Molecular and Celhlar Endocrinology, 84 (1992) 127-135 0 1992 Elseviet Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00

127

MOLCEL 02715

Expression of the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript J.T. Aatsinki, E.M. Pietilg, J.T. Lakkakorpi and H.J. Rajaniemi Biocenter and Department of Anatomy, Vniuersity of Oulu, SF-90220 Oulu, Finland

(Received 10 August 1991; accepted 27 November 1991)

Key words: Alternative splicing; Isoform; Luteinizing hormonc/chorionic (Rat ovarian)

gonadotropin

receptor;

Polymerase

chain reaction;

Summary Luteinizing hormone/chorionic gonadotropin (LH/CG) receptor complementary DNA (cDNA) isoforms were amplified using pseudopregnant rat ovarian total RNA as a template and the primers reaching over the coding regions at both ends in a reverse transcriptase-polymerase chain reaction CRT-PCR). Agarose gel electrophoresis of the PCR products revealed three bands corresponding to about 2.1, 2.0 and 1.8 kilobases (kb). Subcloning of pooled PCR products into EcoRI site of pUCBM20 resulted in 167 clones, from which five different restriction patterns were obtained by digestion with EcoRI and E&III. One clone of each was further characterized. It could be predicted from the nucleotide sequences that the clone rLHR2100 encoded a full-length receptor (a 674 amino acid mature protein), the clone rLHR2075 Iacked part of exon IX (nucleotides 693-717) and encoded a truncated 225 amino acid mature protein, the clone rLHR1950 Iacked exons III and IV (nucleotides 246-395) and encoded a nearly full-length protein (a 624 amino acid mature protein), and the clones rLHR1834 and rLHR1759 lacked the same part of exon XI (nucleotides 960-1225), with exon V (nucleotides 396-470) also absent in the latter, the deletion in exon XI leading both these clones to premature termination. The clone rLHR1834 encoded a 316 amino acid mature protein and rLHR1759 a 291 amino acid mature protein, respectively. The sequence data suggest that all of these isoforms contain the putative signal sequence and are derived from a single copy gene via alternative splicing. These results point further to the fact that the expression of the 90 kDa LH/CG receptor is regulated via an extensive alternative splicing of the receptor gene primary transcript.

Introduction Cloning of porcine, rat and human luteinizing hormone/ chorionic gonadotropin (LH/CG) re-

Correspondence to: Jyrki Aatsinki, M.Sc., Biocenter and Depa~ment of Anatomy, Unive~i~ of Oulu, Kajaanintie 52 A, SF-90220 Oufu, Finland. Fax 3.58-81-337226.

ceptors (Loosfelt et al., 1989; McFarland et al., 1989; Minegish et al., 1990) and other glycoprotein hormone receptors, follicle-stimulating hormone (FSH) receptor (Sprengel et al., 1990) and thyroid-stimulating hormone (TSH) receptor (Parmentier et al., 1989) cDNAs have revealed that these receptors belong to the G protein-coupled receptor superfamily with seven membrane spanning domains. They differ, however, from the

128

other G protein-coupled receptors cloned to date in that they contain a large extracellular domain in the amino (NJ-terminal part of the polypeptide which binds the ligand (Kellokumpu and Rajaniemi, 1985; Tsai-Morris et al., 1990; Xie et al., 1990; Ji and Ji, 1991). Braun et al. (1991) have shown recently that only the leucine-rich repeats l-8 comprising exons I-VIII are required for the high affinity hCG binding site. Loosfelt et al. (1989) reported in addition to the full-length porcine LH/CG receptor cDNA clone three cDNA clones encoding truncated proteins that lack the transmembrane domain, and while our experiments were in progress a rat LH/CG receptor cDNA isoform corresponding to the porcine LH/CG receptor cDNA isoform B and several other isoforms lacking parts of the extracellular domain were also reported (Bernard et al., 1990; Segaloff et al., 1990; Tsai-Morris et al., 1990). All of these isoforms have arisen through alternative splicing of the primary transcript of a single copy gene (Koo et al., 1991; Tsai-Morris et al., 1991). The extensiveness of the alternative splicing of the LH/CG receptor primary transcript and its role in the regulation of LH/CG receptor expression in different physiological states are not known, however. The first step towards elucidating these questions consists of isolation and characterization of the cDNA isoforms of the LH/CG receptor. In the present work we produced rat LH/CG receptor cDNA isoforms by polymerase chain reaction (PCR), which was chosen instead of library screening because of the need to obtain all possible isoforms containing the whole coding region (McFarland et al., 1989). This was achieved with the primers derived from the N- and C-terminus of the LH/CG receptor cDNA used in PCR which moreover contained internal restriction sites in their non-coding regions to facilitate subsequent subcloning. Five cDNA isoforms, including the full-length LH/CG receptor cDNA (McFarland et al., 1990), were characterized. Two were identical to those reported earlier (Bernard et al., 1990; Segaloff et al., 1990; Tsai-Morris et al., 1990), but two represent a completely novel type, lacking exons that comprise part of the hCG binding site, exons 111, IV and V (Braun et al., 1991).

Materials

and methods

Preparation of total RNA

Immature 26-day-old Sprague-Dawley rats were injected S.C. with 40 IU of pregnant mare serum gonadotropin (PMSG) (Diosynth, Oss, Netherlands) followed by 25 IU of human chorionic gonadotropin (hCG) (Diosynth) 54 h later. Seven days after the latter injection they received 500 IU hCG/lOO ~1 phosphate-buffered saline (PBS) (down-regulated) or only 100 ~1 PBS via the tail vein. Three animals were killed prior to and 0.5, 1, 2, 3, 4, 5 and 7 days after hCG or PBS injection and total ovarian RNA was isolated by the guanidinium isothiocyanate/ cesium chloride method (Sambrook et al., 1989) for subsequent reverse transcriptase-polymerase chain reaction CRT-PCR) amplification. Because the alternatively spliced transcripts could change in number or relative amounts during down-regulation and subsequent up-regulation, total RNA isolated from ovaries taken at different time intervals after hCG treatment was used as template in RT-PCR. Reverse transcription and PCR amplification

The reverse primer CAATTTTGGAATTCIAGTGAGTTAACGCTCTCG (R2116) was used in a first strand synthesis by avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, MI, USA) and the forward primer GGGAGCTCGAATTCAGGCTGGCGGGCCATGGGGCGG (F-27) in a second strand synthesis. Both primers are designed to have an internal EcoRI restriction site (mismatched nucleotides are underlined). A 50 pg sample of total ovarian RNA and 100 ng of both primers were mixed in water and denatured at 65°C for 15 min. They were then allowed to cool and 10 ~1 10 X PCR buffer (0.5 M KCI-0.1 M Tris-HCl (pH 8.31-15 mM MgCl,-0.1% gelatin), 2 ~1 10 mM each of deoxynucleoside triphosphate (Boehringer-Mannheim, Germany), 8 U of AMV reverse transcriptase, 1 U of Inhibit-ACE (5 prime-3 prime, USA) and 2.5 U of Taq polymerase (Boehringer) were added to a total volume of 100 ~1. The mixture was overlayed with mineral oil and incubated at 42°C for 1 h. After reverse transcription, the mixture was incubated

129

at 95°C for 5 min. PCR was performed immediately with a DNA thermal cycler (Perkin-Elmer/ Cetus, Norwalk, USA) for 30 cycles. Each cycle consisted of denaturation at 95°C for 1 min, primer annealing at 55°C for 2 min, and extension at 72°C for 10 min + 59 s/cycle. PCR products (25 ~1 of mixture) were size fractionated on 0.8% agarose gels.

2322

)

2027

ä

1078

)

872

)

Characterization of PCR products

The PCR products obtained using total RNA from different time points as a template were pooled, digested with EcoRI and size fractionated by 0.8% agarose gel electrophoresis. The major bands were excised from the gel, pooled and subcloned into the dephosphorylated EcoRI site of pUCBM20 (Boehringer) using T4 DNA ligase (Boehringer). Recombinant plasmids were digested both with EcoRI (Boehringer) and HaeIII (Boehringer) and then size fractionated on 1.5% agarose gels. The structures of clones with different restriction patterns were determined by the double stranded dideoxy sequencing method (Sanger et al., 1977) using the Sequenase Version 2.0 DNA sequencing kit (United States Biochemical Corporation, USA). Computer analyses

Computer searches and sequence analyses were carried out using the programs of the Genetics Computer Group (Devereux, 1989). Hydropathy analyses were carried out according to Kyte and Doolittle (1982). Results

Total RNA from pseudopregnant rat ovaries as a template and primers covering the whole LH/CG receptor coding section were used here in RT-PCR to amplify the LH/CG receptor cDNA isoforms. Agarose gel electrophoresis of the PCR products revealed three bands corresponding to 2.1, 2.0 and 1.8 kb (Fig. 1). Subcloning of pooled PCR products into EcoRI site of pUCBM20 resulted in 167 clones. Digestion of recombinant plasmids with EcoRI and HaeIII revealed five different restriction patterns, one clone of each was sequenced by the double stranded dideoxy sequencing method and further

Fig. 1. Amplification of LH/CG receptor cDNA isoforms using primers R2116 and F-27 and total RNA from the pseudopregnant rat ovary as a template in PCR. Right lane shows the PCR products size fractionated on a 0.8% agarose gel and stained with ethidium bromide. Left lane shows the molecular weight standards (A DNA/Hind111 digest and 4X174 DNA/Hue111 digest).

characterized. In addition to the full-length LH/CG receptor cDNA (rLHR2100) which represented 3% of the recombinant clones four clones corresponding to alternatively spliced transcripts were detected. The clone rLHR1834 represented SO%, the clone rLHR2075 15% and both the clone rLHR1950 and rLHR1759 1% of all the recombinant clones. The nucleotide sequences of the alternatively spliced exons and detected point mutations are shown in Fig. 2. The sequence of the largest clone (rLHR2100) was related to the full-length rat LH/CG receptor cDNA, encoding a 674 amino acid mature protein (75 kDa unglycosylated polypeptide), and the sequence of the second clone (rLHR2075) was identical to it with the exception of a 25 base pair deletion from base position 693-717. This re-

130 F-27 exon I GGGAG_CT~TCAGGC'~GGCGGGCCATGGGGCGGCGAGTCCCAGCTCTGAGACAGCTGCTGGTGCTGGCAGTGCTGCTGCTGMGCCTTCACAGCTGC

1731 AGTCCCGAGAGCTG~'CAGGGTCGCGCTGCCCCGAGCCCTGCGACTGCGCACCGGATGGCGCCCTGCGCTGTCCTGGCCCTCGAGCCGGCCTCGCCAGA~

qtgaqtacaqgqtqccccaq---intros

I

1174 exon kb)---ttttCCtttttcttttcCagATCTCTCACCTATCTCCCTGTC~GTMTTCCATC

(14.7

2451 ACAAGCTTTCAGGGGACTTAATGAGGTCGTCGT~Tgtaagtaagatacttcatat---intron

II

246f II

(2.2kb)---CtttttttcctcttccctagT

exon III 320/ GAAATCTCTCAGAGTGATTCCCTGGAAAGGATAGATAGMGCT~TGCCTTTGAC~CCTCCTCMTTTGTCTGMCTgtaagcatcagctaacggat------

---intron

III

(2.5

,321 exon kb)---tcccccttctctcttcacaqACTGATCCAGMCACC~CCTGCTATACATTGMCCTGGTGCT~TAC~C

3951 CTCCCTCGGTTAAAATACCTgtgagaax,tgttccttata---~r,trcm

IV

(2.1

IV

I396 kb)---tgccctgccctqaatcaaagGAGCATCTGTAACACA

exon v 470/ GGCATCCGAACCCTTCCAGATGTTACGAAGATCTCTCCTCCTCTGMTTT~TTTCATTCTgtaagtatcaggctqattgc---intron

V

1471 exon VI ---ttctcatttctttcccctagGG~TCTGTGAT~CTTACACATMCCACCATACCCGGG~TGCTTTCC~GGGATGMT~CGAGTCTGTCACAC 1546 Tgtgagtacagcctctqga---intron t

VI

(7.2

bp)--

G

I549 kb)---Ctctcctccctcctccag~CTGTATGGAGCCA

61-l/ TGCATTCAATGGGACGACTCTAATCTCTCGCTgtaagtatatgctgatttcc---lntron

(88

exon

VII

1610 VII

(2.7

kb)---qaaactgtctttcCtcacagGGAGC

exon "III 6921 TAAAAGAAAACATCTACCTGGAGAAGATGCACRGTGCACAGTGGAGCCTTCCAGGGGGCCACGGGGCCCAGCATCCTqtgagtacaatggtacagcc---intron I693

VIII

(4.2

C

exon IX CCTGCCGAGCCACGGGCTGGAGTCCATTCAGACGCTC

kb)---CtctCCcCtttqcqqttcagGGATATTTCTTCCACCAAATT -------_~-----_~-~---

_-

8791

078/ T~GGMTTTGCCGAAGAMGAgtqagcaqtgaqcaggagaggq---~ntron

IX

(2.0

AG -_ f!?L!r

kb)---tacctcttctctttcag

l

959, exon x CATTTTTGAAAACTTCTCCAC~TGCGAAAGCACACAGTTAG~GCAGAT~CGAGACGCTqtaagtattcacacac---lntron 1960 ---~~ttaat~cttagagtcgtqqgaacctcttatgtctccctcaaqccaatgtttgtcttaCagTTATTCCGCCATCTTTGAGGAGMTGM~CAGTG

883 TTTTTCATTTTC

X exon

(13.6

kb)

XI

GCTGGGATTATGATTATGGCTTCTGTTCACCCAAGACACTT

CGCTTCCTCATGTGTMTCTCTCCTTT

T_-j226 AC TTTTGCATGGGGCTCTACCTGCTGCTCATTGCCTCCGTGGACTCCC~C~GGCCAGTA~A~

A

AGGCGrTTCAGAGAGATTTCCTTCTGCTGCTGAGCCCAT'I'CGGCTGCTCTAAACGCCGGGCGGAGCTTTACAG~GGMGGMTTTTCTGCATATA~TC CAACTGCAAAAATGGCTTCCCAGGAGCAAGTAAGCCGTCCCAGGCTACCCTGAAGTTGTCCACAGTGCACTGTCMCAGCCCATACCAC~GAGAGCGTTA AC'TCACTAGCATTACAAAAT'IG H,,,,,

to the sequences of alternatively spliced exons underlined by solid or dotted lines. The numbers correspond Fig. 2. Nucleotide nucfeotide sequence of the full-length LH/CG receptor cDNA (McFarland et al., 1989). The intronic sequences of the LH/CG receptor gene are from Koo et al. (1991) and the sequence of intron 10 from Tsai-Morris et al. (1990). Capital letters correspond to exonic sequences and lowercase letters to intronic sequences. The initiator codon and alternative stop codons are overlined. Internal 3’ splice site sequences and branch point sequences are indicated by boxes. Detected point mutations of alternatively spliced transcripts (rLHR1950 and rLHR1759) are indicated above the sequence. Asterisks indicate places where presumed intronic sequences are retained in the isoforms rLHRC1 and rLHRC2. Solid arrow underlinings indicate the positions of the primers used in RT-PCR amplification (F-27 and R2116).

131

sulted in a frame shift leading to a premature termination codon 20 amino acids after the point of divergence. This clone should encode a 225 amino acid mature protein (25 kDa unglycosylated polypeptide). The sequence of the third clone (rLHR1950) was again identical to the rLHR2100 with the exception that a 150 base pair deletion was found at base position 246-395. This clone conserved the same open reading frame and the mature protein should consist of 624 amino acids (69 kDa unglycosylated polypeptide). The predicted polypeptide lacks one conserved potential N-glycosylation site, however, corresponding to asparagine 103 in the full-len~h protein. Three point mutations were also found in this clone. The T at base position 698 was replaced by C, leading to substitution of Thr for Be 233 in the full-length protein, a silent mutation was met at base position 1710, where A was replaced by G, and G was replaced by A at base position 1936 leading to the substitution of Ser for Gly 646 in the full-length protein. The sequence of the fourth clone (rLHR1834) was identical to that of rLHR2100 with the exception of a 266 base pair deletion from base position 9601225 leading to a premature termination codon 22 amino acids after the point of divergence. This clone encoded a 316 amino acid mature protein (35 kDa unglycosylated polypeptide). The sequence of the fifth clone (rLHR1759) was again identical to that of rLHR1834 with the exception of a second deletion from base position 396-470 and one point mutation, A being replaced by G at base position 536, leading to the substitution of Gly for Glu 179 in the full-length protein. The predicted polypeptide also lacks one conserved cysteine, corresponding to amino acid 135 in the full-length protein. The mature protein encoded by this clone should consist of 291 amino acids (32 kDa unglycosylated poIypeptide1. Discussion The present results and some others (Bernard et al., 1990; Segaloff et al., 1990; Tsai-Morris et al., 1990) demonstrate that the primary transcript of the rat ovarian LH/CG receptor exhibits extensive alternative splicing, resulting in at least eleven different transcripts (Fig. 31, the predicted

amino acid sequences of which are shown in Fig. 4. Two of the transcripts encode a nearly fulllength LH/CG receptor. The isoform rLHR1950 encodes a 624 amino acid mature protein and the identical isoforms E and A2 (isoform E/A21 (Bernard et al., 1990; Segaloff et al., 1990) a 612 amino acid mature protein (a 68 kDa unglycosylated polypeptide). These isoforms are spliced through a cassette exon mode without any change in the open reading frame. Exons III and IV are excluded from the isoform rLHR1950 and exon IX from isoform E/A2. The predicted mature protein of the former isoform lacks 50 amino acids inchtding one conserved potentia1 N-glycosylation site (corresponding to asparagine 103 in the fuIl-length protein), and that of the latter one 62 amino acids including two conserved cysteines (corresponding to amino acids 283 and 284 in the full-length protein). According to the hydropathy profiles (data not shown) only the isoforms rLHR1950 and E/A2 can be considered membrane-spanning, whereas the rest may represent soluble proteins or proteins which may be attached to the membrane, e.g. via a glycosyl-phosphatidylinositol membrane anchor (Mayor et al, 1991). It is not yet known whether these putative soluble receptor isoforms are secreted, but there is evidence that an isoform lacking the transmembrane domain expressed in COSl cells is a soluble secreted protein capable of ligand binding (Tsai-Morris et al., 1990). Xie et al. (1990) and Braun et al. (1991) expressed receptor molecules lacking a transmembrane domain in human kidney 293 cells and found that the proteins remained within the cells but were capable of Iigand binding. The isoforms rLHR2075 (identical to isoform B2 in Segaloff et al., 19901, rLHR1834 (identical to isoform B in Bernard et al., 1990, isoform B4 in Segaloff et al., 1990 and one isoform in TsaiMorris et al., 1990) and B3 (Segaloff et al., 19901, which encode truncated mature proteins of 225, 316 and 341 residues, respectively, are spliced by an alternative internal 3’ acceptor site mode, leading to premature termination. Exon IX contains an internal 3’ splice site consensus sequence CAG/G, which is used in the isoform rLHR2075 while the isoform rLHR1834 uses an internal 3’ splice site sequence CAG/A in exon XI which

132

differs from the consensus sequence YAG/G (see review Smith et al., 1989). A putative branch-point sequence TCCTGAC is located 5763 nucleotides upstream of this alternative 3’ splice site. The mammalian branch point consensus sequence YNYTRAY (see review Smith et al., 1989) is also found in the intron X (ttttaat) at almost the same distance (56-62 nucleotides upstream of the 3’ splice site). The isoform B3 uses the 3’ splice site in exon X, which contains the same 3’ splice site sequence as exon XI. All these internal 3’ acceptor sites are preceded by pyrimidine-rich regions which are also important for recognizing alternatively spliced acceptor sites. The isoforms rLHR17.59, EB (Bernard et al., 1990) and Bl (Segaloff et al., 19901, encoding I rLHR2100 (700 aa]

II

Ill

245 246

IV

v

395 396

mature proteins of 291, 254 and 279 residues, respectively, are spliced by a combination of cassette exon and alternative internal 3’ acceptor site mode. The isoform rLHR1759 lacks exon V and is prematurely terminated using an internal 3’ splice site in exon XI, the same one as in isoform rLHR1834. Isoform EB lacks exon IX and is prematurely terminated using the same internal 3’ splice site in exon XI as isoform rLHR1834. Isoform Bl lacks the same exon as isoform EB (exon IX) and is prematurely terminated using the same alternative 3’ splice site in exon X as is isoform B3. The fourth mode of alternative splicing of the LH/CG receptor gene consists of partially retained introns. Segaloff et al. (1990) reported two VI 548

VII 549

VIII 692

IX 693

X

XI

959 960

rLHR2075 1251 aa] rLHR1950 I650 aa) rLHR1834 1342 aa] rLHR1759 1317 aa] rLHREIA2 1638 aa] rLHREB [280 aa) rLHRB1 (305 aa] rLHRB3 (367 aa] rLHRC1 1294 aa) rLHRC2 1183 aa]

I

diagram depicting the organization of the LH/CG receptor cDNA isoforms. Numbers corresponding to Fig. 3. Schematic exon-intron boundaries are indicated in the full-length LH/CG receptor cDNA (rLHR2100) and only alternatively spliced exons are indicated by numbers in the case of the other isoforms. The exons of the LH/CG receptor gene are indicated by Roman numerals above the full-length LH/CG receptor cDNA. Black boxes indicate the coding region of the LH/CG receptor, crossed boxes the different reading frames and white boxes untranslated regions of the alternatively spliced transcripts. The presumed intronic sequences of the isoforms rLHRC1 and rLHRC2 are indicated by asterisks. The lengths of the precursor polypeptides are indicated in parentheses. The isoforms rLHRB1, rLHRB3, rLHRC1 and rLHRC2 are from Segaloff et al. (1990) and isoform rLHREB from Bernard et al. (1990). The isoform E/A2 is common to both groups.

133

I

rLHll2100 rLHR2075 rLHRl950 rLHRlS34 rLHR1759 rIJiRE/AZ rI.HREB rLHRB1 rLHRB3 rLHRC1 rLHRC2

rLHR2100 rLHR2075 rLHR1950 rLHRlS34 rLHR1759 rLHRE/AZ rLHREB rLHRB.1 rLHRB3 rLHRC1 rLHRC2

#

#I

NGRRVPALRQLLVLAVLLLKPSQ~SRCPEPCDCIPSQAFRGWEWKIEISQSDSLERIEANAFDN NGRRVPALRQLLVLAVLLLPSQLPSRELSGSRCPEPCDIPSQAFRGLN~IEISQSDSLERIEANAFDN UGRRVPALRQLLV~VLLLKPSQLQSRELSGSRCPEPCDCIPSQAFRGLNEVVlM.................. NGRRVPALRQLLVLAVLLLKPSQ~SRELSGSRCPEPCDCAP~A~C~P~G~SLTYLP~IPSQAFRGLNEWXiEISQSDSLERI~AFDN MGRRVPALRQLLV~VLLLPSQLQSRELSGSRELSGSRCPEPCDCAP~A~C~P~G~LSLTYLP~IPSQAFRG~~IEISQSDSLERI~AFDN NGRRVPALRQLLVLAVLLLKPSQLQSRELSGSRCPEPCDCLSLTYLPVKVIPSQAFRGLNEVVKIEISQSDSLERI~AFDN NGRRVPALRQLLVUVLLLKPSQ~SRCPEPCDCALSLTYLPVKVIPSQAFRGWEVVKIEISQSDSLERIEANAFDN MGRRVPALRQLLV~VLLLKPSQLQSRELSGSRCPEPCDCLSLTYLPVKVIPSQAFRGLNEVVKIEISQSDSLERIEANAFDN WGRRVPALRQLLVWVLLLKPSQLQSRELSGSRCPEPCDCAIPSQAFRGLNEVVKIEISQSDSLERIWNAFDN MGRRVPALRQLLVLAVLLLKPSQLQSRELSGSRELSGSRCPEPCDCAP~A~CPGP~G~LSLTYLP~IPSQAFRG~~IEISQSDSLERI~AFDN MGRRVPALRQLLVWVLLLKPSQLQSRELSGSRELSGSRCPEPCDCAP~A~CP~PRAG~LSLTYLP~IPSQAFRG~~IEISQSDSLERI~AFDN :.....signal peptide...../........../...........LR-I.........../.........LR-II........../.LR-III...: 100 1 @ # @ # @ LWLSELLIQNTKNLLYIEPGAFTNLPRLXYLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQGlNES~~LYGNGFE~QS~FNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLKYLSICNTGIRTLPDVTKISSSSEFNFILEICDNLHITTIPGNAFQGMNESVTLKLYGNGFE~QS~FNG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..SICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQGINES~~LYGNGFEEVQFNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLXYLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQGINES~~LYGNGFE~QSHFNG EICDNLHITTIPGNAFQGNNNGSVTLXLYGNGFEEvQSHAFNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLXYL......................... LWLSELLIQNTKNLLYIEPGAFTNLPRW(YLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQGIN~S~~LYGNGFE~QS~FNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLKYLSICNTGIRTLPD~KISSSEFNFILEICDNLHITTIPGNAF~~NES~~LYGNGFE~QS~FNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLKYLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAF~~NES~~LYGNGFEEVQS~FNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLKYLSICNTGIRTLPD~KISSSEFNFILEICDNLHITTIPGNAFQG~NESVTLKLYGNGFEEVQSHFNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLKYLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQG~NESVTLKLYGNGFEEVQSHAFNG LLNLSELLIQNTKNLLYIEPGAFTNLPRLKYLSICNTGIRTLPDVTKISSSEFNFILEICDNLHITTIPGNAFQG~NES~L~ :............/.........LR-IV........./..........LR-V.........../..........LR-VI........./.LR-VII...: 200 101 I# @ TTLISLELKENIYLE~SGAFQGATG~~ILDISSTKLQAL~SHGLES~QTLIAL~~Y~LXTL~~K~KFT~LLVATLTY~~H~~AF~LPKKEQNFSFSI TTLISLELKENIYLEKMHSGAFQGATGPSILPCRATGWSPFRISSPCLPTH+ TTLISLELKENIYLEKPMSGAFQGATGPSILDTSSTXLQALPSHGLESIQTLIALSSYSLKTLPSKEKFTSLLVATLTYPSHCCAF~LPKKEQNFSFSI TTLISLELKENIYLEKPMSGAFQGATGPSILDiSSTKLQALPSHGLESIQTLIALSSYSLKTLPSKEKFTSLLVATLTYPSHCCAF~LPKKEQNFSFSI TTLISLELXENIYLE~SGAFQGATG~~ILDISSTKLQAL~SHGLES~QTLIALSSYSLXTL~SKEXFT~LLVATLTY~~H~~AF~LPKKEQNFSFSI TTLISLELXENIYLEKMnSGAFQGATGPSIL..............................................................QNFSFSI TTLISLELKENIYLEKMHSGAFQGATGPSIL.......... .. . . . . .. .. . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . .. ..QNFSFSI TTLISLELKENIYLEKMHSGAFQGATGPSIL . .. . . . . .. . . . . . . . . . .. . . . . . .. . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . .. ..IFHFPFL TTLISLELKENIYLE~SGAFQGATGPSILDISSTKLQALPSHGLESIQTLIALSSYSLXTLPSKEKFTSLLVATLTYPSHCCAF~LPKKEIFHFPFL

rLHR2100 rLHR2075 rLHR1950 rLHRls34 rLHR1759 rLHRE/AZ rLHREB rLHRB1 rLHRB3 rLHRC1 TTLI~LELKENIYLEKPM~GAFQGATGPSILDISSTKLQALP~H~LESIQTLIAL~~Y~LKTL~~KEKFTSLL~~~~~~~SHCCAFRNLPKKEP* :.........../........LR-VIII......../........LR-IX......../......LR-X......./........LR-XI...~..../: 201 300 4 # @ I t x rLHR2100 FENFSKQCESTVRKADNETLYSAIFEENELSGWDYDYGFCSPKTLQCAPEPDAFNPCEDIMGYAFLRVLIWLINI~IFGNLTVLFVLLTSRYKLTVPRF rLHR1950 FENFSXQCESTVRKADNETLYSAIFEENELSGWDYDYGFCSPKTLQCAPEPDAFNPCEDIMGYAFLRVLIWLINI~IFGNLTVLFVLLTSRYXLTVPRF rLHR1834 FENFSKQCESTvRXADNETLLLHGALPAAHCLRGLPNKRPVL* rLHRl759 FENFSXQCESTVRXADNETLLLHGALPAAHCLRGLPNKRPVL* rLHRE/A2 FENFSKQCESTVRKADNETLYSAIFEENELSGWDYDYGFCSPKT~CAPEPDAFNPCEDIMGYAFLRVLIWLINI~IFGNLTVLFVLLTSRYKLTVPRF rLHREB FENFSKQCESTvRXADNETLLLHGALPAAHCLRGLPNKRPVL* rLHRB1 KTSPNNAKAQLEKQITRRFIPPSL~SVAGIMI~S~P~S~LQNQMLSTP~ILW~PS~S~ fLHRB3 KTSPNN~QLEKQITRRFIPPSL~SVAGIMI~S~P~S~~NQMLSTPVKILW~PS~S~ :.....LR-XII.../..........LR-XIII . . . . . . . . ../........LR-XIV......../.........TM-l........./.ICL-l../. 301 400 I

LnCNLSFADFCNGLYLLLIASVDSQTKGQYYNHAIDWQTGSGCGMGFFTVFASELSVYTLTVITLERWHTITYAVQLDQKLRWUiAIPIMU;GWLFSTL LnCNLSFADFCnGLYLLLIASVDSQTXGQYYN~IDWQTGSGCG~GFFTVFASELSVYTL~ITLER~TITYAVQL~K~~IPIMLGGWLFSTL LnCNLSFADFCMGLYLLLIASVDSQTKGQYYN~IDWQTGSGCG~GFFTVFASELSVYTLTVITLERWHTITYAVQL~KLRL~IPIMLGGWLFSTL : . . . . . . ..TM-2......../.......ECL-l......../... . . . ..TM-3........../.......ICL-2......./.....TM-4..... 401 500 I rLHR2100 IATnPLVGISNYPMVSICLPMDVESTLSQVYILSILILWWAFWICACYIRIYFAVQNPELTAPNKDTKIAK~ILIFTDFTCMPISFFAISMFKV rLHR1950 IATMPLVGISNY~SICLPNDVESTLSQVYILSILI~A~ICACYIRIYFAVQNPELTAPNKDTKI~~ILIFTDFTC~PISFFAISMFKV rLHRE/AZ IAT~PLVGISNYMKVSICLPMDVESTLSQVYILSILIL~A~ICACYIRIYFAVQNPELTAPNKDTKI~~ILIFTDFTC~PISFFAISMF~ :......./.......ECL-2......./........TM-5.........J........ICL-3........./..........TM-6........./.: 501 600 II x I rLHR2100 PLITVTNSKILLVLFYPVNSCANPFLYAIFTKAFQRDFLLLLSRFGCCKRRAELYRRKEFSAYTSNCKNGFPGASKPSQATLXLSTVHCQQPIPPRALTH+ rLHR1950 PLI~NSKILLVLFYPVNSCANPFLYAIFT~FQRDFLLLLSRFSCCKRRAELYRRKEFSAYTSNCKNGFPGASKPSQATLKLSTVHCQQPIPPRALTH+ rLHRE/A2 PLI~NSKILLVLFYPVNSCANPFLYAIFTKAFQRDFLLLLSRFGCCKRRAELYRRKEFSAYTSNCKNGFPGASKPSQATLKLST~CQQPIPPRALTH' :.ECL-3./.........TM-7....... ./intracellular domain................................................: 601 700 rLHR2100 rLHR1950 rLHRE/A2

Fig. 4. Alignment of the predicted primary structures of the LH/CG isoforms, indicated in single-letter code. Dots indicate missing amino acids. Amino acids coded after frame shift are underlined as are altered amino acids resulting from point mutations. Asterisks denote stop codons. Potential sites for N-linked glycosylation are denoted by (@). Cysteines conserved between species (rat, porcine and human) are denoted by (#). Fourteen leucine-rich repeats (LR-I-XIV) and seven putative transmembrane segments (TM-l-7) are represented, also three intracellular loops (ICL-l-3) and three extracellular loops (ECL-l-3). The isoforms rLHRB1, rLHRB3, rLHRC1 and rLHRC2 are from Segaloff et al. (1990) and isoform rLHREB from Bernard et al. (1990). The isoform E/A2 is common to both groups.

134

isoforms (Cl and C2) containing 95 and 66 nucleotides, respectively, of presumptive intronic sequences, Cl containing this foreign sequence immediately after exon IX and C2 immediately after exon VI. These isoforms encode 268 and 157 amino acid mature polypeptides, both terminating after an in-frame terminator codon at the beginning of the intronic sequences. The sequences of alternatively spliced exons and consensus sequences for the 3’ sptice sites and branch points are shown in Fig. 2. Whether the isoforms cloned in our laboratory lacking different parts of the ligand binding regions formed by the N-terminal leucine-rich repeats l-8 (Braun et al., 1991) corresponding to exons I-VIII exhibit ligand binding remains to be elucidated with expression studies. Isoform rLHR1950, lacking exons III-IV, and isoform rLHR1759, lacking exon V, may be incapable of ligand binding. In turn all the isoforms, except for C2, which contain the intact N-terminal leucinerich repeats 1-8, are probably capable of ligand binding. A comparison of truncated LH/CG receptor isoforms between species suggests that alternative splicing is highly conserved in mammals. The rat clone rLHR1834 is almost identical to the porcine isoform B (Loosfelt et al., 1990) and they share the same divergence point, although rat LH/CG receptor isoforms homologous to the porcine C and D isoforms have not yet been detected. The rat isoform E/A2 (Bernard et aI., 1990; Segaloff et al., 1990) is almost identical to the human truncated LHjhCG receptor isoform (Minegish et al., 1990) and their divergence points differ by only one amino acid. Alternative splicing may lead to crucial changes in protein function, changes in protein location, deletion of protein activity, modification of protein activity, novel protein activities, and changes in RNA stability and translational efficiency (Smith et al., 1989). The extensiveness of the alternative splicing of the LH/CG receptor primary transcript points to the fact that it should play a significant role in the regulation of LH/CG receptor gene expression and also in the appearance of hormonal effects in different physiological states. The putative soluble isoforms capable of ligand binding could regulate free hormone

Ievels in the blood while the one or more isoforms that are incapable of ligand binding could modulate the hormone responsiveness of the target cells by complexing the active receptor to inactive heterodimer(s). This type of modulation of hormone responsiveness by a splicing variant of a receptor has been proposed for the thyroid hormone receptor (Koenig et al., 1989). Ueno et al. (1991) have recently reported that a soluble truncated PDGFj3 receptor capable of ligand binding inhibited signal transduction in the native PDGF## receptor, possibly by forming a heterodimer with it. Whether LH/CG receptor isoforms capable of ligand binding could analogously modulate LH/CG receptor function remains to be elucidated. Acknowledgements

We would like to thank Paula Soininen and Alian Haimakainen for expert technical assistance. This work was supported by grants from the Academy of Finland and the Sigfrid Jus6lius Foundation. References Bernard, M.P., Myers, R.V. and Moyle, W.R. (1990) Mol. Cell. Endocrinol. 71, R19-R23. Braun. T., Schofield, P.R. and Sprengel, R. (1991) EMBO J. 10, 1885-1890. Devereux, J. (1989) The GCG Sequence Analysis Software Package, Version 6.0, Genetics Computer Group, Inc., University Research Park, 57.5 Science Drive, Suite B, Madison, WI, 53711, USA. Ji, I. and Ji, T.H. (1991) Endocrinology 128, 2648-2650. Kellokumpu, S. and Rajaniemi, H.J. (1985) Endocrinology 116, 70’7-714. Koenig, R.J.. Lazar, M.A., Hodin, R.A., Brent, GA., Larsen, P.R., Chin, W.W. and Moore, D.D. (1989) Nature 337, 659-661. Koo, Y.B., Ji, I., Slaughter, R.G. and Ji, T.H. (1991) Endocrinology 128, 2297-2308. Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Thi, M.T.V.N.-L, Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, B., Garnier, J. and Milgrom, E. (1989) Science245, 525-528. Mayor, S., Menon, A.K. and Cross, G.A.M. (1991) J. Cell Biol. 114, 61-71.

135 McFarland, KC., Sprengel, R., Phillips, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff. D.L. and Seeburg, P.H. (1989) Science 245, 494-499. Minegish, T., Nakamura, K., Takakura, Y., Miyamoto, K., Hasegawa, Y., Ibuki, Y. and Igarashi, M. (1990) Biochem. Biophys. Res. Commun. 172, 1049-1054. Parmentier, M., Libert, F., Maenhaut, C., Lefort, A., Gerard, C., Perret, J., Van Sande, J., Dumont, J.E. and Vassart, G. (1989) Science 246, 1620-1622. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, 2nd edn., pp. 7.19-7.21, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Nat]. Acad. Sci. USA 74, 5463-5467.

Segaloff, D.L., Sprengel, R., Nikolics, K. and Ascoli, M. (1990) Recent Prog. Norm. Res. 46, 261-303. Smith, C.W.J.. Patton, J.G. and Nadal-Ginard, B. (1989) Annu. Rev. Genet. 23, 527-517. Sprengel, R., Braun, T., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1990) Mol. Endocrinol. 4, 525-530. Tsai-Morris, C.H., Buczko, E., Wang, W. and Dufau, M.L. (1990) J. Biol. Chem. 265, 19385-19388. Tsai-Morris, C.H., Buczko, E., Wang, W., Xie, X.-Z. and Dufau, M.L. (1991) J. Biol. Chem. 266, 11355-11359. Ueno, H., Colbert, H., Escobedo, J.A. and Williams, L.T. (1991) Science 252, 844-848. Xie, Y.-B., Wang, H. and Segaloff, D.L. (1990) J. Biol. Chem. 265, 21411-21414.

CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript.

Luteinizing hormone/chorionic gonadotropin (LH/CG) receptor complementary DNA (cDNA) isoforms were amplified using pseudopregnant rat ovarian total RN...
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