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MOLECULAR CLONING OF A TRUNCATED ISOFORM OF THE HUMAN FOLLICLE STIMULATING HORMONE RECEPTOR’ J&g Gromoll, Thomas Gudermann and Eberhard Nieschlag* Institute of Reproductive Medicine of the University, Steinfirter Straje 107, D-4400 Mtinster, Germany

Received

September

15,

1992

Summary: Northern blot hybridization of human testicular poly (A)+ RNA to a human follicle stimulating hormone receptor probe revealed the existence of multiple mRNA transcripts. In order to investigate whether alternative splicing of the receptor occurs in the human testis we amplified the extracellular and the transmembrane domain of the human testicular follicle stimulating hormone receptor by reverse transcription polymerase chain reaction and subcloned the resulting DNA fragments. Sequence analysis of the recombinant clones revealed the existence of a truncated isoform of the human follicle stimulating hormone receptor which is spliced through a cassette exon mode without a change in the open reading frame, thereby deleting exon IX from the coding region of the receptor. 0 1992Academic PrP*s,Inc.

The gonadotropin receptors, i.e. the follicle stimulating hormone receptor (FSHR) and the lutropin-choriogonadotropin receptor (LHKG-R), belong to the superfamily of G protein coupled receptors which are characterized by the common structural feature of seven transmembrane domains (1). The glycoprotein hormone receptors, however, diverge structurally from other G protein-coupled receptors in that they contain a large extracellulat domain in the amino-terminal part of the polypeptide (2, 3) which is required for the interaction with complex glycoprotein hormones (4, 5). Recent studies on the gene organization of the gonadotropin receptors revealed that a region of the receptors comprising the seven transmembrane domains is encoded by one large exon whereas the extracellular portion of the receptors is composed of 9 and 10 exons for the FSHR 1 Sequence data from this paper have been deposited with the EMBWGenBank Libraries under Accession No. X-68044. * To whom correspondence should be addressed. The abbreviations used are-: bp, base pairs; FSH, follicle stimulating hormone; FSHR, FSH receptor; hCG, human chorionic gonadotropin; kb, kilobases; LH/CG, lutropinchoriogonadotropin; LH/CG-R, LH/CG receptor; PCR, polymerase chain reaction; RTPCR, reverse transcription PCR.

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and LH/CG-R, respectively (6, 7, 8). Northern blot hybridization analysis of rat ovarian and testicular RNA with an LH/CG-R probe reveals multiple mRNA transcripts (9, 10, 11 ). Expression of the smallest transcript (1.8 kb), which presumably represents a truncated isoform of the LH/CG-R acting as a secreted hormone binding protein, is greater in the testis than in the ovary and, in contrast to ovarian tissue, is not downregulated in the testis after LH or hCG administration (12). This differential regulation of multiple LH/CG-R transcripts in the testis may represent an important mechanism to modulate the cellular effects of gonadotropins. The characterization of various truncated isoforms of the LWCG-R resulting from extensive alternative splicing of the primary transcript serve to complement the above mentioned findings (13). As far as the FSHR is concerned, information is scant. Northern blot analysis of rat ovarian (14) and testicular RNA (15) indicated different patterns of mRNA transcripts. No information is available on FSHR mRNA transcripts in the human testis and truncated isoforms of the FSHR as a result of alternative splicing have not yet been demonstrated. Whether alternative splicing events are restricted to the LH/CG-R or represent a general principle to modulate the expression of glycoprotein hormone receptors remains to be elucidated. In the present study we analysed the expression of FSHR mRNA in human testes by Northern blot hybridization to investigate whether multiple mRNA transcripts of the human FSHR could be identified. Amplification of human testicular mRNA by reverse transcription polymerase chain reaction (RT-PCR) with oligonucleotide primers specific for the human FSHR cDNA, followed by sequencing of subcloned products, was performed to characterize potential isoforms of the human FSHR and to address the issue of whether they could be the result of alternative splicing events. Materials and Methods

Poly (A)+ enriched RNA was obtained from testes of three patients with prostate carcinoma undergoing orchidectomy. For Northern blot analysis 5-20 pg of human testicular poly (A)+ RNA were subjected to electrophoresis in 1% agarose-formaldehyde gels and transferred to nylon membranes by vacuum blotting in 20 x SSC. A 503 bp fragment of the extracellular domain of the human FSHR (prepared as described in 16) cloned into pBluescript SK(-) was used as template for in vitro transcription by T7 RNA (modified after 17). The 503 bp fragment displays less than 50 % nucleotide homology to the human LH/CG-R cDNA (18). The specific activity of r*P]CTP labelled probe was approximately lxlOscpm/pg cRNA. Blots were hybridized (50% formamide, 5x SSC, 5x Denhardt’s solution, 1% SDS) for 16-18 h at 55OC. Final washing conditions were: O,l% SSC, 0,196 SDS, 65OC. Blots were exposed to Kodak X-Omat film for l-3 days at -70°C. The reverse primer Rl (corresponding to base 1005-1026, plus external J@?rI restriction site and TC) and the reverse primer R2 (corresponding to base 2160-2179, plus external QzI restriction site and TC) based on the nucleotide sequence of the human FSHR (19) were used for first strand cDNA synthesis. 2.5-5 c(g poly (A)+ RNA served as template for avian myeloblast virus (AMV) reverse transcriptase. PCR was performed with forward primer Fl (corresponding to base -16 - +5, plus external &&I restriction site and TC) and Rl. In a second set of experiments the forward primer F2 (corresponding to base 1019-1038, plus external ECORI restriction site and TC) and R2 were used. The reaction mixtures were incubated using a Hybaid temperature c cler according to the following programme: 5 initial cycles (94 “C, 50 set; 52 “C, 90 set; 7 I “C, 3min) followed by 30 cycles (94 “C, 50 set; 55 1078

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“C, 1 min; 72 “C , 2 min). The program was preceded by a 4 rain denatu&on step at 94 “C and followed by a final extension step at 72 “C for 10 min. PCR products were sizefractionated on 1 % agarose gels, fragments were isolated, purified and subchned into ~&Eicri~ SK(-). Roth strands of the subcloned DNA inserts were directly sequen& by the ldideoxy ihain &mination method (20) applying the primer walk technique (21) and using alkali-denatured plasmid DNA as template. Results and Discussion To study the expression of human FSHR mRNA, RNA was extracted from the testes of three patients undergoing orchidectomy. Northern blot hybridization of testicular poly (A)+ RNA with a cRNA probe corresponding to the extracellular domain of the human FSHR indicated the existence of three predominant mRNA transcripts (2.2, 2.5 and 6.5 kb) and of three minor mRNA species (1.3, 1.6, 2.0 and 4.2 kb) (Fig. 1). The most abundant mRNA in the human testis had a size of 6.5 kb. Although the relative abundance of the 1.3 and 4.2 kb transcripts varied between different RNA preparations, faint bands at 1.3 and 4.2 kb could always be discerned. Northern blot analysis of rat ovarian RNA revealed four FSHR transcripts (1.8, 2.5, 4.2 and 7.0 kb) (14) and two transcripts could be discerned in rat testicular RNA (2.6 and 4.5 kb) (15). In contrast to our observations, only three mRNA transcripts of 2.5, 4.2 and 7.0 kb could be identified in RNA prepared from human follicular phase ovary, the 4.2 kb transcript being most abundant (22). Multiple FSHR mRNAs in human and rat reproductive tissues may arise from different sites and lengths of polyadenylation and/or alternative splicing of the FSHR primary transcript. The reason for the differences in the transcript pattern of the human ovarian and testicular FSHR still remains to be elucidated. However, it might reflect a tissue-specific regulation of FSHR mRNA expression. To investigate whether alternative splicing of the primary FSHR transcript occurs in the human testis, amplification of testicular poly (A)+ RNA by RT-PCR with oligonucleotide primers specific for human FSHR cDNA was performed. In order to reduce the introduction of PCR-based sequence errors we decided to amplify the complete coding region of the human FSHR as two overlapping fragments: primers FUR1 were to yield a product of 1042 base

6.5 4.2 -

-28s

Figure 1, Northern blot analysis of human FSHR mRNA in human testis. 20 and 5 pg of poly(A)+ RNA were resolved through agarose-formaldehyde gels, transferred to n km filters, and hybridized to a r2P]CTP labeled cRNA probe of the extrac&uIar domain o r the human FSHR. Filters were washed and exposed to pho@raphic film for l-3 days at -70 “C. The size of tzuucripts is indicated in kilobases(kb). Migration distancesof the 28s and 18s ribosomal RNAs of a parallel total RNA sampleare indicated.

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pairs (bp) corresponding to the extracellular domain of the human FSHR (18), primers F2/R2 gave rise to a product of 1160 bp corresponding to the 3’ region of the extracellular as well as the whole transmembrane domain of the FSHR. Poly (A)+ RNA was prepared from testes of three different subjects and two independent RT-PCR reactions were performed for each RNA preparation. The resulting PCR products were subcloned into EcoRIIKpnI cut pBluescript SK (-) and at least two clones per ligation were sequenced in both directions. As expected, primers F2/R2 yielded a distinct 1160 bp product. Restriction digests of recombinant plasmids only revealed one restriction pattern. The DNA sequence of the subcloned fragment showed 100% identity to the published ovarian clone (18). Agarose gel electrophoresis of PCR reactions using primers Fl/Rl, however, revealed two major bands of 856 and 1042 bp (data not shown). The latter fragment could be shown to correspond to the extracellular domain of the human FSHR. Sequence comparison between our and the published ovarian clone (19) lead to the identification of four different amino acids at the following positions: 112 (Thr to Asn), 197 (Ala to Glu), 198 (Val to Leu) and 307 (Ala to Thr) (Fig.2). Our sequence is identical to the human testicular FSHR cDNA published recently (22). The DNA sequence of the 856 bp fragment was identical to the 1042 bp product with the exception of a 186 bp deletion between nucleotide position 668 and 855. The nucleotide deletion did not lead to a shift in the open reading frame nor to premature termination of the polypeptide but resulted in a deletion of 62 amino acids (Fig.2).

hPBKR/E

- #ULLLVSLLAFLl5LGSGCEKRICECSNRVFLCQKSKVTKIPSDLPRNAIE

hPSHR1042

- MALLLVSLLAPLSLGSGCHKRICECSNRVFLCQESKVTEIPSDLPRNAIE

hFSHR/

-

LRFVLTKLRVIQKGAFSGFGDLKKIEISQNDVLKVIKADVFSNLPKLHEI

-

LRFVLTKLRVIQKGAFSGFGDLEKIEISQNDVLKVIKADVFSNLPKLEEI

E

hFSAR1042

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

-50

-100

l

hFSER/

E

-

RIBKANNLLYINPEAFQNLPNLQYLLISNTGIKELPDVKKIEsLQKVLLD

IIIIIIIIIIIIIIIIIIIIlllllllillllllllllIIIIIIIIIIII

hFSHR1042

- RIEKANNLLYINFKAFQNLPNLQYLLISNTGIKRLPDVRKIHSLQKVLLD

-150

hFSHR/E

-

IQDNINIBTIERNSFVGLSFE8VILHLNKNGIQEIKNCAFNGTQLD&L

hFSHRlO42

-

IQDNINIHTIERNsFVGLSFESVILULNKNGIQEIENCAFNGTQLDELNL

hFSHR/E

-

sD-EELPNDVPBGABGPVIL---------------------------

hFSHR1042

-

SDNN'NLEELPNDVFRGASGPVILDIBRTRIHSLPSYGLENLKKL~STY

hFSHR/E

-

-----------------------------------BELBPpILRQ~

hFSHR1042

- NLKKLPTLEKLVALK?ZASLTYPS?l~AFANWRRQIsELHPICNKPILRQE

hFSHR/E

- VDYRTQ~RGQRSSLAEDNESSYSRGFDHTYTEFDYDLCNEVV

hFSHR1042

- VDYl4TQTRGQRSSLAEDNESSYSRGFDMTYTEFDYDLCNEW

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

-200

IIIIIIIIIIIIIIIIIIIIIII

-250

IIIIIIIIIIIIIII

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

-300

-342

Firmre 2, Predicted amino acid sequence of the truncated (hFSHR/E) and the full-length (hFSHR 1056) extracellular domain of the human testicular FSHR as amplified with primers FURl. The putative signal peptide is underlined (3). Amino acids that differ from those of the

human ovarian FSHR (19) are highlighted with asterisks. Conserved cysteine residues at positions 275 and 276 are underlined (=).

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rFSRREX9

-

hFSHR1042

-

ATQTTTTCCACQQAQCCTCT~AC~Q~~~AQATAT~CMGMCA

rFSHREX9

-

AAQQTCCATTCCTTACCAAACCATQQTTTAQAAAATCTQAAQAAQCTQAQ

hFSHR1042

-

AGGATCCATTCCCTGCCTAGCTATGQCTTAQAMATCTTAAGAAGCTGAG

rFSHREX9

-

GGCCAGGTCAACATACCGCTGGMAAAQCTCCCTAATCTQGACMGTTTG

hFSHRlO42

-

GGCCAQQTCGACTTM!AACTTAAAAAA

rFSHREX9

-

TCACCCTCATQQAQQCCAGCCTCACCTACCCt!AQCCACTGCTGTQC!TTTT

hFSHR1042

-

TCQCCCTCATQQAAQCCAGCCTCACCTATCCCAQCCATTGCTGTQC~T

rFSHREX9

-

QCAAACTTGAAGCQGCAAAT

hFSHR1042

-

GCAAACTGGAGACGGCAAATCTCTGAGCTTCATCCAATTTGCAACAAATC

COMMUNICATIONS

AQATATCTCAAQQACA

I I IIIIIIII

I II I I III1

IIIIIIIII

II III

II IIIIIIIIII IIIIIII

II

IIIIIIIIIII

lllll~llll~~~lll

IIIIIIIIIIIIII

II

IIIIII

IIIIIIII

IIIIIIII

IIIII

III

IIIIIIIIIII III

QQAAAAGCTTG

IIIIIIII

-684

-734

III III

-704

-834

-864

Fkure 3, Sequencecomparison of exon IX of the rat FSHR (rFSHREX9) with the full-length extracellular domain of the human testicular FSHR as amplified with primers FUR1 (hFSHR1042). DNA sequenceswhich are absent in the truncated isoform E of the human FSHR are underlined (nucleotide position 669 to nucleotide position 854).

Recently the structural organization of the rat FSHR gene has been described (8), whereas the genomic organization of the human FSHR gene is still unknown. We therefore compared exonic sequences of the rat FSHR with our clones of the human FSHR. Sequence alignments revealed an identical length and a 83.7% identity of the 186 bp deletion to rat exon IX (Fig.3). The truncated clone continues with sequences which can be allocated to exon X, the remaining 3‘ portion of which was amplified as an overlapping fragment with primers F2/R2. In the truncated clone a leucine precedes the 62 amino acid deletion (Fig. 1). It has been demonstrated for both gonadotropin receptors that the amino acid which resides at nearly each intron/exon junction is either a leucine or an isoleucine (8). In our truncated FSHR clone the deletion interrupts the codon for leucine between the second and third nucleotide. This finding is consistent with an intron phase 2 which has been described for all of the introns in the FSHR gene (8). A comparison of truncated LHKG-R isoforms between species reveals that alternative splicing is highly conserved in mammals (13, 18). Taking all these facts into account we concluded that the truncated clone of the human FSHR represents an isoform of the human FSHR which is spliced through a cassette exon mode by which exon IX is removed (Fig.4). The truncated isoform of the human FSHR corresponds to the A,/E isoform of the LH/CG-R (23, 24) from which exon IX is removed by alternative splicing. We therefore refer to the truncated clone of the human FSHR as the E isoform. According to hydropathy profiles (data not shown) the FSHR/E isoform can be considered membrane-spanning and therefore the possibility exists that the truncated FSHR mRNA is actually translated and the protein is functionally expressed. Deletion of exon IX would lead to the removal of two highly conserved vicinal cysteine residues at positions 275 and 276 which are regarded as crucial for the three-dimensional folding of the large extracellular domain of the glycoprotein hormone receptors. An antibody directed against a peptide sequence of exon IX is able to stimulate progesterone synthesis of porcine granulosa cells without competing 1081

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hFSHR (695 aa)

hFSHR/E (633 aa)

Fieure 4. Model of alternative splicing of the human FSHR transcript through a cassetteexon mode. Filled boxes represent exons which are numbered I to X. Nucleotide sequenceslost in the splicing processare representedby an open box. The length of the human FSHR (hFSHR) and of &form E (hFSHR/E) are indicated in amino acids (aa). Nucleotide positions at splice junctions of exon VIII to IX and of exon IX to X are indicated.

with labeled FSH for binding to receptor (25). It is concluded that a conformational change elicited by binding of an antibody to peptide sequences encoded by exon IX of the FSHR leads to receptor activation. Expression studies are currently being performed to define the contribution of exon IX to ligand binding and receptor activation characteristics of the human FSHR. Extensive alternative splicing of the primary transcript of a gonadotropin receptor has been demonstrated for porcine (26) and rat LH/CG-R (13). In the present study we demonstrate for the first time that alternative splicing events are not restricted to one glycoprotein hormone receptor but also occur in human testicular FSHR. The physiological role of alternative splicing of gonadotropin receptors is still unclear. The expression of truncated &forms which act as secreted hormone binding proteins or of membrane-spanning isoforms with altered binding and receptor activation characteristics may represent a general mechanism to modulate target cell responsiveness to gonadotropins. It will be of interest to see whether splicing events of the human FSHR are under hormonal control and if similar mechanisms can also be demonstrated in the human ovary. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Ni 130/l 1, Projekt A6). The authors wish to thank M. Lanwehr for skillful technical assistance and S. Nieschlag, M.A., for language editing. References 1.

2. 3. 4. 5.

Dohlman, G.D., Caron, M.G. and Lefkowitz, R.J. (1987) Biochemistry 26,26572664. McFarland, K.C., Sprengel, R., Phillips, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L. and Seeburg, P.H (1989) Science 245,494-499. Sprengel, R., Braun, T., Nikohcs, 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. Xie, Y.-B., Wang, H. and Segaloff, D.L. (1990) J. Biol. Chem. 265, 21411-21414. 1082

* Voh

6. 7.

i:-

12: 13. 14. :::

17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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Koo, Y.B., Ji, I., Slaughter, R.G. and Ji, T.H. (1991) Endocrinology 128, 2297-2308. Tsar-Morris, C.H., Buczko, E., Wang, W., Xie, X.-Z. and Dufau, M.L. (1991) J. Biol.Chem. 266, 11355-l 1359. Heckert, L.L., Daley, I.J. and Griswold, M.D. (1992) Mol. Endocrinol. 6, 70-80. LaPolt, P.S., Oikawa, M., Jia, X.C., Dargan, C. and Hsueh, A.J.W. (1990) Endocrinology 126, 3277-3279. Segaloff, D.L., Wang, H. and Richards, J.S. (1990) Mol. Endocrinol, 4. 1856-1865. Wang, H., Ascoli, M. and Segaloff, D.L. (1991) Endocrinology 129, 133-138. LaPolt, P.S., Jia, X.-C., Sincich, C. and Hsueh, A.J.W. (1991) Mol. Endocrinol, 5. 397-403. Aatsinki, J.T., Pietili, E.M., Lakkakorpi, J.T. and Rajaniemie, H.J. (1992) Mol. Cell. Endocrinol. 84, 127-135. LaPolt, P.S., Tilly, J., Aihara, T., Nishimori, K. and Hsueh, A.J.W. (1992) Endocrinology 130, 1289-1295. Heckert, L.L. and Griswold, M.D. (1991) Mol. Endocrinol. 5, 670-677. Kliesch, S., PenttilH, T.L., Gromoll, J., Saunders, P.T.K., Nteschlag, E. and Parvinen, M. (1992) Mol. Cell. Endocrinol., 84, R45-R49. Gudermann, T., Nichols, C., Levy, F.O., Bimbaumer, M. and Bimbaumer, L. (1992) Mol. Endocrinol. 6, 272-278. Minegish, T., Nakamura, K., Takakura, Y., Miyamoto, K., Hasegawa, Y., Ibuki, Y. and Igarashi, M. (1990) Biochem. Biophys. Res. Commun. 172, 1049-1054. Minegish, T., Nakamura, K., Takakura, Y., Ibuki, Y. and Igarashi, M. (1991) B&hem. Bmphys. Res. Commun. 175, 1125-1130. Sanger, F., Nichlen, S. and Coulson, A.R. (1977) Proc. Nat. Acad. Sci. USA, 74, 5463-5467. Gudermann, T., Bimbaumer, M. and Bimbaumer, L. (1992) J. Biol. Chem. 267, 44794488. Tilly, J.L., Aihara, T., Nishimori, K., Jia, X.-C., Billig, H., Kowalski, K.I., Perlas, E.A. and Hsueh, A.J.W. (1992) Endocrinology 131, 799-806. Bernard, M.P., Myers, R.V. and Moyle, W.R. (1990) Mol. Cell. Endocrinol. 71, R19R23. Segaloff, D.L., Sprengel, R., Nikolics, K. and Ascoli, M. (1990) Recent Prog. Horm. Rcs. 46. 261-303. LaBarbera, A.R., Lessard, J.L., Balasubramaniam, A., Kessel, B., Song, D.Y. and Patterson, K. (1992) Biol. Reprod. 46, 63, abstract 332. Loosfelt, H., Misrahi, M., Atger, M.. , Salesse, R., Thi, M.T., Jolivet, A., GuichonMantel, A., Sar, S., Jallal, B., Garmer, J. and Milgrom, E. (1989) Science 245, 525c79

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