Composite structure of the human thyrotropin receptor gene

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STRUCTURE OF THE HUMAN TI-IYROTROPIN RECEPTOR GENE

Babette Gross, Micheline Misrahi, Sokhavuth Sar and Edwin Milgrom*

Institut National de la Sante et de la Recherche Medicale unite 135, Hormones et reproduction, 94270 Le Kremlin-BicCtre, FRANCE Received

April

19,

1991

The exon/intron organization and the structure of the 5’ flanking region of the human thyrotropin receptor gene (hTSH-R) were determined. The hTSH-R gene spans more than 60 kb and is split into ten exons. The extracellular domain is encoded by the first nine exons and part of the last exon, whereas the transmembrane and intracellular domains are encoded in totality by the last exon. The leucine-rich repeats of the extracellular domain are encoded as monomers or multimers by separate exons. The TSH receptor gene seemsto have arisen by insertion of a DNA sequence encoding repeated leucine-rich elements between the regions encoding the extracellular and the transmembrane domains of a proto-receptor gene ressembling the intronless l3 adrenergic receptor genes. Primer extension and Sl mapping experiments identified three transcription start sites. In the hTSH-R gene, the main site was located 157 bp upstream from the start of translation. The promoter region is very GC-rich and contains multiple SPl, ETF and AP2 binding site consensussequences. 0 1991Academic Press,1°C.

Thyrotropin (TSH) is the primary hormone that regulates thyroid cell function and proliferation (1). Binding of TSH to its receptor activates cyclic AMP (CAMP) and the phosphoinositol cascade through membrane-associated G proteins (1,2). A family of G-protein coupled receptors has been identified whose members (3-S) are characterized by the common structural feature of 7 transmembrane domains known to be involved in signal transduction and in binding small ligands (9). As LH/CG-R (10, 11) and FSH-R (12), the TSH-R is related to the other G-protein coupled receptors in that it contains 7 transmembrane regions (13-17). However, in contrast to G protein-coupled receptors that bind small ligands, receptors of this group (i.e. LH/CG-R, FSH-R, TSH-R) have a large glycosylated extracellular domain that is involved in ligand binding (18, 19). This characteristic makes the pituitary glycoprotein hormone receptors members of a specific subfamily of G-protein coupled receptors. Moreover, the TSH receptor (hTSH-R) is of interest because it can be the target of auto-immune reactions. Auto-antibodies directed against the TSH receptor are directly responsible for the pathogenesis of Graves’ disease and idiopathic myxoedema (20). To further our understanding of both normal and abnormal thyroid functions, we determined the organization of the human TSH receptor gene and its 5’ flanking region. * To whom correspondence should be addressed.

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Isolation and characterization of human genomic clones: A human genomic library (800 000 clones) constructed in the hage EMBL3 (211, was screened with a nick-translated full length human TSH-R cI! NA probe (16). Six positive clones were isolated through three rounds of screening and analysed by hybridization with oligonucleotides derived from the hTSH-R cDNA sequence. Selected restriction fragments of genomic clones were subcloned into Bluescri t vector (Stratagene). Continuous overlapping subclones were obtained and sequence B on both strands. Analysis of the 5’ end of the LTSH-R mRNk Poly(A)+RNA was prepared from human thyroid and goiter. Two primers were used: a 23 mer corresponding to nucleotides 15-37 upstream from the initiation codon (primer 2, 5’GGGGCTATTTCTCCATCCTCCGA-3’) and a 25 mer starting 14 b upstream from cpr C-3’). A 696 b the initiator ATG ( rimer 1, S-GCCGGCCTCATITKCACGGGA single-stranded D NpA robe initiating at end-labeled primer 1 and ending at the SaPI site, 687 bp upstream From the initiator ATG, was synthesized using a Ml3 vector, and purified on a 6% polyacrylamide-urea el. Thyroid poly(A)+mRNAs and probe or primers were hybridized. Extension wit % reverse transcriptase or digestion with Sl nuclease were performed as described (22), except that the Sl nuclease concentration used was 2000 to 8000 u/ml. DNA fragments were analyzed on a 15% polyacrylamideurea gel. RESULTS

AND DISCUSSION

Exon/intron organization of the BTSH-R gene The human TSH-R is split into 10 exons with sizes of 327, 72, 75, 75, 75, 78, 69, 78, 189, and more than 1412 bp respectively (fig. 1). Each intron/exon boundary (tab. 1)

El A

I

E2

E3 E4E5 289 385403416

10R

TM

11 I2 6X2 708 761

AT0

C 1 ,-I

hGhTF+I-I-R 2 ,I!

IoObp 3 ,-,

4 ,-, 5h-3

N

Fig. 1. Organization of the human TM-I-R gene. (A) Structure of the human TSH rece tor corn arison with LH and FSH rece tars. The human TSH receptor (hTSH-R) has t een’ dlvl -8 ed into domains according to tKe extent of homology with the porcine LH receptor ( LH-R) and the rat FSH receptor (rFSH-R). The extracellular part is divided in El-S. l%l. 1sthe transmembrane domain with its seven dashed membrane spans. ‘Ike

GhTSH-R 1 to 6 are the EMBW genomic clones used for sequencing. 680

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Table 1. Intron/exon boundaries of the hTSH-R gene. The sequence of each of the intron/exon boundaries is shown and the position of the intron with respect to the amino acid sequence (triplet code) is indicated. The highly conserved GT and AG sequences, at the donor and acceptor sites respectively, are underlined. DONOR

ACCEPTOR INTRONS

exon l--ACT.CT Thr Leu 56 57

gtgagtacccgggag...ttgttattttcaca

exon 2--AGA.AT Arg Ile 80 81

gtaagtttttttaat...ttttctgacattca

CTAC Ile Tyr 81 82

exon 3--CAF.$T

SaagtacaaggaaaJtgtaacttataca

A.GAA Ile Glu 106 107

105 106

GAAG

Leu Lys 57 58

exon 4--TTC.CT Phe Leu 130 131

gaagtattaaatcc...ttttcattaattta

T.GGC Leu Gly 131 132

exon 5--ATA.(7T Ile Leu 155 156

aaagtatgcacaca...ccctctctcttgcg

T.GAA Leu Glu 156 157

exon 6--ACA.CT Thr Leu 181 182

gIgagtattaccagt...ctctgttggttgta

GAAG Leu Lys 182 183

exon 7--GCT.GT Ala Val 204 205

gQagtaagacgata...tctctctccctcta

T.TAC Val Tyr 205 206

exon 8--TTG.CT Leu Leu 230 231

aagtaagacatac...ttctctgcctccca

G.GAC Leu Asp 231 232

exon 9--AGA.GG

aaagtggcagggac...tctgttgccttgca

A.ATC Gly Ile 294 295

3

$2

corresponds to a canonical splice consensus sequence (23). The coding sequence of the exons is identical to the cDNA sequence of the hTSH-R (17) except for: Ala.&GCC)-> Ala.&GCT),

Glu&GAG)--

> As~7~7(GAc)

and

LYS~~(AAG)--

> Asn,(AAC).

The mutations giving the same amino acid or the same class of amino acids may represent allelic variations. The coding region of hTSH-R gene was estimated to span

more than 60 kb. This evaluation was obtained mapping analyses (fig. 1). In situ hybridization chromosomal localization in 14q3 1 (24,25).

by restriction and oligonucleotide experiments have shown a single

Two different regions form the hTSH-R gene

The human gene of TSH-R displays an original feature. Nine exons encode part of the extracellular

intracellular

N-terminal

domain whereas

the totality of the transmembrane

domains is coded by a single long exon. This gene organization 681

and

is to be

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derivation of the hTSH-R gene from a proto-receptor gene. On top is showna schematicrepresentationof a proto-receptor genesimilar to the intronlessBadrenergicreceptor genes.The stripedregion representsthe extracellular part (denoted E) of the proto-receptor. Underneath,the hTSH-R geneisre resented.It 1sdivided into 10 exonswhoselimits are given by vertical bars.The extracellpular part of the receptor is composed,in the followin order, of: the signalpeptide (dashedregion), the E region derived from the extracelKular part of the proto-receptor (striped region) and the 16 leucine-richrepeats.Theserepeatsare representedby black squares.The arrow shows the position of the intron presentin the rat LH-R geneand absentin the hTSH-R gene (31). Dotted linesshowthe domainsof the proto-receptorfound in the hTSH rece tor. Black regionsrepresentthe sevenmembranespansof the proto-receptor and hTSR -R genes. TM and I are the transmembraneandintracellulardomains,respectively. Fig. 2. Putative

compared to other G-protein coupled receptor genes.Most of these genesare completly devoid of intronic structures (4, 5). This is the case for adrenergic and muscarinic receptor genes. For such receptors, which bind relatively small ligands and have a small extracellular domain (about 25 residues), a single exon encodes the totality of the protein. Moreover, the transmembrane and intracellular domains of the l3 adrenergic receptor have very similar sizes to the corresponding region of the hTSH receptor encoded by a single exon. These observations may suggestthat this domain of hTSH-R has evolved from a proto-receptor which spans seven times the membrane and that showsa similarity with a l3 adrenergic-type receptor. In contrast, the extracellular part of hTSH-R is highly mosaic and includes a successionof leucine-rich repeats found in the family of leucine-rich glycoproteins (LRG) (26). This long extracellular domain is involved in binding a large glycoprotein heterodimer, i.e., TSH (19). Hence, it is tempting to suggestthat the N-terminal region of hTSH-R derives from the grafting of the leucine-rich repeats onto the proto-receptor. The location of this insertion may be hypothesized by examining the segmentsadjacent to the leucine-rich repeat region. A leucine-rich repeat highly conserved in all three receptors (LH/CG-R,

FSH-R, TSH-R)

is found just prior to the first transmembrane segment whereas, this motif is absent in other G-protein coupled receptors. In the sameway, the first exon of the hTSH-R gene can be considered as a composite. It contains: the signal peptide, a domain spanning 15 residues, and a first leucine-rich repeat. The 15 residues-domain displays no homology but has a similar size when compared to the extracellular domain of the 13adrenergic receptor. This domain could be considered as originating from the extracellular part of the proto-receptor. Thus, the LRR composed domain of hTSH-R would have been inserted between the extracellular and transmembrane domains of the proto-receptor (see fig. 2). 682

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To date, several types of G-protein coupled receptors can be defined according to the presence and the position of introns in their genes. This classification is provisional because few genes of these receptors have been entirely cloned. The proteins of the first group have a short extracellular domain and their genes are intronless. This group includes adrenergic and muscarinic receptors (4, 5). Proteins in the second group also have a short extracellular domain, but their genes contain introns. The D2-dopamine receptor (27) and the substance P receptor (8) have introns in the transmembrane domain. The rhodopsin (3) and the substance K receptor (7) have introns in the transmembrane domain and at the junction with the intracellular domain. So far, only one member of this group has been found with an intron in the extracellular part: the RTA (Rat Thoracic Aorta) an unidentified rat protein (28). In contrast, proteins in the third group have a large extracellular domain (from 335 to 415 residues) which binds large glycoprotein hormones. The extracellular domain is encoded by many exons. This group includes TSH-R, and probably LH/CG-R and FSH-R. Exons correspond

to monomers or multimers

of leucine-rich

repeats

It has previously been hypothesized that the N-terminal part of the receptor has similarities to the leucine-rich glycoprotein family (26). This idea is now strongly supported by the organization of the 5’ half of the hTSH-R gene and in particular by the division into exons whose limits correspond to the limits of monomers or multimers of the repeats. The structure of the 5’ end of the hTSH-R gene confirms that the extracellular domain could have been constituted by successive duplications from a single sequence, thus bordering leucine-rich motifs regularly located, sometimes with loss of introns. We have not found any correlation between the limits of exons and the limits of leucine-rich repeats for two LRG genes whose structures have been determined, namely apolipoprotein A-I (29) and chaoptin (30). Exons of hTSH-R

and domains of sequence homology between hTSH-R,

LH-R

and FSH-R

TSH-R and LH-R were the first proteins of this family whose sequences were determined. Comparison of these sequences led us to propose the organization into domains of variable homology (see fig. 1) (16). Recently, the sequence of the FSH receptor has been established (12). When it was compared to the TSH-R and regions of variable homology scored the same domains were defined as when TSH-R and LH-R were compared. Moreover, in most cases these domains and especially in the extracellular region of hTSH-R correspond to discrete exons (fig. 1). Such defined domains might have been submitted to different selective pressures during evolution suggesting that they have different functional roles. These observations suggest that the overall organization of the LH-R and FSH-R genes may be similar to that of the hTSHR gene. However, partial sequencing of a genomic clone of the rat LH-R has shown the presence of an intron which does not exist in the hTSH-R gene (31) (in fig. 2, the position of this intron is shown by an arrow). 683

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l 78*100

II

I

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bp

III 1 2

GACTl

Cl

-12

Fig. 3. Determination of the transcription start sites.(A) showsthe primers used in primer extension experiments,the direction and the length of the extended products (icdicated,by dotted arrows).The probe usedfor Sl nucleasemappingoriginating at primer 1 ISshownunderneath. (B) I: Sl mappingwith the probe m the presenceof thyroid mRNAs (lane 1) and control tRNA (lane 2). II: cDNA extensionwith primer 2 in the presenceof tRNA (lane 2) and thyroid mRNA.s(lane 1). III: primer extension experimentwith primer 1 and thyroid mRNAs (lane 1). GACT lanes represent the four sequencingreactionsinitiated with the homologous primersand run m parallel.11,12and13are the three transcriptionstart sites.

Characterization of the promoter region of the human TSH receptor gene (i) Determination of the transcription initiation sites by SI nuclease mapping and primer extension experiments.Two major protected fragments and a cluster of multiple protected fragments were detected by Sl mapping. These three start sites of transcription are designated I,, I, and I,, with I, being the most 5’ of the three (fig. 3). Tbe same fragments were also found in primer extension experiments. Primer 1 only allowed the detection of the most proximal site, I,. This could be explained by stops of the reverse transcriptase due to secondary structures in this GC-rich region. But the two sites I, and I, were clearly detected with primer 2, localized upstream from primer 1. The major start site I, is localized at a guanine residue, 157 bp upstream from the 684

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-530 GTCGACTCAA

-520 CCACCGGAGT

-510 GGCCCCTGCA

-500 GTTGGATAGC

-490 AACGAGAATC

-470 GCAGGGCGX

-460 GGCTTCGGCC

-450 GCACCGCGGG

-440 CTAGCCAGGG

-430 -420 CTGCGTGCCC . . . . .GCCTCTGACC ...

-410 CTCAGCAGAG

-400 GTGTCTCTGG

-390 CCAGGAGGAG

-380 CTGAAGTTCT

-370 GCAGGACATT

-360 GGTCCGCCCG . .... ..

-350 CGGACAGTCC

-340 ACTCCGCGGG

-330 GACTTTCTCT

-320 GGATAAGGAG

-310 TGCGTGCGAG

-290 GCAGACAGGG ...

-280 TGTCTAGAAG

-270 GCTACACGCT

-260 AGGGAAGGTG

-250 GCTCCTTGGA

-300 TGGCTCCCAG . .. . .. . -24c T?TA.L:.SAGG

-230 AGGAMGGAG

-220 GGGGCATCTA

-210 AACTAGGCTT

-200 TGGAGAGAAC

-170 -160 GTGGGGGGGC GGGCTGGAAA . . . . . . . . z -110 -100 CAGACGGAGC GGACTGCGTG

-150 ACAGAGGGGA

-140 CAGCCAGGW

-190 -189 TAATGGGAGG GGCGCCCGGG m. . .. .. . . . -130 -120 TGGTGTTGGG TGTCAGG'ZM,

-90 GGTCCAGCCA

-80 AGWGTGA

-70 AGCAAGCAGA

-60 CTTGTTTGGG

-50 -40 TCAAGGTTGC CTAGGGAAGC . .. . .. . .. . . . 10 20 TCCACAGTGG TGAGGTCACA

-30 GGAGCACTTA

-20 AGTGCCTCTT

-10 TTTCCCCTTC

30 GCCCCTTGGA

40 GCCCTCCCTC

50 TTCCCACCCC

+1 TCCAGCCTCC A 60 TCCCGCTCCC

100 TGCAGAGCTG A A 160 m

110 AGAATGAGGC

120 GATTTCGGAG

l *******

Fig.

AND

-

70 GGGTCTCCTT

80 TGGCCTGGGG

90 TAACCCGAGG

130 GATGGAGAAA

140 TAGCCCCGAG

150 TCCCGTGGAA

Sequence of the promoter

-480 CTCCAGGGGT

and of the 5’ flanking region of the LTSH-R

gene. The

initiator codon ATG is boxed at the end of the sequence.The three transcription initiation sitesare indicatedby closedtriangles.The most5’ one is noted nucleotide + 1. In the promoter region, the modified TATA box and the modified CAAT box at positions-43 and -86 bp, respectively,are indicatedby a thin line. Consensus sequences for the SPl binding site at positions-427, -361, -168 bp are underlined by dashes. Multiple ETF consensus sequences, found at positions-513,-480,-338,-225,-172,-149,11, + 28, + 53, + 83, are underlined.A CRE consensus siteat position + 17 1sunderlined by stars.The four consensus sequences for the AP2 binding site are underlined by dots at positions-300,-181,-46 and +79 bp.

initiation codon ATG. The second site I, starts at a guanine, 80 bp from the initiator ATG. The third site I, is not precisely defined and includes two major starts at positions 57 and 52 bp from the ATG. (ii) ,4nalysis of the promoter and of the S’flanking region of the hTSH-R gene. The sequence of the 534 bp upstream from the first transcription initiation site I, has been determined (nucleotide + 1 on figure 4). This 5’ flanking region is very GC-rich (61%). There are no sequences corresponding to a consensus CAAT box or a TATA box. However, degenerated TAGGGAA and CAAGGAAAGT boxes respectively, may be defined at positions -43 and -86 bp. Three potential binding sites for the transcription factor SPl were located at positions -427, -361, -168 bp (32). Multiple ETF binding consensus sequences were found at positions -513, -480, -338, -225, -172, -149, -11, +28, + 53, + 83 (33). These characteristics are similar to the promoter regions of genes for EGF receptor, the insulin receptor, H-Ras I, and insulin-like growth factor II (34). Between the major transcription initiation site and the minor sites, there are potential sites for regulatory transcriptional elements such as CREB, AP2 and ETF. This 685

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observation raises the possibility of two differentially regulated transcription units (35). In human thyroid, TSH-R is regulated by TSH and CAMP (2). In the rat thyroid FRTL-5 cell line, TSH-R messenger RNA is down-regulated by CAMP (36). In the case of LH-R and of the 82 adrenergic receptor, CAMP has been shown to exert a dual effect, increasing receptor mRNAs at low concentration and decreasing it at high concentration (37, 38). Therefore, we searched for cis-acting elements activated by CAMP. The sequence of the hTSH-R promoter displays a single CRE consensus site with one mismatch, at position + 17 (39). Four consensus sequences at positions -300, -181, -46 and +79 bp could constitute binding sites for the nuclear factor AP2, which is able to induce gene transcription mediated by both CAMP and phorbol esters (40). Three of the AP2 consensus sites have one mismatch each, three of them are inverted. Two AP2 consensus sequences overlap ETF sites and one overlaps a TAAGGGAA consensus sequence. Such overlaps between binding sites for regulatory proteins and transcription factors have been shown to explain transcription repression mechanisms in several systems (41). TSH receptor is expressed mostly in the thyroid (42). Hence, we looked for cis elements found in promoters of thyroid-specific genes such as the thyroglobulin (43) and the thyroid peroxydase (44) which are both controlled by TSH and CAMP. Within the 535 bp of the 5’ flanking region, we did not find any binding site for the thyroidspecific nuclear proteins l-IF-1 or T-IF-2 (45). Functional studies of these potential regulatory regions will provide a thorough understanding of the regulation of the hTSH-R gene expression. Moreover, the availability of the complete gene structure for hTSH-R may provide additional information about structural or functional domains within the glycoprotein hormone receptors. ACKNOWLEDGMENTS

We thank P. Dessen for computer analysis. We thank M. Rauch for oligonucleotides synthesis. BG received a grant from the Institut de Formation Superieure BioMCdicale. REFERENCES

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