Proc. Nati. Acad. Sci. USA Vol. 90, pp. 408-412, January 1993 Neurobiology Cloning of another human receptor (5-HT1F): A fifth 5-HT1 receptor subtype coupled to the inhibition of adenylate cyclase NIKA ADHAM, HUNG-TEH KAo*, LEE E. SCHECHTER, JONATHAN BARD, MICHAEL OLSEN, DEBORAH URQUHART, MARGARET DURKIN, PAUL R. HARTIG, RICHARD L. WEINSHANK, AND THERESA A. BRANCHEKt Synaptic Pharmaceutical Corporation, 215 College Road, Paramus, NJ 07652 Communicated by Eric Kandel, September 28, 1992 ABSTRACT An intronless gene encoding an additional 5-HTD10/p receptors. Primers 3.17 and 5.5 [(5'-TGGAATTC- human serotonin (5-1T) 5-HTificee receptor subtype was iso- TGYGYIATHKCICTGGAYMGSTA-3') and (5'-CATIA- lated from a human genomic library with probes obtained from VIRIIARIGGDATRWARAAIGC-3')] were used to amplify degenerate PCR primers used to amplify 5-liT-receptor- 5 ,ug of poly(A)+ RNA from rat brain that was reverse- specific sequences. The highest degree of homology was found transcribed by reverse transcriptase (avian myeloblastosis with the 5-HT1E subtype (70%) and the 5-HTlDa (63%) and virus). PCR was done on single-stranded cDNA as follows: 5-HTlDp3 (60%) receptors. RNA for this gene was detected in 940C for 1 min, 500C for 2 min, and 72TC for 3 min for 40 the human brain but was not detected in kidney, liver, spleen, cycles. After PCR, 90 A.l of the reaction was phenol/ heart, pancreas, and testes. High-affinity (Kd = 9.2 nM) chloroform-extracted and precipitated, treated with T4 DNA 3H-labeled 5-HT binding was detected. Competition studies polymerase, and digested with EcoRI before separation on a revealed the following rank order of potencies for 1% agarose gel. The DNA fragment was isolated from the gel, ligands: 5-HT > >> 5-carboxyamidotryptamine kinased, and cloned into pBluescript. Recombinant clones > 8-hydroxy-2(di-1-propylamino)tetralin > . 5-HT were analyzed by sequencing. One clone, designated S51, produced a dose-dependent inhibition of forskolin-stlmulated was chosen for further study. cAMP accumulation (EC50 = 7.9 nM) in transfected cells. Cloning and Sequencing. A human lymphocyte genomic These properties distinguish this receptor from any previously library (Stratagene) was screened by using the rat S51 frag- characterized and establish a fifth 5-HT1.15&. receptor subtype ment as a probe. The probe was labeled with 32P by the (5-HT1F) coupled to the inhibition of adenylate cyclase. method of random priming (18) and hybridized (19). For subcloning and further Southern blot analysis, DNA was The diverse physiological actions of serotonin (5-HT) are inserted into pUC18 (Pharmacia). Nucleotide sequence anal- mediated by at least four receptor classes: 5-HTlike, 5-HT2, ysis was done by the Sanger dideoxynucleotide chain- 5-HT3, and 5-HT4 (1-3). The 5-HT1like class appears the most termination method (20) on denatured double-stranded plas- heterogeneous. Four human genes encoding 5-HT1.like recep- mid templates with Sequenase (United States Biochemical). tors have been cloned: 5-HT1A (4, 5), 5-HTDa (6, 7), 5-HT1D, Expression. The coding region of clone hL16a was cloned (7-9), and 5-HT1E (10-12). The 5-HT1B receptor has been into vector pcEXV-3 (21), and stable cell lines were obtained cloned (13-15) and is homologous to the 5-HT1Dp receptor by cotransfection (19). One LM(tk-) cell line, L-1F-3, was (14). These 5-HT1like receptors all share an amino acid selected for binding studies; one NIH 3T3 stable cell line homology of at least 50o, high affinity for 3H-labeled 5-HT N-1F-6 was selected for functional studies. ([3H]5-HT), and coupling to the inhibition of adenylate cy- clase activity as a primary coupling pathway. Many addi- Membrane Preparation. Membranes were prepared from tional 5-HT-mediated functional responses do not seem to stably transfected LM(tk-) cells as described (19). Protein correlate with any ofthese cloned receptor subtypes (16, 17). concentrations were determined by the method of Bradford We have used a PCR cloning approach to isolate a human (22). gene that encodes another 5-HT1-lke, receptor, termed 5-HT1F, Radioligand-Binding Studies. [3H]5-HT-(20-30 Ci/mmol; with a pharmacological profile distinct from any serotoner- New England Nuclear; 1 Ci = 37 GBq) binding was done as gic receptor yet described.t Like the other 5-HTl ike recep- described (7). Competition experiments were done by using tors, 5-HT1F couples to the inhibition of adenylate cyclase 10-12 concentrations of drug and 4.5-5.5 nM [3H]5-HT. activity, and this response is blocked by methiothepin. De- Nonspecific binding was defined by 10 A.M 5-HT. Binding tection ofthe mRNA encoding this receptor in the human brain data were analyzed by nonlinear-regression analysis (7). IC50 indicates that binding studies using [3H]5-HT as a ligand values were converted to Ki values using the Cheng-Prusoff require reevaluation. Furthermore, the high affinity of the equation (23). All experiments were done in triplicate. sumatriptan at this subtype indicates a Adenylate Cyclase Activity. Adenylate cyclase activity was possible role of the 5-HT1F receptor in . determined in initial experiments in LM(tk-) cells, as de- scribed (14). A weak inhibition of forskolin-stimulated ade- EXPERIMENTAL PROCEDURES nylate cyclase (FSAC) (25-30%o) was obtained. Stable cell PCR. The third (III) and fifth (V) transmembrane (TM) Abbreviations: TM, transmembrane; 5-CT, 5-carboxyamido- domains of the following receptors were used to synthesize ; 5-HT, serotonin (5-hydroxytryptamine); [3H]5-HT, 3H- degenerate primers: 5-HT1A, 5-HT1c, 5-HT2, and the labeled 5-HT; FSAC, forskolin-stimulated adenylate cyclase. *Present address: Department of Psychiatry and Behavioral Sci- ences, Stanford University Medical Center, Stanford, CA 94305. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe nucleotide sequence reported in this paper has been deposited in accordance with 18 U.S.C. §1734 solely to indicate this fact. in the GenBank data base (accession no. L04962).

408 Neurobiology: Adham et al. Proc. Natl. Acad. Sci. USA 90 (1993) 409

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...... N .... 5 - H T, ,: U- L- C-- N 5...... N-.-- V:T.-.-...... F-N -E F: O A S 377 5-HT, ; ..L.... IN .DL . I T NE F K Q A F H. K L R F K C T S 390 ...... 5-HTA N. LGVSKY...... N$L LNVI.. AYY C R 421 FIG. 1. Comparison of 5-HT1F receptor-deduced amino acid sequences with other 5-HT receptors. The seven putative membrane-spanning domains (TM I-VII) are indicated by lines and Roman numerals. Homologies between the 5-HT1F receptor and other receptors are noted by shading. ~~~~~~~~~~~~~~~~~~~~. lines were produced in NIH 3T3 cells and were used for all charged nylon membrane (ZetaProbe, Bio-Rad). Filters were subsequent experiments. hybridized and washed under high stringency. Tissue Localiation Studies. Human tissues (National Dis- In Situ Hybridization. In situ hybridization was done as ease Research Institute) were homogenized, and the total described (25) by using male Hartley guinea pigs (300-350 g). RNA was extracted (24). cDNA was prepared from 5 ,ug of A fragment ofthe guinea pig 5-HT1F receptor gene was cloned total RNA with random hexanucleotide primers (500 pmol) by homology and sequenced. Forty-five-base oligoprobes using Superscript reverse transcriptase (BRL) in PCR reac- synthesized to the 4,5 loop and 5'-untranslated regions were tion buffer (Cetus) containing 1 mM dNTPs, at 42°C for 1 hr. 3'-end-labeled with deoxyadenosine 5'-[y[35S]thioltriphos- An aliquot of the first-strand cDNA was diluted (1:5) in a phate to a specific activity of 4 x 109 Ci/mmol. The nucle- 50-IAI PCR reaction mixture (200 ,uM dNTPs, final concen- otide sequences were as follows: 5'-GTGATGCTTGAT- tration) containing 1.25 units of Taq polymerase and 1 uM of GATGCACTCATCATCTCGGCTTGTCCCCTGGTG-3' primers from the sense strand (5'-TCTATTCTGGAGGCAC- and 5'-TAGCAGTTCCTCTGAGGTCAAGTTTTGATCA- CAAGGAAC-3') and from the antisense strand (5'- GAAGAGTTTAAGAA-3'. Sense probes, melting tempera- TGTTGATGGGTCAGATAAAGACTT-3'). The PCR prod- ture, and RNase pretreatment were used as controls. Sec- ucts were run on a 1.5% agarose gel and transferred to tions were exposed to Kodak X-Omat AR film for 1 week or 410 Neurobiology: Adham et al. Proc. Natl. Acad. Sci. USA 90 (1993) A Table 1. Apparent dissociation constants (Ki values) of various 10 drugs for cloned 5-HT1F and 5-HT1E receptors. Ki, nM Ki, nM Compound 5-HT1F 5-HT1E* L6C1 5-HT 10± 2.0 11 ± 1.0 ± 0. Sumatriptan 23 11 2520 ±135 Free n = Methylergonovine 31 ± 11 89 ± 4.0 I 10.6 - B cm *mgopnrotein 34 ± 4.9 228 ± 16 5-MeO-DMT 37 ± 1.5 528 ±32 mE 1-NP 54 ± 3.8 207 ± 69 0.~~~~~~~~ 92 ± 11 1270 ± 233 0.0~~~ 171 ± 28 599 ± 39 0 2 3 4 5 6 7 a-Me-5-HT 184 ± 35 121 ± 13 pmo[/mgBound, of protein NAN 190 203 ± 13 ND 0 50 100 1150 341 ± 71 ND Free [3H]5-HT, nM E50 0. | Poraoo 2-Me-5-HT 413 ± 5.6 817 ± 203 Methiothepin 652 ± 41 194 ± 4.0 B 5-CT 717 ± 71 7875 ± 284 101 t0 o5-HT TFMPP 1002 ± 85 6293 ± 259 averaged .5%.1-NP, 1-(1-naphtyl~piperaz 5-CT,1-NP 5-MT 1166 ± 197 3153 ± 1041 0 &Yohimbine DP-5-CT 1613 ± 817 >10,000 ain.Metergoline DOI 1739 ± 84 2970 ± 592 I *~~~~~~~~~~~~a5-CT 8-OH-DPAT 1772 ± 38 3333 ± 310 LO50 ~~~~~~~~o8-OH-DPAT Tryptamine 2409 ± 103 2559 ± 827 S >10,000 >10,000 CD Spiperone >10,000 5051 ± 689 C) >10,000 >10,000 0. C') ; >10,000 >10,000 11 10 9 8 7 6 4 3 2 >10,000 ND -log [competitor] (M) K1 values were determined from IC50 values by computer-assisted Foa. 2. (A) Determination ofKdeand B. of [3H]5-HT for the nonlinear-curve analysis using the Cheng-Prusoff equation (23). 5-HTwFreceptor expressed in LM(tk-) cells. (B) Determination of Values are means ± SEM from at least three separate experiments. affinity constants of serotonergic ligands for the 5-HTlF receptor. DOI, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; 8-OH- Each data point is the mean of triplicate determinations, and SDs DPAT: 8-hydroxy-2-(di-1-propylamino)tetralin; 5-MeOT, 5-meth- averaged <5%. 1-NP, l-(l-naphthyl)piperazine; 5-CT, 5-carboxy- oxytryptamine; 5-MeO-DMT, 5-methoxy-N,N-dimethyltryptamine; amidotryptamine; 8-OH-DPAT, 8-hydroxy-2-(di-1-propylamino)te- a-Me-5-HT, a-methyl-5-hydroxytryptamine; 2-Me-5-HT, 2-methyl- tralin. 5-hydroxytryptamine; 1-NP, 1-(1-naphthyl); TFMPP, N-(m-trifluoromethylphenyl)piperazine; 5-MT, 5-methoxytrypt- coated with amine; DP-5-CT, dipropyl-5-carboxyamido-tryptamine. ND, not de- Kodak NTB-2 emulsion/2% glycerol, 1:1, for 2 termined. weeks. *Published values (12).

RESULTS Saturable, high-affinity [3H]5-HT binding was seen with membranes from cells We synthesized degenerate oligonucleotide primers on the prepared LM(tk-) (Fig. 2A). Analysis ± ± basis of sequences corresponding to the third and fifth TM of saturation data indicated a Kd of 9.2 0.99 nM (mean segments of 5-HT receptors. The primers were designed to SEM, n = 4) and a binding density (Bmax) of 4.4 ± 0.36 amplify only 5-HT-specific sequences by annealing to the sequence the nucleotide encoding protein sequence AFY- 100 *5-HT (F)IP in TM V. The presence of an alanine (A) rather than a serine (S) at the amino end of this sequence distinguishes the 0 + METHIOTHE)N (1 M) closely related adrenergic and receptor fami- lies from the serotonergic receptor family. After 30 amplifi- ~C* cation cycles, agarose gel electrophoresis revealed a clear pattern of cDNA species of -250 base pairs. Individual cDNAs were cloned into pBluescript and subjected to se- 0 quence analysis. One clone, designated S51, was observed to U CL encode a distinctive receptor. We then screened a human f 0 - genomic lymphocyte library with the PCR fragment S51. Isolation of the full-length coding region was obtained from a genomic clone designated hL16a. The primary amino acid sequence of clone hL16a is shown in Fig. 1. A long open reading frame can encode a protein 366 amino acids in length, having a relative molecular weight (Mr) of -42,000. Com- parison of this protein sequence with previously character- FIG. 3. 5-HT concentration-effect curves are represented with- ized neurotransmitter receptors indicates that hL16a is most out (-) and with (0) methiothepin (1.0 ,u&M). Data were normalized to closely related to the recently characterized 5-HT1E receptor 100%o relative to forskolin-stimulated values in the absence of (10-12) (Fig. 1). The TM amino acid identity ofhL16a to other to derive E~a and EC50 values. The antagonist Kb was estimated by known 5-HT1 receptors is as follows: 5-HTlE (70%), 5-HT1Da the method of Furchgott (26):antagonist/M(EC50Kb = dose of in the (63%), 5-HT1D,0 (60%), and 5-HTlA (53%). presence of antagonist/control EC5t)- 11. Neurobiology: Adham et al. Proc. Natl. Acad. Sci. USA 90 (1993) 411 other indole derivatives, excluding sumatriptan, had poor Brain affinity. Other compounds including 8-hydroxy-2-(di-1- propylamino)tetralin (5-HT1A), spiperone (5-HTlA/5-HT2), ketanserin (5-HT2), 1-(2,5-dimethoxy-4-iodophenyl)-2- Testes aminopropane (5-HTic/5-HT2), pindolol (5-HT1A/5-HT1B), and zacopride (5-HT3) had very poor affinity. Endometrium The functional coupling of clone hL16a to adenylate cy- *Mesentery clase inhibition was tested in intact NIH 3T3 cells (Bmax 1 pmol/mg of protein). 5-HT (10 ,uM) had no effect on either Myometrium basal activity or FSAC in untransfected or mock transfected Pancreas NIH 3T3 cells, indicating an absence ofendogenous cyclase- Kidney coupled 5-HT receptors in untransfected cells. Addition of 10 Liver ,tM forskolin increased the basal cAMP release (0.036 ± Spleen 0.0050 pmol/ml per 10 min) by -10-fold. 5-HT inhibited the response (Fig. 3) with an EC50 value of 7.9 + 0.76 nM and a Heart maximum percentage inhibition of FSAC (Emx value) of 74 H20 + 4.6 (mean ± SEM, n = 10). The 5-HT response (1 ,uM) was completely blocked by the nonselective antagonist me- Control thiothepin (10 ,uM). This antagonism was surmountable (Fig. 3), indicating probable competitive antagonism. The dose shift produced by methiothepin yielded an apparent Kb of438 + 14 nM, consistent with the K, for this compound (Table 1). FIG. 4. Human tissue distribution of RNA coding for 5-HT1F No direct effect of methiothepin was seen. No other com- receptor gene. Total RNA was converted to single-stranded cDNA pound tested in this study was an antagonist. In addition, no by random priming with reverse transcriptase. cDNAs were ampli- evidence for coupling ofthis receptor to inositol phospholipid fied by PCR using 5-HTF-specific PCR primers. PCR products were turnover was detected at a dose of 10 ,M 5-HT. run on a 1.5% agarose gel, blotted onto nylon membranes, hybridized Expression of the 5-HT1F transcripts was analyzed from to internal gene-specific oligonucleotides, and washed under high PCR-RNA blots and in situ hybridization studies. By PCR, stringency. Positive controls represent gene-specific recombinant we detected 5-HT1F receptor mRNA in the human brain, plasmids; water (H20) served as a negative control. PCR amplifica- uterus (endometrium and myometrium), and mesentery (Fig. tion and Southern blotting of RNA samples not treated with reverse transcriptase were negative. 4) but did not detect it in kidney, liver, spleen, heart, pancreas, or testes. In in situ hybridization experiments, we pmol/mg of protein (mean SEM, n = 4). No specific observed 5-HT1F transcripts in lamina V of frontal cortex [3H]5-HT binding was seen in untransfected host cells. 5'- (Fig. SA) in large pyramidal cells (Fig. SD). Moderate labeling Guanylyl imidodiphosphate produced a dose-dependent in- was also detected over layer VI nonpyramidal neurons. In hibition (IC50 = 243 + 115 nM, Imps = 56 + 3.2%; mean + both layer V and layer VI, the labeling was most evident in SEM, n = 3) of the specific [3H]5-HT binding. Specific dorsal sensorimotor neocortex and in cingulate and retro- [3H]5-HT binding was displaced in a monophasic manner by splenal cortices (Fig. SC). The pyramidal cells in the piriform a collection of structurally diverse serotonergic ligands (Fig. cortex were heavily labeled, as were large neurons in the 2B). 5-HT displayed the highest affinity (Table 1). 5-CT and raphe nuclei (Fig. 5E). Hippocampal pyramidal cells in CA1-CA3 were moderately labeled, as were the granule cells in the dentate gyrus, and some neurons in the nucleus of the solitary tract. Little labeling was found in the thalamus and hypothalamus.

DISCUSSION The pharmacological profile obtained from cells expressing gene hL16a (Table 1) indicates that it encodes a member of the 5-HTllike receptor subfamily, as 5-CT is an agonist, and methiothepin is an antagonist (27). As for the 5-HT1E recep- tor, its closest genetic relative (Fig. 1), the affinity for these compounds is relatively low. Nonetheless, the high affinity for sumatriptan, 5-methoxy-N,N-dimethyltryptamine, meth- ysergide, and other compounds from diverse structural classes (Table 1) distinguishes this additional receptor from the 5-HT1E subtype (r = 0.45). We have classified it as a FIG. 5. 5-HT1F receptor mRNA in guinea pig brain coronal 5-HT1F receptor on the basis ofits distinctive pharmacology: sections. (A) Antisense oligonucleotide probe (4,5 loop) was used. the low affinity of the 5-HT1F receptor for 5-CT but the high An identical pattern was seen with the 5'-untranslated probe (data affinity of 5-HT1F receptor for sumatriptan. This activity not shown). Hybridization densities are high in layer V of cerebral clearly distinguishes it from any other 5-HT1 fike-binding site cortex (V) and in CA1-CA3 of the hippocampus (HC). (B) Control or functional response model. contralateral hemisphere of a section adjacent to that in A. No The cloned human 5-HT1F receptor was found to couple to hybridization was seen by using a sense probe of identical specific the inhibition of adenylate cyclase, as do other cloned activity. (C) Section hybridized with the antisense probe. The dorsal members of the receptor This raphe (DR) is densely labeled. (D) Hybridization (antisense probe) is 5-HT1like family. coupling detected in layer V of sensorimotor cortex. Arrowheads indicate mimics their accepted functional responses in native tissue heavily labeled pyramidal cells. (E) Hybridization (antisense probe) preparations (28-30, except the native 5-HT1E receptor for in layer V of sensorimotor cortex sectioned through the dorsal raphe. which a functional correlate has not been reported). The Arrowheads indicate large, heavily labeled neurons. (x 130 in D and S-HT1F receptor represents the fifth cloned 5-HT receptor E.) that shows coupling to the inhibition of adenylate cyclase. 412 Neurobiology: Adham et al. Proc. Natl. Acad. Sci. USA 90 (1993) The only "5-HT1 receptor" that does not couple to adenylate bilka, T. S., Francke, U., Lefkowitz, R. J. & Caron, M. G. cyclase is the 5-HTic receptor (31), which is now considered (1987) Nature (London) 329, 75-79. 5. Fargin, A., Raymond, J. R., Lohse, M. J., Kobilka, B. K., a member ofthe 5-HT2 subfamily (2). Why so many different Caron, M. G. & Lefkowitz, R. J. (1988) Nature (London) 335, 5-HT1 receptor subtypes have been maintained in the genome 358-360. is not at all clear. Possible explanations include different 6. Hamblin, M. W. & Metcalf, M. A. (1991) Mol. Pharmacol. 40, midpoints for activation by 5-HT under physiological condi- 143-148. tions, different desensitization rates, different efficiencies or 7. Weinshank, R. L., Zgombick, J. M., Macchi, M., Branchek, kinetics of coupling to adenylate cyclase inhibition, coupling T. A. & Hartig, P. R. (1992) Proc. Natl. Acad. Sci. USA 89, 3630-3634. to diverse adenylate cyclases, different developmental reg- 8. Demchyshyn, L., Sunahara, R. K., Miller, K., Teitler, M., ulation, coupling to separate ion channels, or coupling to Hoffman, B. J., Kennedy, J. L., Seeman, P., Van Tol, ion-channel regulation rather than adenylate cyclase regula- H. H. M. & Niznik, H. B. (1992) Proc. Natl. Acad. Sci. USA tion as a primary physiological pathway. 89, 5522-5526. Localization of transcripts for this receptor indicates a 9. Jin, H., Oksenberg, D., Ashkenazi, A., Peroutka, S. J., Dun- relatively selective tissue distribution. Of tissues reported can, A. M. V., Rozmahel, R., Yang, Y., Mengod, G., Palacios, J. M. & O'Dowd, B. F. (1992) J. Biol. Chem. 267, 5735-5738. here, the 5-HT1F receptor was detected only in the brain, 10. Levy, F. O., Gudermann, T., Birnbaumer, M., Kaumann, uterus, and mesentery. The possible role of this receptor in A. J. & Birnbaumer, L. (1992) FEBS Lett. 296, 201-206. uterine or vascular function is intriguing. Future studies 11. McAllister, G., Charlesworth, A., Snodin, C., Beer, M. S., defining the specific cell type(s) in these tissues that express Noble, A. J., Middlemiss, D. N., Iversen, L. L. & Whiting, P. the receptor may provide insight into its function in the (1992) Proc. Natl. Acad. Sci. USA 89, 5517-5521. periphery. In the brain, the expression had a limited distri- 12. Zgombick, J. M., Schechter, L. E., Macchi, M., Hartig, P. R., Branchek, T. A. & Weinshank, R. L. (1992) Mol. Pharmacol. bution compared with that ofother 5-HT receptors (9). In the 42, 180-185. neocortex, labeling of layer V pyramidal neurons may indi- 13. Voigt, M. M., Laurie, D. J., Seeburg, P. H. & Bach, A. (1991) cate a functional role for the 5-HT1F receptor protein in the EMBO J. 10, 4017-4023. integration of sensorimotor (somatodendritic; frontal cortex) 14. Adham, N., Romanienko, P., Hartig, P., Weinshank, R. & or afferent information associated with limbic functions (so- Branchek, T. A. (1992) Mol. Pharmacol. 41, 1-7. or in 15. Maroteaux, L., Saudou, F., Amlaiky, N., Boschert, U., Plas- matodendritic; cingulate/retrosplenial cortex), spinal sat, J. L. & Hen, R. (1992) Proc. Nat!. Acad. Sci. USA 89, cord processes (axonal). Strong labeling was also detected in 3020-3024. hippocampal pyramidal cells, in several thalamic nuclei, and 16. Branchek, T., Zgombick, J., Macchi, M., Hartig, P. & Wein- in the dorsal raphe. The detection oftranscripts for this gene shank, R. (1991) in Serotonin: Molecular Biology, Receptors, in the dorsal raphe nucleus indicates a possible role as an and Functional Effects, eds. Saxena, P. & Fozard, J. R. autoreceptor. This localization is shared by both the 5-HTID' (Birkhauser, Basel), pp. 21-32. and receptors (9). Such heterogeneity of autorecep- 17. Hartig, P. R., Branchek, T. A. & Weinshank, R. L. (1992) 5-HT1Ds Trends Pharmacol. Sci. 13, 152-159. tors has recently been described (32). 18. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, The cloned 5-HT1F receptor does not appear to be any of 6-13. the 5-HT1-like receptor subtypes defined in a variety of 19. Weinshank, R. L., Zgombick, J. M., Macchi, M., Adham, N., isolated tissue preparations by in vitro response assays. Lichtblau, H., Branchek, T. A. & Hartig, P. (1990) Mol. However, such preparations contain heterogeneous popula- Pharmacol. 38, 681-688. tions of 5-HT1.jk. receptors and are primarily studied in 20. Sanger, S. (1977) Proc. Nat!. Acad. Sci. USA 74, 5463-5467. nonhuman tissues. The elucidation of the physiological role 21. Miller, J. & Germain, R. N. (1986) J. Exp. Med. 164, 1478- of the its autoreceptor 1489. 5-HT1F receptor, including possible 22. Bradford, M. (1976) Anal. Biochem. 72, 248-254. function, awaits the discovery of selective compounds, most 23. Cheng, Y. C. & Prusoff, W. H. (1973) Biochem. Pharmacol. likely through the use of heterologous expression systems 22, 3099-3108. expressing each individual subtype. 24. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) in Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold We thank Drs. M. Macchi, E. Gustafson, and D. Wang for helpful Spring Harbor, NY), 2nd Ed., pp. 7.19-7.22. discussions; B. Dowling, B. Ellerbrock, L. Gonzalez, A. Kokki- 25. McCabe, J. T. & Pfaff, D. W. (1989) in Gene Probes, ed. Conn, nakis, J. McHugh, M. Smith, H. Lichtblau, and S. Nawoschik for P. M. (Academic, San Diego), pp. 98-126. their excellent technical assistance; and J. Xanthos for sequence 26. Furchgott, R. F. (1972) in Catecholamines, eds. Blaschko, H. comparison studies. We thank E. Lilley and G. Moralishvili for & Muscholl, E. (Springer, Berlin), pp. 283-335. assistance with the illustrations and E. Marton forassistance with the 27. Bradley, P. B., Engel, G., Feniuk, W., Fozard, J. R., Hum- manuscript. Human samples were obtained from the National Dis- phrey, P. P. A., Middlemiss, D. N., Mylecharane, E. J., Rich- ease Research Institute. This work was partially funded by Small ardson, B. P. & Saxena, P. R. (1986) Neuropharmacology 25, Business Innovation Research Grants lR43NS28616 and 563-576. 2R44NS27789 and by Eli Lilly and Company. 28. De Vivo, M. & Maayani, S. (1986) J. Pharmacol. Exp. Ther. 238, 248-253. 1. Frazier, A., Maayani, S. & Wolfe, B. B. (1990) Annu. Rev. 29. Schoeffier, P., Waeber, C., Palacios, J. M. & Hoyer, D. (1988) Pharmacol. Toxicol. 30, 307-348. 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