European Journal o f Pharmacology - Moh'ctlhtr Pharmacoh)gy Section, __7 "~-~ ( 1~))~) ~ "~ 153- I'~2 (t', 1992 Elsevier Science Publishers B?v. All righls rescr,,,cd 0922-4V16/92/$05.00
153
E J P M O L 9113511
Genomic organization, coding sequence and functional expression of human 5-HT 2 and 5-I-tT A receptor genes Nico J. Stam ~', F r a n s V a n H u i z c n b, C a r o l i n e V a n A i c b c c k ', Jola,~ ~ Brands ", Rein Dijkcma '~ J c r o e n A.D.M. T o n n a e r b and W i e b c Olijve '~ !)~7~t:,,'tments o f " lliotectmoloey and Ri~ cl emir'If'y, arid " ,~,tetlropllarmacolo~,'v, ()tT~anoll hm'rnational i3. l':. Oss. Netherlands
Received 12 February 1992. revised MS received 22 May 1992. acceplcd 2 June 1992
The fanfily of scrotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Hcrc wc report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HTta reccptor st,btypcs. Both gcncs cncodc functional receptors for 5-11T. that differ considcrably in gcnomic structure primary amino acid scqucnce, pharmacology and signal transduction. The 5-HT~A receptor transfcctants displayed a single high affinity site for thc agonist [~H](+)-8-hydroxy-2-(di-n-propylamino)tclralin HBr ([3H]8-OH-DPAT) and a pharnmcoMgical profile spccific for the 5-HTIa receptor. In these transfcctants. 5-HT mediated a dose-dependent inhibitkm of forskolin-stimulatcd cAMP Icvcls. Cells expressing the 5-ITT2 receptor cxhibitcd high affinity binding for the anlag~mist [31t]kctanserin with a 5-HT. rcccptor spccific pharmacological profile, In these cells 5-HT activated phsopholipasc C in a dosc-dcpcnclc~t manner. The 5-HTz receptor displayed a genomic organization quite different from lhc 5-HTbx, 5-HT m and 5-HT m receptor ~;ubtypcs. While d~ese receptors arc encoded by one single cxon, the 5-HT2 receptor is er~codcd by three cxons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification. 5-HTz receptors (human); 5-1-1TIAreceptors (human): G-protein coupling: Kctanscrin: ( ] _ ~j - ~o, - i ~ 1.*1"~I-" . . . . . . . . ..~. ~. . . _. t , t ; ........ ~__,-~'~vl'~m-~ha) Ct "a 1 ttBr (8-OH-DPAT)
1. Introduction Thc neurotransmittcr serotonin or 5-hydroxytryptaminc (5-HT) clieits a widc array ~f physiological effects by binding to several receptor subtypes (Fraser ct al.. 199(I; Peroutka, 19901. These rcceptors bind 5-HT with diffcrent affinities, couple to different intracellular second messenger systems, and are expressed in a tissue specific fashion in cells of the central and periphcral nervous system. The 5-HT rcceptor subtypes have been classified on basis of their pharmacological characteristics and functional effects into four major classcs: 5-HTl-like, 5-HT 2, 5-HT3 and 5-Hq:~. The first class is thought to comprise at least five different submembers, termed 5-HTb,,, 5-HTu~, 5HT w, 5-HTu) and 5 - H T j p All 5-HT receptor subtypes, with the exception of the 5-HT 3 receptor which tbrms an ion channel, are linked to specific second messenger systems through guanosine triphosphatc
Correspondence to: Dr. N,J. Stare, ()rgamm Intl. B.V,. Dept. Biotechnok)gy and Biochemistry. P.O. Box 20, 5340 BIt ()ss, Nett~cro lands. Tel. 04120q127711: Fax 04120-fi2617.
binding proteins (G-proteins: Fraser et al., 1990; Pcroutka, 1990). In the past few years, several cDNA:~ and genes encoding receptor subtypes for 5-HT have been cloned, characterized and functionally expressed in eukaryotic cells. These studies indicated that these receptors share several structural characteristics with members of the large family of G pro~ein-eoupied recepl~ors (Lameh et al., 199(,1). They contain sew.'n putative transmembrane domains which are predicted to form a pocket for small ligands. Specific charged amino acid residues, located at the inner part of this pocket are thought to act as counter-ions for the ce,rresponding charged ligand (Findlay and Eliopoulos, 1990; Lameia et al., 199(.I; Hibert et at., 1991,1. Alignment of the receptor amino acid sequences indicates that highest sequence similarities between these (3 prolein-coupled receptors are found within these putatiwz transmembrane domains, especially between receptors that bind identical or structurally related compounds. A sc~ of oligonucleotide probes derived from the transmembrane segments of cloned receptor subtypes for 5-HT were used to isolate genes encoding human 5-HT receptor subtypes. Cloning of the genes for all 5-HT receptor subtypes is thought to be of importance
15-I
for several reasons. First, the cloning and expression of the individual genes in cells that do not express endogenous 5-HT receptors may enable us to answer questions concerning receptor activation and signal transduction. Second, the availability of sequences of several receptors that bind figands with varying affinities may provide new insights on the structural basis for ligand binding. Finally, the elucidation of the nucleic acid sequence and the genetic organization of the gc,,zs ~,~,:,~cni an interesting ~;pportunity ~.~ examine several evolutionary aspects of this multi-member receptor family. Moreover, such rnolecular data open the possibility It) validate and if necessary to revise the pharmacological nomenclature and classification scheme (Hartig, 1989). During the preparation of this paper, a eDNA sequence wa~ published identical to our 5-HT, receptor sequence (Saltzman et al., 1991). However, this genc was not expressed and therefore its gene product was not pharmacologically characterized. Here we report the genomic organization, coding scquenec and the functional exp~rcssion of the full-length human 5-HT, gene and compare its characteristics with the 5-1"H:~A receptor gene.
GACCG), tile sequence of which was derived from a consensus sequence corresponding to the putative third transmcmbrane-spanning domain of previously cloned G protein-coupled receptors for serotonin (Hartig et al., 1990). The probes ptmr5 and ptmr7 correspond to the fifth (nucleotides 865-897) and seventh (nucleotides 1683-1713) transmembrane segment of the rat 5-HT, eDNA as described by Pritchett et al. (1988). In order to determine whether the 5' terminal part ,ff the 5-HT= receptor gene contained an additional intron, this region was amplified from both cDNA and genomic DNA using oligonucleotide primers pl and p2 with the polym.erase, chain reaction (PCR) technique. Primers pl and p2 were positioned at the sequences 23 bp upstream ( - 23 to 4) and 262 bp downstream (262 to 29(I) of the translational start site of the human 5-HT 2 (fig. 2), respectively (Saltzman et al., 1991). The PCR conditions were as described by Sambrook et al. (1989). Primer pl contains an adaptor at the 5'-end to introduce a BamH! site 26 bp upstream of the A T G initialor codon.
2.3. Construction of both 5-HT1. a and 5-HT, expression i'eclors
2. Materials and methods
2. l. General nlethods General recombinant DNA procedures were performed as dcscribed (Sambrook et al., 1989). Oligonuclcotides were synthesized by an Applied Biosystems automated DNA synthesizer. Restriction endonuclcases wcrc purchased from Pharmacia. Radiolabelcd nuclcotides were from Du Pont (NEN). Dideoxynucleotide scquencing of selected restriction fragments of gcnomic and cDNA clones was performed by the chain-termination method using Scqucnasc version 2.0 (United Statcs Biochemical Corp.). Z2. Screenhtg o f libraries and PCR A human genomic DNA library prepared from placenta DNA partially digested with Sau3A restriction endonuelease and cloned into the EMBL3 bacteriophage vector was obtained from Clontech Laboratories (Palo Alto, CA, USA). A human cortex eDNA library (randomly and (dT) primed) in lambda Zap 1I vector was obtained from Stratagcne (La Jolla, CA, USA). Approximately 1.5 × l ( I 6 phage clones of both libraries were screened under iow stringency conditions with a mix of three oligo probes (ptmr3, ptmr5 and ptmr7) using standard procedures (Sambrook et al., 1989). Oligo ptmr3 is a degenerated oligonucleotide probe ( 5 ' - T C C A T C A T G C A C C T C T G C G C C A T C T C G CTG-
The expression vector pKCRE that was used to express the 5-HT~A and 5-HT 2 receptor genes into 3T3 cells differs from pKCR (O'Hara et al., 1981) in two aspects: (i) the 4 kb pBR322 moiety of pKCR has been replaced by the analogous 2.9 kb fragment from pBR327, and (it) a 80 bp EcoRI-Bglll fragment was removed from the coding part of the third exon of the rabbit /3-globin gene (Van Ooyen et al., 1979). In case of the human 5-HT~,x receptor, the entire coding sequence including somc 5' and 3' flanking sequences was excised from the 4 kb EcoRl fragment of gcnomic clone 1AI by BamHl and partial Hinfl digestions (scc fig. !). The resulting fragment was blunt-cndcd and inserted into the BamHl site of the cxprcssion vector pKCRE, which had been blunt-ended with the Klcnow fragment of DNA polymcrase (termed pE-5HT2). Genomic and eDNA clones were used to construct a full-length 5-I-IT~ receptor gcne (designated h 5-HT 2) and this procedure is schcmatically depicted in fig. 1. To express the 5-1-I"I"2 receptor the full-length 5-HT2 sequence was subcloned into the BamHl site of the exprcssion vector p K C R E (termed pE-5HT=). 2.4. Cell culture and gene tran.~fection Swiss 3T3 cells wcre incubated at 37 ° C in a humidified atmosphere (5% CO z) as a monolayer culture in DMEM (Ham's FI2) supplemented with 10% fetal calf serum (Hyclonc), 62.5 g g / m l each of penicillin and
155
streptomycin, and 2 mM glutamine. To establish cells that stably express the 5-HT~A and 5-HT 2 receptors 3T3 cells were transfected with the corresponding expression constructs. Cells were transfected by calcium phosphate-mediated transfection using the plasmid pAG60 (Colb~'.re-Garapin et al., 1981) as a selectable marker. Cells were selected and maintained in the presence of the neomycin analogue G418 (300 #t;/ml; Gibco, NY, USA). Subsequently, pools of G-418 resistant cells were expanded and tested for binding of either ['HId-OII-DPAT or [3H]ketan:,cd, in case of the 5-HT~a or 5-HT, receptor, respectively. For both receptor subtypes several single eel! ciones were generated by limiting dilution from pools of G418-resistant cells.
2.5. Pharmacolo~#' 15. i, Lis.and binding A f t e r d i s l o d g i n g o f t h e t r a n s f e c t e d 3 T 3 cells fl'om the dishes by scraping, cells were counted, pelleted, and frozen at - 8 0 ° C in aliquots of 10 7 cells. Frozen cell pellets were thawed and resuspended in ice-cold Tris-HCl buffer and homogenised using a PotterElvehjem homogeniser (five strokes at 1000 rpm) and diluted 5 x 10~ cells/ml. For saturation- and competition-binding experiments, 400 #1 of this homogenate w a s added to 50 /xl of 10-fold c o n c e n t r a t e d l i g a n d (fina! c o n c e n ! r a t i o n 2 n M ) a n d
5 ( / i l l o f d i s p l a c e r (1-1(I
p,M) or b u f f e r . T h e r e a c t i o n w a s t e r m i n a t e d by r a p i d filtration over GF/B (Whatman) filters and immediate
A t-/6:8-kg~
gen.lA1
,
gen.ZBl
,,.~
~ H ~'"
Sa
153
E J P M O L 9113511
Genomic organization, coding sequence and functional expression of human 5-HT 2 and 5-I-tT A receptor genes Nico J. Stam ~', F r a n s V a n H u i z c n b, C a r o l i n e V a n A i c b c c k ', Jola,~ ~ Brands ", Rein Dijkcma '~ J c r o e n A.D.M. T o n n a e r b and W i e b c Olijve '~ !)~7~t:,,'tments o f " lliotectmoloey and Ri~ cl emir'If'y, arid " ,~,tetlropllarmacolo~,'v, ()tT~anoll hm'rnational i3. l':. Oss. Netherlands
Received 12 February 1992. revised MS received 22 May 1992. acceplcd 2 June 1992
The fanfily of scrotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Hcrc wc report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HTta reccptor st,btypcs. Both gcncs cncodc functional receptors for 5-11T. that differ considcrably in gcnomic structure primary amino acid scqucnce, pharmacology and signal transduction. The 5-HT~A receptor transfcctants displayed a single high affinity site for thc agonist [~H](+)-8-hydroxy-2-(di-n-propylamino)tclralin HBr ([3H]8-OH-DPAT) and a pharnmcoMgical profile spccific for the 5-HTIa receptor. In these transfcctants. 5-HT mediated a dose-dependent inhibitkm of forskolin-stimulatcd cAMP Icvcls. Cells expressing the 5-ITT2 receptor cxhibitcd high affinity binding for the anlag~mist [31t]kctanserin with a 5-HT. rcccptor spccific pharmacological profile, In these cells 5-HT activated phsopholipasc C in a dosc-dcpcnclc~t manner. The 5-HTz receptor displayed a genomic organization quite different from lhc 5-HTbx, 5-HT m and 5-HT m receptor ~;ubtypcs. While d~ese receptors arc encoded by one single cxon, the 5-HT2 receptor is er~codcd by three cxons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification. 5-HTz receptors (human); 5-1-1TIAreceptors (human): G-protein coupling: Kctanscrin: ( ] _ ~j - ~o, - i ~ 1.*1"~I-" . . . . . . . . ..~. ~. . . _. t , t ; ........ ~__,-~'~vl'~m-~ha) Ct "a 1 ttBr (8-OH-DPAT)
1. Introduction Thc neurotransmittcr serotonin or 5-hydroxytryptaminc (5-HT) clieits a widc array ~f physiological effects by binding to several receptor subtypes (Fraser ct al.. 199(I; Peroutka, 19901. These rcceptors bind 5-HT with diffcrent affinities, couple to different intracellular second messenger systems, and are expressed in a tissue specific fashion in cells of the central and periphcral nervous system. The 5-HT rcceptor subtypes have been classified on basis of their pharmacological characteristics and functional effects into four major classcs: 5-HTl-like, 5-HT 2, 5-HT3 and 5-Hq:~. The first class is thought to comprise at least five different submembers, termed 5-HTb,,, 5-HTu~, 5HT w, 5-HTu) and 5 - H T j p All 5-HT receptor subtypes, with the exception of the 5-HT 3 receptor which tbrms an ion channel, are linked to specific second messenger systems through guanosine triphosphatc
Correspondence to: Dr. N,J. Stare, ()rgamm Intl. B.V,. Dept. Biotechnok)gy and Biochemistry. P.O. Box 20, 5340 BIt ()ss, Nett~cro lands. Tel. 04120q127711: Fax 04120-fi2617.
binding proteins (G-proteins: Fraser et al., 1990; Pcroutka, 1990). In the past few years, several cDNA:~ and genes encoding receptor subtypes for 5-HT have been cloned, characterized and functionally expressed in eukaryotic cells. These studies indicated that these receptors share several structural characteristics with members of the large family of G pro~ein-eoupied recepl~ors (Lameh et al., 199(,1). They contain sew.'n putative transmembrane domains which are predicted to form a pocket for small ligands. Specific charged amino acid residues, located at the inner part of this pocket are thought to act as counter-ions for the ce,rresponding charged ligand (Findlay and Eliopoulos, 1990; Lameia et al., 199(.I; Hibert et at., 1991,1. Alignment of the receptor amino acid sequences indicates that highest sequence similarities between these (3 prolein-coupled receptors are found within these putatiwz transmembrane domains, especially between receptors that bind identical or structurally related compounds. A sc~ of oligonucleotide probes derived from the transmembrane segments of cloned receptor subtypes for 5-HT were used to isolate genes encoding human 5-HT receptor subtypes. Cloning of the genes for all 5-HT receptor subtypes is thought to be of importance
15-I
for several reasons. First, the cloning and expression of the individual genes in cells that do not express endogenous 5-HT receptors may enable us to answer questions concerning receptor activation and signal transduction. Second, the availability of sequences of several receptors that bind figands with varying affinities may provide new insights on the structural basis for ligand binding. Finally, the elucidation of the nucleic acid sequence and the genetic organization of the gc,,zs ~,~,:,~cni an interesting ~;pportunity ~.~ examine several evolutionary aspects of this multi-member receptor family. Moreover, such rnolecular data open the possibility It) validate and if necessary to revise the pharmacological nomenclature and classification scheme (Hartig, 1989). During the preparation of this paper, a eDNA sequence wa~ published identical to our 5-HT, receptor sequence (Saltzman et al., 1991). However, this genc was not expressed and therefore its gene product was not pharmacologically characterized. Here we report the genomic organization, coding scquenec and the functional exp~rcssion of the full-length human 5-HT, gene and compare its characteristics with the 5-1"H:~A receptor gene.
GACCG), tile sequence of which was derived from a consensus sequence corresponding to the putative third transmcmbrane-spanning domain of previously cloned G protein-coupled receptors for serotonin (Hartig et al., 1990). The probes ptmr5 and ptmr7 correspond to the fifth (nucleotides 865-897) and seventh (nucleotides 1683-1713) transmembrane segment of the rat 5-HT, eDNA as described by Pritchett et al. (1988). In order to determine whether the 5' terminal part ,ff the 5-HT= receptor gene contained an additional intron, this region was amplified from both cDNA and genomic DNA using oligonucleotide primers pl and p2 with the polym.erase, chain reaction (PCR) technique. Primers pl and p2 were positioned at the sequences 23 bp upstream ( - 23 to 4) and 262 bp downstream (262 to 29(I) of the translational start site of the human 5-HT 2 (fig. 2), respectively (Saltzman et al., 1991). The PCR conditions were as described by Sambrook et al. (1989). Primer pl contains an adaptor at the 5'-end to introduce a BamH! site 26 bp upstream of the A T G initialor codon.
2.3. Construction of both 5-HT1. a and 5-HT, expression i'eclors
2. Materials and methods
2. l. General nlethods General recombinant DNA procedures were performed as dcscribed (Sambrook et al., 1989). Oligonuclcotides were synthesized by an Applied Biosystems automated DNA synthesizer. Restriction endonuclcases wcrc purchased from Pharmacia. Radiolabelcd nuclcotides were from Du Pont (NEN). Dideoxynucleotide scquencing of selected restriction fragments of gcnomic and cDNA clones was performed by the chain-termination method using Scqucnasc version 2.0 (United Statcs Biochemical Corp.). Z2. Screenhtg o f libraries and PCR A human genomic DNA library prepared from placenta DNA partially digested with Sau3A restriction endonuelease and cloned into the EMBL3 bacteriophage vector was obtained from Clontech Laboratories (Palo Alto, CA, USA). A human cortex eDNA library (randomly and (dT) primed) in lambda Zap 1I vector was obtained from Stratagcne (La Jolla, CA, USA). Approximately 1.5 × l ( I 6 phage clones of both libraries were screened under iow stringency conditions with a mix of three oligo probes (ptmr3, ptmr5 and ptmr7) using standard procedures (Sambrook et al., 1989). Oligo ptmr3 is a degenerated oligonucleotide probe ( 5 ' - T C C A T C A T G C A C C T C T G C G C C A T C T C G CTG-
The expression vector pKCRE that was used to express the 5-HT~A and 5-HT 2 receptor genes into 3T3 cells differs from pKCR (O'Hara et al., 1981) in two aspects: (i) the 4 kb pBR322 moiety of pKCR has been replaced by the analogous 2.9 kb fragment from pBR327, and (it) a 80 bp EcoRI-Bglll fragment was removed from the coding part of the third exon of the rabbit /3-globin gene (Van Ooyen et al., 1979). In case of the human 5-HT~,x receptor, the entire coding sequence including somc 5' and 3' flanking sequences was excised from the 4 kb EcoRl fragment of gcnomic clone 1AI by BamHl and partial Hinfl digestions (scc fig. !). The resulting fragment was blunt-cndcd and inserted into the BamHl site of the cxprcssion vector pKCRE, which had been blunt-ended with the Klcnow fragment of DNA polymcrase (termed pE-5HT2). Genomic and eDNA clones were used to construct a full-length 5-I-IT~ receptor gcne (designated h 5-HT 2) and this procedure is schcmatically depicted in fig. 1. To express the 5-1-I"I"2 receptor the full-length 5-HT2 sequence was subcloned into the BamHl site of the exprcssion vector p K C R E (termed pE-5HT=). 2.4. Cell culture and gene tran.~fection Swiss 3T3 cells wcre incubated at 37 ° C in a humidified atmosphere (5% CO z) as a monolayer culture in DMEM (Ham's FI2) supplemented with 10% fetal calf serum (Hyclonc), 62.5 g g / m l each of penicillin and
155
streptomycin, and 2 mM glutamine. To establish cells that stably express the 5-HT~A and 5-HT 2 receptors 3T3 cells were transfected with the corresponding expression constructs. Cells were transfected by calcium phosphate-mediated transfection using the plasmid pAG60 (Colb~'.re-Garapin et al., 1981) as a selectable marker. Cells were selected and maintained in the presence of the neomycin analogue G418 (300 #t;/ml; Gibco, NY, USA). Subsequently, pools of G-418 resistant cells were expanded and tested for binding of either ['HId-OII-DPAT or [3H]ketan:,cd, in case of the 5-HT~a or 5-HT, receptor, respectively. For both receptor subtypes several single eel! ciones were generated by limiting dilution from pools of G418-resistant cells.
2.5. Pharmacolo~#' 15. i, Lis.and binding A f t e r d i s l o d g i n g o f t h e t r a n s f e c t e d 3 T 3 cells fl'om the dishes by scraping, cells were counted, pelleted, and frozen at - 8 0 ° C in aliquots of 10 7 cells. Frozen cell pellets were thawed and resuspended in ice-cold Tris-HCl buffer and homogenised using a PotterElvehjem homogeniser (five strokes at 1000 rpm) and diluted 5 x 10~ cells/ml. For saturation- and competition-binding experiments, 400 #1 of this homogenate w a s added to 50 /xl of 10-fold c o n c e n t r a t e d l i g a n d (fina! c o n c e n ! r a t i o n 2 n M ) a n d
5 ( / i l l o f d i s p l a c e r (1-1(I
p,M) or b u f f e r . T h e r e a c t i o n w a s t e r m i n a t e d by r a p i d filtration over GF/B (Whatman) filters and immediate
A t-/6:8-kg~
gen.lA1
,
gen.ZBl
,,.~
~ H ~'"
Sa
