Human Serotonin Id Receptor Is Encoded by a Subfamily of Two Distinct Genes: 5-Ht1da and 5-HT1DP RICHARD L

Home , Tryptamine

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 3630-3634, April 1992 Pharmacology Human serotonin iD receptor is encoded by a subfamily of two distinct genes: 5-HT1Da and 5-HT1DP RICHARD L. WEINSHANK*, JOHN M. ZGOMBICK, MARY J. MACCHI, THERESA A. BRANCHEK, AND PAUL R. HARTIG Synaptic Pharmaceutical Corporation,t Paramus, NJ 07652 Communicated by Eric R. Kandel, January 17, 1992 (receivedfor review September 16, 1991)

ABSTRACT The serotonin iD (5-HT1D) receptor is a phar- MATERIALS AND METHODS macologicaily defied binding site and functional receptor site. Observed variations in the properties of 5-HTID receptors in Cloning and Sequencing. A human placental genomic li- different tissues have led to the speculation that multiple brary (Stratagene) was screened with the 1.3-kilobase (kb) receptor proteins with slightly different properties may exist. HindIII-Sph I genomic DNA fragment from the canine RDC4 We report here the cloning, deduced amino acid sequences, clone (12). The probe was labeled with 32P by random priming pharmacological properties, and second-messenger coupling (15). Hybridization and Southern blot analysis were per- of a pair of human 5-HTID receptor genes, which we have formed as described (16). For subcloning and further South- designated 5-HTID, and 5-HTDp due to their strong similar- ern blot analysis, DNA was inserted into plasmid pUC18. ities in sequence, pharmacological properties, and second- cDNA clones were isolated by screening a human hippoc- messenger coupling. Both genes are free of introns in their ampal AZAPII cDNA library (Stratagene) with an oligonu- coding regions, are expressed in the human cerebral cortex, cleotide probe derived from the canine RDC4 sequence (17) and can couple to inhibition of adenylate cyclase activity. The (corresponding to amino acids 165-188 in transmembrane pharmacological binding properties ofthese two human recep- region IV). Overlapping oligomers were labeled with tors are very similar, and match closely the pharmacological [32P]dATP and [32PjdCTP by synthesis with the large frag- properties ofhuman, bovine, and guinea pig 5-HTID sites. Both ment of DNA polymerase. Hybridization was performed as receptors exhibit high-affinity binding of sumatriptan, a new described (16). DNA fragments cloned in the AZAPII vector anti-mIgae medication, and thus are candidates for the were excised by helper phage and recircularized to generate pharmacological site of action of this drug. subclones in the pBluescript (Stratagene) phagemid vector. Nucleotide sequence analysis was done by the dideoxynu- cleotide chain-termination method (18) on denatured double- The serotonin iD (5-HT1D) receptor was initially character- stranded plasmid templates with Sequenase (United States ized by radioligand binding procedures using membranes Biochemical). derived from the bovine caudate nucleus (1). In physiological assays, the has been Expression. The entire coding region of the 5-HTID, or 5-HTID receptor subtype shown to 5-HTDf3 receptor gene was modulate vascular tone (2) and to inhibit subcloned into the expression neurotransmitter vector pcEXV3 (16) or pSVL (Pharmacia). Stable cell release through its role as a presynaptic heteroreceptor (3, 4) lines were generated by cotransfection with pcEXV3-5-HT1,D or or as a terminal autoreceptor (5). The 5-HT1D receptor is and known to be a G based pSVL-5-HTD3 pGCcos3neo, using the calcium phos- protein-coupled receptor upon its phate method. Cells were grown as functional coupling to inhibition and the monolayers and selected adenylate cyclase (6) for antibiotic resistance (16). Stable transfectants were sensitivity of high-affinity agonist ([3H]5-HT) binding to screened to guanine nucleotides the specifically bind [3H]5-HT, and two clones, (7). Sumatriptan, only 5-HTlD- and were selected for selective yet identified has been to 5-HTD1 5-HTlDp, pharmacological ligand (8, 9), reported characterization. No specific of mem- effectively treat acute binding [3H]5-HT to migraine (10). branes from sham-transfected cells was detected. were We have identified a Cells recently previously cloned G protein- grown as monolayers to 100% coupled receptor that to the confluency before membranes displayed high homology 5-HT1A were harvested for binding assays (16). Freshly prepared receptor, RDC4 (11), as a canine receptor 5-HT1D subtype membranes for radioligand binding assays were prepared (12). In the current study, we have used this RDC4 gene as from stably transfected cells (12). a probe for the isolation of its human homolog from a human Radioligand Bing Studies. Binding studies were con- hippocampus cDNA library. As described below, this human ducted clone has been and with [3H]5-HT (24-30 Ci/mmol; DuPont-NEN; 1 Ci = sequenced, transfected, characterized 37 as the and and has been shown to encode a human GBq) radioligand, 5-HTlD assay conditions in 5-HT1D receptor the absence of masking ligands were as described subtype. A preliminary report describing some of (12). properties Occupancy studies were performed with [3H]5-HT concen- this clone has been published (13). Subsequently, a report trations from to appeared describing a human clone that is ranging 0.25 50 nM, and competition studies 5-HT1D receptor were performed with 5 nM [3H]5-HT. Binding data were very similar in pharmacological properties (14). We have analyzed by computer-assisted nonlinear regression analysis designated this first human 5-HT1D receptor gene, which is (ACCUFIT and gene RDC4 in the dog, as 5-HTlDa. In addition, we have ACCUCOMP, Lundon Software, Chagrin Falls, cloned a highly homologous, yet distinct, 5-HT1D receptor OH). IC50 values were converted to Ki values by using the (5-HTlDp) from a human genomic library. These two 5-HT1D Cheng-Prusoff equation (19). Drugs were obtained as de- subtypes represent two closely related members of a sub- scribed (12). family of 5-HT1D receptors.t Abbreviation: 5-HT, serotonin (5-hydroxytryptamine). *To whom reprint requests should be addressed. The publication costs ofthis article were defrayed in part by page charge tFormerly Neurogenetic Corporation. payment. This article must therefore be hereby marked "advertisement" tThe sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. M81589 and M81590). 3630 Downloaded by guest on September 28, 2021 Pharmacology: Weinshank et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3631 Functional Studies. Intracellular cAMP levels were deter- sequence analysis. Complementary portions of the clones mined (12) by radioimmunoassay (Advanced Magnetics, were then ligated at an internal Bgl II restriction site (corre- Cambridge, MA). Dose-response curves for 5-HT and suma- sponding to nucleotides 624-629) to obtain a full-length triptan were obtained by using Y-1 cells stably transfected clone, designated 5-HTlDa. Overall, 88% amino acid se- with the 5-HT1D9 receptor gene. Functional response curves quence conservation was observed between the dog RDC4 were fit to a four-parameter logistic equation to obtain EC50 sequence and the human receptor over 377 amino acids. An and Emau values (GraphPAD InPlot, San Diego, CA). additional genomic clone, hpll, was also isolated and characterized. A comparison ofthe deduced amino acid sequence ofclone hpll with previously characterized neurotransmitter RESULTS receptors indicates that the protein product of hpll is a We have screened a human genomic placental library (Strat- memberofthe G protein-coupled receptor family. Clone hpll agene) with a 1.3-kb Hind III-Sph I restriction fragment contains an uninterrupted open reading frame that encodes a derived from the canine genomic clone RDC4 (12). Two ofthe protein of 390 amino acids. clones obtained, hp8 and hp84, were nonoverlapping partial Amino acid sequence comparison shows that hpll is clones that represented human homologs ofthe canine RDC4 closely related to, but distinct from, the 5-HT1,D receptor receptor. A human hippocampus cDNA library was subse- (Fig. 1). The overall amino acid identity between 5-HT1,D and quently screened with an oligonucleotide probe derived from hpll is 63%, with 77% amino acid identity within the trans- transmembrane region IV of the canine clone RDC4. Two membrane regions alone. This high amino acid homology, overlapping clones were isolated and characterized by DNA particularly in the transmembrane regions, is characteristic of

liT,,,. MikMPs L N sA E G E.PL P :A 15 5-HT M.EE P..Aac A P.- P P PA SE T P. 23 5-HT^, M D V L S P.G a G N N I T S P P A P 18 5-HTc M V N L G N A V R S L L M H L I G L L V w Q F D S I S P VA A 32 5-HT2 M E I L C E D N S L SS I P N S L M O L G D G P R L Y H N D F N S R D A N T S E A SN W T I DAE 50

5-%....SN RSCN A T ET SE A W DPRT L SALK I SL AV VL sV.IITL.ATl .SNA'-VV'L T-T.: 62 5-HT,2# NL S':S'A P.S ON':C S'A KY ODDSY S LP VK V'LLVM L L AL -T.L.A-T T L SNAl VIAT:'1 73 5-AT1A FE T G GN T T'G I.:SV TVSV S ...... VI T SLLLS.GT L I FC''A V L:GN: A C:V:VA-A 60 5-HT2c. I VTD T FNS S'D GG RL F FP D GVSN W P AL SI V VI. I IMT IdGONI. LVIMN A 78 5-AT, Nt R T N L S C E G Y L P P T C L S L H L S E K N W S A L L 'T T V V II I .T. I A G N I L V.I.:4MA 98

5-HT,,, L L T ft K L PA -Y L I G'S L A T J J) L IL 11VALSM V N L .C 11 5~T,,[, V T ft ~Tft K LU T P ~A N Y L L A S L A V -T: D L: L VX S I L. V N4 P 1. 5 T M:YV-T. V. .TGS NV 0 V V C- 122 5-HTI2 I ALERSL N V ANWY:L- I G.S.L AV T L M'VS:V L:VL P MA AL:OQ'V LN .KW.T.:LSGS-VTJ~C- 109 5-HT~c VL A A M L V. 0 L L V 14 P'L S 'L L A L V D YVWV P L'PRfYVL.C 12 5-AT2 VSL E kKI0N A TN YFFLM S LA AbM:L L GF.LVTM P V S'MLT LY.SV RW PLPSKL. 4 IIII -911~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~... 5 ,,AT 0.. I L S S S I T CCTA AS J L H L C V'A A L::S fR:F W.A 4T S A: L' E T§I K ftR .: T 'A GKA AA I N.L: ::A.I 161 OAT0 F V L S S 5 T C C ~:T: A S~IL H L C V 0 A L`0 RYtVVA I T'0 A.v EY:5VS:A:: R.: T P 'K ft A. A -V: M A:-LV 172 5OAT, AL F IA L ~V L::C':CTJ~S -.SILLHLC'A I-A IORVYVW:A'I .T 0-P I D:Y-:V KRT P:RPRA .LIO:S~L` 158 5AHTn P VWI''S L V L FST-A S.I MHLC A IS L'DRY VA I RkP IE HSR FN SRTKAIKII 178 5-AT A IVWI YLDYL FST ASIM HLC A:'IS L.SNYVVA IS0N P I'H FN S RTK AFLKI. IA198 I IT T IV-4 5A-HT,;, V WAI SI CIS I-P.P L-F .AWR'.QA kA QE E MS DCL V N TS0. S'YT.IYSTCGA F'Y' 1208 OAT, WVVVF SI:S I S.L:P. P'FPF.WER.SA KA E E E V SS~C V V.'NTD.ILVY.T VYSTVS: .-A.F'Y:F219 OAT2VVLISFL1SI~~~~~~~~P.P MLGW.P SDDCIKH.SGT' STFAF 205 OAHT VVWA I Si GTSV 'VPIT-P'VIG LRDE SK'V FV NNTT CV L NDP N ..FL'I G'S FVA:F':-IF226 OAHT, VVWT S VSGI S NP I P-V-FG L D DDS0KVF .K E GS CL LA D 0N .F-VOL I SSFVAPI24

5OAT,. P S V LL II Y G.R IYRA ANRWRI L .N P PS LYGK R FTTA'HLVT G.SAGSSL 253 5-HT.[ P .-T LL LIA L Y Y VEA RS RI L K. TP NRT GK RL TR-A LITEDSPGSTS'SV 267 OAHT P L LL M LVL GRIF A ARF RI R KTV K KVE K TG ADTRH GA SPAP PKKSVN 254 OA .P LTIMVITOLLLIRVLRRSTLMTLRD.ATEEEANMS LN FLNC CCKKNGS270 OAHT P LT'I M VITYF LTIK SL SKEAT LC V S DL ST RA KLA .FS S FLPSSSLS.S'EK 293

5 HT ~ .CS LNS S LEGMISHAS AGS PL FF A V KI KL A 0SAL-E...... 287 5T T I NSR V PD-VP SES .GDS PV YV.WO V KV RV S0ALL E...... 300 OAHT_ G E SGS RNAWERL .G VE SK A GGA L CA N GAV RGD DDGAAL:E.VI E VARVGNSKEH 303 5OATI E EE NA PNP NP DQKP RRKKK E KRP R GT M ...... 303 O AT, L F 5 Rt S I ARft P 0GSYVA GRft ...... T M 0 ...... 313

H-...... R KfRtIDSA ARE 'k A'T KI' .'GIO GA' F 310

,...... K K KL MA ARE 'K A T K TLGIIL A: F 323 O H LPL PS E A GPT PC A PA S F EARKAE R NAAEAK R KMA LA RE RKTV KT L G I I M T'' 353 ...... AlI NNEK KA SKXVL G IVFFVF 322 .OT...... SI SNES KACK VL GI'VFFL F 332

OA Tj. II C WLP F FV V SLV L:P IC RISC .IW HP ALFD FFTWLGSOLNW85LINWPIIYTTV 308 OAHT IV C WL PPFFI I SLVMKP I CK DA C.WVH L A IPFDPFFTWILGYL NSLI NP.I1TIN371 OHTI, IL C WLPPFF IV ALV LPFPCE S S C .M PTL L0GA II NWLGYSVWSNSLL NPVL-YAY 401 OAHT, L IMWC P FF I T NIL SVL C GKA C N DKLM E KLL NV F VWIGYV CSGINWP LV..T'L 372 5HT, V V MVWCPPFF IT NIM A VIC K ES C E NV IGA L L V FVWIGYL S SAV NPLVYTL 382

OAHT 1,,F N E F RDA FSK IVP FR KA S...... 377 OAHT,, S NE DFK SA FAK LIRPFK CT S...... 390 OAHT, F NK 0 F NA FK.KIIK CN FCR0...... 421 SAHT, PNK IYfRRA FS KTYL RC DYK PD K KP .POVERS1PNROVAA T AL S GfRE L NOVNI YfRT 421 OAHT, PNKTOYR SA FS RY.:ISCQYKE NR K PL L I LV T I PALAYVKS S QL DV GDK K NS432

5Tj. 377 T.,? 3900 OAHT,, N E RVA RK A ND PE P GI EA V E NL EL PV NP SAVVS ER I SS V 460 5-HT 0 E D A ft 5 T V 0 D C S M V T L G K 4 Q S E E N C T D N A T V N A K V SC V 471 FIG. 1. Comparison of the 5-HTD. and 5-HTDg receptor deduced amino acid sequences with those of other serotonin receptors. The seven putative membrane-spanning domains (I-VII) are indicated by overbars. Homologies between the 5-HTDD receptor and other receptors are noted by shading. In the amino-terminal region, consensus sequences for N-linked glycosylation sites are underlined. Selected invariant residues are highlighted by arrows. Downloaded by guest on September 28, 2021 3632 Pharmacology: Weinshank et al. Proc. Natl. Acad Sci. USA 89 (1992) the relationship between closely related subtypes. Further- more, the pharmacological binding properties and second- messenger coupling of this clone are very similar to the properties of the 5-HT1D<, clone (see below). We have therefore named this second receptor 5-HTlDs. A comparison of the human 5-HT1,D and 5-HTDp receptor sequences to all other known serotonin receptors is shown in Fig. 1. All

r , . . serotonin receptors contain certain structural features that are invariant, including the aspartic residues of transmem- 2 4 6 Bound, pmnol/mg brane regions II and III, the Asp-Arg-Tyr sequence at the end of transmembrane region III, and the conserved proline O 10 20 30 40 50 60 residues of transmembrane regions IV-VII (20). Clonal cell lines stably expressing the or 2.0 5-HTID1 5-HTDp receptor gene were generated from murine LM(tk-) fibro- blasts. Membranes harvested from cells transfected with

-06 either the 5-HTD1 or the 5-HTDp gene displayed an appar- 1.5 ently homogeneous population of high-affinity, saturable [3H]5-HT binding sites (Fig. 2). The equilibrium dissociation I-.~~~~~~~~02 constants (Kd) of [3H]5-HT for the 5-HT1,D (Kd = 5.1 ± 0.4 nM; n = 5) and 5-HTlDp (Kd = 4.3 ± 0.5 nM; n = 5) receptors were similar. The density (Bmt) of binding sites was 2-fold 0 1 2 higher in the 5-HTD1 receptor cell line (B., = 4.0 + 0.5 Bound, pmol/mg pmol/mg) than in the 5-HTlDg3 receptor cell line (B. = 2.3 ± 0.4). Specific binding was >85% of total [3H]5-HT binding at the Kd concentration of radioligand, for both clones. Free fHJ5-HT, nM The pharmacological profiles of both clones were determined in parallel FIG. 2. experiments from analysis ofcompetition of Determination of equilibrium dissociation constant (Kd) [3H]5-HT binding (Table 1). The rank order of potency of of [3H]5-HT for cloned human 5-HT11< (A) and 5-HTDwp (B) receptors. Membranes harvested from stable transfectants were incubated these ligands to compete for the [3H]5-HT binding site is with 9-10 concentrations of [3H]5-HT (0.25-50 nM) in absence or consistent with a 5-HTID receptor pharmacological profile for presence of 10 ,uM 5-HT for 30 min at 37°C in the dark. Each data both clones: 5-carboxamidotryptamine > 5-HT > yohimbine point is the mean of triplicate determinations; standard deviations > 8-hydroxy-2-(di-n-propylamino)tetralin > spiperone > za- averaged <5%. Kd and B. values were determined by nonlinear copride. Compounds exhibiting high affinity for both the regression analysis, and are illustrated in the form ofa Scatchard plot 5-HTD. and 5-HTDs clones and the pharmacologically de- (Insets). Calculated Kd and B. values are 4.5 nM and 5.8 pmol/mg fined 5-HT1D receptor in native brain tissue included for clone 5-HT1,D and 5.0 nM and 2.0 pmol/mg for clone 5-HT1Dp. tryptamine derivatives (5-carboxamidotryptamine, 5-HT, Table 1. Apparent dissociation constants (Kj) and Hill coefficients (nH) of serotonergic ligands for the cloned human 5-HT1D, and 5-HTDs receptors in comparison to the pharmacologically defined 5-HT1D receptor from native brain membrane preparations 4 Drug* Human 5-HTD1 receptor Human receptor Bovine 5-HTDp membranest No. Name Ki, nM nH Ki, nM nH Ki, nM 1 Lysergol 0.63 ± 0.10 1.04 + 0.12 1.19 ± 0.13 0.87 + 0.04 ND 2 5-CT 0.70 + 0.06 0.% ± 0.03 1.6 ± 0.2 0.81 + 0.03 2.5 3 CGS-12066B 2.9 + 0.3 0.91 ± 0.04 2.1 + 0.3 0.91 ± 0.07 32 4 Sumatriptan 3.4 ± 0.3 0.80 ± 0.04 7.7 ± 0.5 0.88 ± 0.07 29 S Methysergide 3.6 + 0.6 1.00 ± 0.02 25 ± 5 0.91 ± 0.03 4 6 5-HT 3.9 ± 0.3 0.93 ± 0.02 4.3 ± 0.9 0.94 ± 0.01 4 7 5-MeOT 4.8 ± 0.3 1.02 ± 0.02 34 + 6 0.95 ± 0.01 4 8 1-NP 7.4 ± 0.6 1.05 ± 0.04 12 ± 2 1.05 ± 0.03 15 9 DP-5-CT 13 + 1 0.97 ± 0.09 42 ± 3 0.91 ± 0.02 63 10 Rauwolscine 16 ± 2 0.90 + 0.05 40 ± 7 0.87 ± 0.06 20 11 Yohimbine 22 ± 2 0.91 ± 0.04 27 ± 3 0.97 ± 0.04 79 12 TFMPP 64 ± 5 0.95 + 0.03 114 ± 18 1.00 + 0.02 282 13 Tryptamine 86 + 6 0.95 ± 0.02 521 + 82 0.91 + 0.05 40 14 DPAT 120 ± 11 0.90 ± 0.05 260 ± 32 0.91 ± 0.07 1260 15 mCPP 216 + 20 0.93 ± 0.05 361 ± 31 0.93 ± 0.01 1550 16 2-Methyl-5-HT 915 ± 68 0.90 ± 0.02 860 ± 109 0.95 ± 0.09 398 17 Spiperone 995 ± % 0.86 ± 0.03 >10,000 ND 5000 18 Quipazine >1000 ND >1000 ND 1380 19 Pindolol 4100 ± 500 0.91 ± 0.04 4900 ± 300 0.91 ± 0.02 6300 20 Zacopride >10,000 ND >10,000 ND ND K; values and Hill coefficients are means + SEM from three to seven determinations. ND, not determined. *Numbers correspond to those in Fig. 3. 5-CT, 5-carboxamidotryptamine; 5-MeOT, 5-methoxytryptamine; 1-NP, 1-naphthylpiperazine; DP-5-CT, N, N-dipropyl-5-carboxamidotryptamine; TFMPP, 1-[3-(trifluoromethyl)phenyllpipera- zine; DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; mCPP, 1-(3-chlorophenyl)piperazine. tData from ref. 9. Downloaded by guest on September 28, 2021 Pharmacology: Weinshank et al. Proc. NatL. Acad. Sci. USA 89 (1992) 3633 5-methoxytryptamine), ergoline analogs (lysergol and meth- were completely blocked by the nonselective antagonist ysergide), and a2-adrenergic antagonists (rauwolscine and methiothepin (10 juM) in both transfected cell lines (data not yohimbine). Sumatriptan was one of the most potent com- shown). In the absence of forskolin, 5-HT did not stimulate pounds for both clones (K1 = 3.4 nM for 5-HT1TE; K1 = 7.7 adenylate cyclase in these transfected cells. In addition, 5-HT nM for 5-HTlD3). With the exception of CGS-12066B, all (1 ;LM) was unable to produce any alterations in phosphoti- compounds tested exhibited higher affinity for the 5-HT1,< dylinositol metabolism. In untransfected cells, 5-HT did not receptor than for the 5-HTlDp receptor (Table 1). This con- stimulate or inhibit cAMP accumulation. sistent difference in affinity values was maintained when the expression densities were reversed (i.e., BX, for 5-HT1Dc = 1 pmol/mg of protein; Bm. for 5-HTDs = 2.1 pmol/mg of DISCUSSION protein). Structurally diverse compounds that discriminated In the current study, two separate human 5-HTlD receptor (>7-fold) between these closely related subtypes included genes (5-HTjEr and 5-HTDp) have been isolated and char- methysergide, 5-methoxytryptamine, tryptamine, and spip- acterized. The dog homolog of the 5-HT1Dc, receptor, known erone. High correlations were obtained between the apparent as the RDC4 sequence (11), was the first 5-HTE1, receptor affinities of compounds at the pharmacologically defined gene to be isolated, although its identity as a 5-HTlD receptor 5-HT1D receptor from native brain membranes and each of was only recently recognized (12). The second human 5-HT1D the cloned receptor subtypes (Fig. 3 A and B). In addition, a receptor to be cloned and characterized, the 5-HTDp recep- very high correlation coefficient (r = 0.96) was obtained in tor, exhibits pharmacological and functional properties that the comparison of the cloned 5-HT1,D, and 5-HTDs subtypes are strikingly similar to those ofthe 5-HTlDa receptor. The rat (Fig. 3C). homolog ofthe human 5-HTDp receptor was recently cloned, The functional coupling of these cloned 5-HTID receptor characterized, and shown to encode a 5-HT1l receptor site subtypes to second-messenger responses was determined in (21, 22). This result confirmed the predicted relationship of intact cells stably expressing each one ofthese genes (Fig. 4). the rat 5-HT1l and human 5-HTlD sites (23). The fact that Forskolin (10,4M) produced a 3- to 5-fold increase in cAMP sufficient pharmacological differences were present to have above basal values. 5-HT (1 ,tM) inhibited forskolin- originally led to the naming of the 5-HTlD and 5-HT1l sites stimulated cAMP accumulation by 70 ± 4% in transfected as separate receptor subtypes is a reflection of the unusual cells expressing the 5-HT1,D receptor and by 92 ± 1% in degree of species variation shown by the 5-HTlDp receptor. transfected cells expressing the 5-HTlDs receptor. The EC50 The binding properties of these two 5-HT1D receptor for 5-HT was 2.49 ± 0.65 nM for 5-HT1TE and 1.8 ± 1.2 nM subtypes are so similar that a linear correlation coefficient (r) for 5-HTlDp (Fig. 4). Sumatriptan was found to produce of 0.96 was calculated in the comparison of log Ki values of agonist responses (EC50 = 3.2 ± 0.67 nM for 5-HTlDa and 5.2 19 compounds at these two receptor sites. It is interesting that ± 1.2 nM for The responses to 5-HT or sumatriptan the 5-HT1Ea receptor displayed higher affinity for all struc- 5-HTD,8). tural classes of compounds tested than did the 5-HT1D8 10 receptor. Although the 5-HT1,D cell line had a higher expres- IAv sion level than did the cell this difference does 9 r G 7>9 5-HTDp line, II not account for the differences in binding affinity. Specifi- I7a- 110 cally, when the receptor densities of two cell lines were I 100 0 reversed and studied in parallel for a selection of compounds 160 1 showing the largest differences in affinity values, the 5-HTDc °17 receptor still showed higher affinity for these compounds 5 6 7 8 9 l1 0 plO, Clone 5-HT1Da

'D *0 .E0 > -0- cL = _x T Is 0-0I0

pKi, Clone 5-HTlDp 10 0" 10 9 8 1= C 0 9 32I V- r aO.96 6032 -log [drug] (M) a 184 A ~~1 FIG. 4. Concentration-response curve for the inhibition of for- S 12 9O7 skolin-stimulated cAMP production by 5-HT (o) and sumatriptan (o) in clonal cell lines stably expressing the 5-HTD1 (A) or 5-HT1D3 (B) 0Y13 receptor gene. Curves shown were fitted to a four-parameter logistic equation to obtain response parameters. Calculated EC50 values for 5 6 7 8 9 10 5-HT and sumatriptan in the experiments shown were 1.7 nM and 2.4 pKi, Clone 5-HTiDa nM in the 5-HTEa cell line and 1.3 nM and 3.3 nM in the 5-HT1Ds cell line. Each data point is the mean of triplicate determinations; FIG. 3. Correlations between affinity constants (pK; values) of standard deviations averaged <5% of the mean. The point preceding serotonergic ligands for the cloned human 5-HT1,D (A) or 5-HT1Dp the break in the curve represents the mean of triplicate measure- (B) receptor and the pharmacologically defined 5-HT1D receptor. ments of forskolin-stimulated cAMP accumulation in the absence of Correlation between pK, values of the same ligands for the cloned agonist. Basal cAMP levels were subtracted from all cAMP values human 5-HTlD0 and 5-HT1Dq receptors is also shown (C). Numbers obtained by using forskolin and were normalized to 100%o relative to correspond to compounds listed in Table 1. The correlation coeffi- forskolin-stimulated cAMP levels in the absence of agonist. The cient (r) is listed in each panel. experiment was replicated twice with similar results. Downloaded by guest on September 28, 2021 3634 Pharmacology: Weitishank et al. Proc. Nad. Acad. Sci. USA 89 (1992) than did the 5-HTlD, receptor. The strong similarity in in part by Grant 1R43NS27789 from the National Institutes ofHealth properties of the 5-HT1,D and 5-HTIDfl receptors makes it and in part by Eli Lilly and Company. difficult to determine whether any of the multiple physiolog- ically or pharmacologically defined 1. Heuring, R. E. & Peroutka, S. J. (1987) J. Neurosci. 7, 894- 5-HTID subtypes matches 903. the properties of these genetically defined receptors. That 2. Humphrey, P. P. A., Feniuk, W., Perrin, M. J., Connor, these two cloneos are not sufficient to explain the known range H. E., Oxford, A. W., Coates, I. H. & Butina, D. (1988) Br. J. of5-HT10 subtypes means that additional 5-HTID-like recep- Pharmacol. 94, 1123-1132. tor subtypes may remain to be cloned. 3. Bond, R. A., Craig, D. A., Charlton, K. G., Ornstein, A. G. & Similar to the 5-HTID receptor in native tissues, both Clarke, D. E. (1989) J. Auto. Pharmacol. 9, 201-210. cloned human receptors were found to couple to 4. Molderings, G. J., Werner, K., Likunga, J. & Gothert, M. 5-HT1D (1990) Naunyn-Schmiedeberg's Arch. Pharmacol. 342, 371- adenylate cyclase inhibition. Neither produced a phosphati- 377. dylinositol response; The EC"0 values obtained for com- 5. Schlicker, E., Fink, K., Gothert, M., Hoyer, D., Molderings, pounds at each receptor.subtype closely matched the affinity G., Roschke, I. & Schoeffier, P. (1989) Naunyn-Schmiede- constants for binding. These results contrast with the similar berg's Arch. Pharmacol. 340, 45-51. comparison of agonist affinities for binding and function of 6. Hoyer, D. & Schoeffier, P. (1988) Eur. J. Pharmacol. 147, the 145-147. 5-HTID receptor in the calf substantia nigra (9). It is likely 7. Herrick-Davis, K., Titeler, M., Leonhardt, S., Struble, R. & that the high degree of agonist activity in these heterologous Price, D. (1988) J. Neurochem. 51, 1906-1912. expression systems relative to those reported in the literature 8. Peroutka, S. J. & McCarthy, B. G. (1989) Eur. J. Pharmacol. can be attributed to differences in receptor reserve. Since the 163, 133-136. 5-HT1A receptor has been shown to activate potassium 9. Waeber, C., Schoeffier, P., Hoyer, D. & Palacios, J. M. (1990) channels in hippocampal neurons through a pertussis toxin- Neurochem. Res. 15, 567-582. 10. Humphrey, P. P. A., Feniuk, W., Motevalian, M., Parsons, sensitive G-protein (24), it will be interesting to determine A. A. & Whalley, E. T. (1991) in Serotonin: MolecularBiology, whether the 5-HTlD receptor subfamily shares this property. Receptors, and Functional Effects, eds. Saxena, P. & Fozard, The presence in the human genome oftwo different 5-HTlD J. R. (Birkhauser, Basel), pp. 421-429. receptor genes that encode receptors which are remarkably 11. Libert, F., Parmentier, M., Lefort, A., Dumont, J. E. & similar in their pharmacological selectivity, second- Vassart, G. (1989) Science 244, 569-572. messenger coupling, and amino acid sequence cannot be 12. Zgombick, J. M., Weinshank, R. L., Macchi, M., Schechter, L. E., Branchek, T. A. & Hartig, P. R. (1991) Mol. Pharmacol. attributed to the trivial explanation that only one gene is 40, 1036-1042. expressed. The 5-HTID receptor was isolated from both 13. Branchek, T., Zgombick, J., Macchi, M., Hartig, P. & Wein- human (present study) and dog (11) cDNA libraries. The shank, R. (1991) in Serotonin: Molecular Biology, Receptors, 5-HTlDp- receptor was expressed in the human brain, since and Functional Effects, eds. Saxena, P. & Fozard, J. R. mRNA encoding this receptor was detected by PCR ampli- (Birkhauser, Basel), pp. 21-32. fication of cDNA derived from human cortex not 14. Hamblin, M. W. & Metcalf, M. A. (1991) Mol. Pharmacol. 40, (data 143-148. shown). Thy possibility that these two 5-HT1D receptors are 15. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, expressed in different brain regions or in different cells within 6-13. the same region needs to be investigated. Other possible 16. Weinshank, R. L., Zgombick, J. M., Macchi, M., Adham, N., explanations include different rates of desensitization, cou- Lichtblau, H., Branchek, T. A. & Hartig, P. R. (1990) Mol. pling to different G proteins, or different ratios of high- and Pharmacol. 38, 681-688. low-affinity agonist states in native tissues. 17. Libert, F., Parmentier, M., Lefort, A., Dumont, J. E. & Vassart, G. (1990) Nucleic Acids Res. 18, 1916. The 5-HTID receptor has been implicated in a variety of 18. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. important clinical problems, but only one partially selective Acad. Sci. USA 74, 5463-5467. ligand`for the 5-HTID receptor site, sumatriptan, is available. 19. Cheng, Y. C. & Prusoff, W. H. (1973) Biochem. Pharmacol. This new anti-migraite cnompound has been shown to interact 22, 3099-3108. with 5-HT1D (and 5-HTiB) sites in animal preparations, with 20. flartig, P. R., Kao, H.-T., Macchi, M., Adham, N., Zgombick, apparent dissociation constants ranging from 17 to 251 nM (8, J., Weinshank, R. & Branchek, T. (1990) Neuropsychophar- 25, 26). The two cloned human receptor subtypes macology 3, 335-347. 5-HT11, 21. Voigt, M.M., Laurie, D.J., Seeburg, P.H. & Bach, A. (1991) both exhibit high-affinity dissociation constants (3.4 nM and EMBO J. 10, 4017-4023. 7.7 nM) for sumatriptan, and therefore both the 5-HT1, and 22. Adham, N., Romanienko, P., Hartig, P., Weinshank, R. L. & 5-HTlDs receptors are likely targets for the therapeutic Branchek, T. (1992) Mol. Pharmacol. 41, 1-7. actions and side effects of this drug. 23. Hoyer, D. & Middlemiss, D. M. (1989) Trends Pharmacol. Sci. 10, 130-132. We acknowledge the excellent technical assistance provided by 24. Andrade, R., Malenka, R. & Nicholl, R. A. (1986) Science 234, Mr. Marcelino Dizon, Ms. Barbara Dowling, Ms. Anastasia Kokki- 1261-1265. nakis, Mr. Harvey Lichtblau, and Ms. Michelle Smith. We thank 25. Schoeffter, P. & Hoyer, D. (1989) Naunyn-Schmiedeberg's Drs. Nika Adham, Jonathan Bard, and Lee Schechter for experi- Arch. Pharmacol. 339, 675-683. mental support. We thank Mr. Ernest Lilley and Mr. George 26. Sumner, M. J. & Humphrey, P. P. A. (1989) Br. J. Pharmacol. Moralishvili for producing the illustrations. This research was funded 98, 29-31. Downloaded by guest on September 28, 2021