Molecular Neurobiology: A Practical Approach. Vol. 2. IRL Press/Oxford University Press, Oxford, UK.

Received on Auglust

15, 1991;

revised on September 30, 1991

Acknowledgements We would like to thank Drs W.Wisden, R.Sprengel and P.Giershik for their insightful discussions, S.-Grunewald for dedicated assistance with tissue culture and A.Herold for expert technical assistance with sequencing. M.M.V. is a recipient of an Alexander von Humboldt Fellowship and D.J.L. holds a European Science Exchange Fellowship awarded by the Royal Society (London). This work was supported by the Bundesministerium fuir Forschung and Technologie, grant No. BCT 364 Az 231/7291, the Deutsche Forschungsgemeinschaft (SFB 317/B9) and the Fonds der Chemischen Industrie to P.H.S.

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