Proc. Nati. Acad. Sci. USA Vol. 90, pp. 408-412, January 1993 Neurobiology Cloning of another human serotonin 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 serotonergic 1% agarose gel. The DNA fragment was isolated from the gel, ligands: 5-HT > sumatriptan >> 5-carboxyamidotryptamine kinased, and cloned into pBluescript. Recombinant clones > 8-hydroxy-2(di-1-propylamino)tetralin > spiperone. 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. antimigraine drug sumatriptan at this subtype indicates a Adenylate Cyclase Activity. Adenylate cyclase activity was possible role of the 5-HT1F receptor in migraine. 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 tryptamine; 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 5 - H T M D F L N S S D Q N LT S E L L N RMPSK ;S -1 M N I T N C T T E A S M A; R P K T I T E K M 5-HT, M S P L N Q S A E 4 L P Q E A S N R S L N A T E T S E A W D P R T L Q AL 39 c -n 5 - HT, 0 M E E P G A Q C A P P P P A G S E T W V P Q A N L S S A P S O N C S A K D Y I Y Q D S I S L PW:.O KV 5 - H T. Ar M D V L S PGQGNNTTSPPAPFEIT GN TTGr 'S5UV' TVS4 C i9' -. .. :. 5-HI1 , . V S- L-- :- -- L : F. -n L A - Y-XR K-L HHP A N Y I C$sL A-VTXD F- L VAVY 7L4 .. ..................... 5-HTI1 ..L. I C M i.X..tt- V I L...T...T.. L L A & - .......K.- ...-" .V ..N...L.LM.A$. ...... -:.. --....--------- - S L A ...V I ..L..A V L A F 5 HI;),,LT V...... S. .L .L L.T.R.. KL)...... ...... ..t..:...T...GSLATT.LT.......:P....A...PA #:Y - LVS 89 I t.-. L V M L...L....A L I A..X-..A.-.-t....T .. L S F A 5- H T L. N........A .. I. RT~~KtA~~TPANYL1A........O... 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