Vol.

189, No. 3, 1992

December

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30, 1992

1645-1652

Pages

ISOLATION AND CHARACTERIZATION OF cDNAs ENCODING THE RAT PITUITARY GONADOTROPIN-RELEASING HORMONE RECEPTOR Ursula B. Kaiser, Dayao Zhao, Guemalli R. Cardona, and William Division Howard Received

of Genetics, Department

W. Chin

of Medicine, Brigham and Women’s Hospital,

Hughes Medical Institute and Harvard Medical School, Boston, MA 02115 November

2,

1992

Rat pituitary cDNAs encoding the full peptide coding sequence of the rat gonadotropin-releasing hormone receptor were isolated and characterized. The deduced amino acid sequence encodes a protein of 327 residues with seven putative transmembrane domains characteristic of the family of G-protein coupled receptors. It is 95% identical at the amino acid level with the mouse gonadotropin-releasing hormone receptor. An mRNA of 4.5 Kb was identified in the rat pituitary, ovary, and testis, and in murine cir3 cells. In addition, a larger mRNA species of 5.0 - 5.5 Kb was present in these rat tissues, and a smaller mRNA species of 1.8 Kb was present in the rat pituitary and ovary, and in aT3 cells. The receptor mRNA levels were increased in the female rat pituitary after ovariectomy compared to levels in intact female rats. 0 1992Academic ~~~~~~~ ~~~~

Regulation hormone

(LH)

reproductive including

of the biosynthesis and

and secretion of the gonadotropins,

follicle-stimulating

function. It is accomplished gonadotropin-releasing

in a pulsatile

manner,

(FSH),

influenced

whereas

factors,

a hypothalamic

decapeptide.

portal circulation

and transported

to the anterior

where

it binds

to specific

high affinity

number

high concentration,

receptors

of LH and FSH secretion. Thus, GnRH is a and responsiveness

by its own ligand. Low concentration,

regulation,

normal

of multiple

humoral link between neural and endocrine components of reproductive of GnRHR

for

(GnRH),

(GnRHR) and serves as the major regulator The regulation

luteinizing

is critical

by the complex interaction

hormone

GnRH is released into the hypophysial pituitary

hormone

pulsatile

continuous

function (I, 2).

is complex,

and is

GnRH causes receptor up-

GnRH induces

receptor

down-

regulation and desensitization (3). The ability of GnRH analogs to both activate and inhibit the hypothalamic-pituitary-gonadal axis has led to their clinical application in the treatment

of a variety of clinical disorders,

prostatic carcinoma, and endometriosis

such as infertility,

precocious

puberty,

(4).

The abbreviations used are: LH, luteinizing hormone; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; m, mouse; r, rat; OVX, ovariectomized; PCR, polymerase chain reaction; Kb, kilobase. 0006-291X192

1645

S4.00

Copyright 0 1992 by Academic Pren. Inc. Ail rights of reproduction in crny form reserved.

Vol.

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No.

BIOCHEMICAL

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GnRH,

acting

through

AND BIOPHYSICAL

the GnRHR,

stimulates

RESEARCH COMMUNiCATI0N-S

several

putative

“second

The cellular messengers”, which are thought to participate in signal transduction. responses to GnRHR activation include phosphoinositide turnover, calcium mobilization,

protein kinase C activation,

and arachidonic

acid release (5,6). However,

little is known about the molecular mechanisms of this signal coupling. Isolation of cDNAs encoding a mouse GnRHR (mGnRHR) cell line, aT3, has recently been reported

(7, 8). The mGnRHR

from the gonadotrope is predicted

to be a 327

amino-acid protein belonging to the family of G-protein coupled receptors that possess seven putative transmembrane domains (9). We report here the isolation and characterization

of cDNAs encoding rat GnRHR (rGnRHR)

RNA. The rGnRHR

is highly

acid level. The availability GnRHR in the rat pituitary well-characterized.

similar to the mGnRHR,

derived from rat pituitary

with 95% identity

of this cDNA should facilitate

studies of the regulation

in uivo, a model for reproductive

Using this cDNA

at the amino of

studies which has been

it will also be possible to study the molecular

mechanisms of ligand binding, signal transduction

and receptor auto-regulation.

Materials and Methods Animals and Cell Culture. Intact adult female and male Sprague-Dawley rats and 21-day post-ovariectomy (OVX) adult female rats (CD strain; 200-225 g; Charles River Breeding Laboratories, Wilmington, MA) were used. aT3 cells, generously provided by Dr. Pamela Mellon (lo), were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.25 mM glucose. Preoaration of RNA. Animals were sacrificed by decapitation. Pituitaries, ovaries, testes, and liver from intact rats, and pituitaries from OVX rats, were removed by dissection, quick-frozen on dry ice, and homogenized in 4 M guanidinium thiocyanate. Tissue culture cells were harvested with a rubber policeman in 4 M guanidinium thiocyanate. Total RNA was prepared by centrifugation through 5.7 M cesium chloride (11). Rat placental RNA was generously provided by Dr. Joseph Majzoub. RNA concentrations were estimated by measuring the A260. Polvmerase Chain Reaction (PCR) Cloning of rGnRHR. Two oligonucleotide primers were constructed based on the mGnRHR sequence. The primers were selected to flank the amino acid coding region of the mouse GnRHR cDNA and were modified to include restriction enzyme sites for BamHI and XbaI. The primer sequences were as follows: 5’-ACGCGGATCCCTTGAAGCCTGTCC’ITGGAGAAAT-3’ and 5’-GGCTTCT AGAATCTGAGTTCTTGTGTAGTCTCCC-3’. To generate cDNAs for use as PCR templates, two pg each of total pituitary and aT3 cell RNA were reversed transcribed as previously described (12). Two d of each reaction mixture was used as a template in a 100 d PCR reaction mixture. PCR amplification was carried out as previously described (12) for 40 cycles (94C, one minute; 56C, one minute; 72 C, three minutes). Control samples for each RNA from which reverse transcriptase had been omitted were run in parallel. Fifteen d of each PCR reaction product were subjected to electrophoresis on a 1% agarose gel for analysis of amplification results. The amplified products from rat pituitary cDNA from two separate PCR reactions were digested with BarnHI and XbaI, purified by agarose gel electrophoresis, and cloned into the BamHI and XbuI sites of pBluescript KS(+) (Stratagene, La Jolla, CA). Both clones were fully sequenced by the double-strand dideoxy chain termination method (13).

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Library Construction and Screening. cDNA, synthesized from rat pituitary mRNA using both random and oligo(dT)primers, was ligated to EcoRl adaptors and lractionated to eliminate sequences smaller than 500 basepairs, ligated into the hZAPI1 vector (Stratagene), and packaged. The primary library was amplified once, and ‘106 independent plaques were screened by standard filter hybridization techniques (14) using the PCR-generated GnRHR cDNA. After two successive rounds of screening, two positive plaques were isolated and purified. pBluescript SK(-) containing the insert was excised in vifro by helper phage R408 (Stratagene). Double-stranded cDNA from one of the clones was sequenced using the dideoxy chain termination method (13). Northern Blot Analvsis. Twenty pg of total RNA from rat ovary, testis, placenta, liver, intact adult female pituitary, OVX pituitary, and from murine uT3 cells were denatured, subjected to electrophoresis on a 1.2% agarose gel, and transferred to a nylon membrane (15). Each blot was hybridized with a full-length [32P]dCTP-labeled rGnRHR cDNA probe, using conditions previously described (16). Blots were washed and subjected to autoradiography. For the comparison of GnRHR mRNA levels in normal and OVX adult females, total RNA loading was assessedby hybridization of the same blot with a rat cyclophilin [32P]dCTP-labeled cDNA probe and normalization of the rat GnRHR mRNA to cyclophilin mRNA (17). The mRNA levels were estimated on the basis of the intensity of the hybridization signal measured by densitometric scanning (Molecular Dynamics, Sunnyvale, CA).

Results and Discussion Rat GnRHR cDNA Isolation, Seauencing, and Analvsis. Amplification by PCR of rat pituitary and mouse aT3 cell cDNAs using the mGnRHR primers described above resulted in a 1 Kb basepair product in both samples. Corresponding RNA samples treated identically except for the omission of reverse transcriptase resulted in no detectable amplification product, ruling out contamination. Two clones of 1.4 Kb each were obtained from the rat pituitary cDNA library. The PCR products from two separate PCR reactions using a rat pituitary cDNA template, and one clone isolated from the rat pituitary cDNA library, were subcloned and completely sequenced. Figure 1 depicts the nucleotide and deduced amino acid sequences of the rGnRHR, and provides a comparison with the mGnRHR. Like the mouse, the rGnRHR encodes a 327-amino acid protein with seven putative membrane spanning domains based on hydrophobicity analysis, characteristic of the family of G-protein coupled receptors (Figure 2). Within the coding region, there is 93% identity between the nucleotide sequences of rGnRHR and mGnRHR, and 95% identity between the amino acid sequences. The translation initiation site was defined by analogy with the mGnRHR. Potential N-glycosylation sites and potential phosphorylation sites were conserved (Figure 2) (18, 19). Transmembrane spanning domains 2 and 7 were entirely conserved, supporting the hypothesis that these regions may be important for ligand binding, as the structures of rat and mouse GnRH are identical. In contrast, three amino acid differences are present in the N-terminal extracellular domain, suggesting that this region may be less important for ligand binding. Like the mGnRHR, the rGnRHR lacks a typical intracellular C-terminus. 1647

Vol. 189, No. 3, 1992 MOUSe Rat

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CACGAGGTTCAGTTACGATAAAAACATCAGCAGTAACAGAGACTCGACTCTTGAAGCCCGTCCTTGGAGAAAT-1 ,

Mouse Rat

------------------A----------------------C----------------G-------------------ATG GCT AAC AAT GCG TCT CTT GAG CAG GAC C A A AAT CAC TGC TCA GCC ATC A A C A A C AGC MANNASLEQDQNHCSAINNS

60

MOUSe Rat

--------T----T----------------------------------G-----------------------------C ATC CCC CTG A C A CAG GGC AAG CTC CCG AC? CTA ACC TTA TCT GGA AAG ATC CGA GTG ACG IPLlQGKLPTLT LSGKIRVT

120

MOUSe Rat

------------------------------------------------------T---C---------T-G-------GTG ACT TTC TTC CTT TTC CTA CTC TCT ACT GCC TTC ART GCC TCT TTC VTFFLFLLSTAFNASFLYKL

TTG GTA AAG CTG

i80

CAG AGG

-----A--------T---------------------------------------------------------------TGG ACC CAG AAG AGG AAG A A A GGA A A A AAG CTC TCA AGG ATG RAG GTG CTT T?A Q 3 W T Q K R K K G K K L S R M K V L L

240

MOUSe Rat

------------------------------G---G-----------G---------------A---------------AAG CAT TTG ACC TTA GCC AAC CTC CTT GAG AC? CTA ATC GTC ATG CCG CTG GAT GGG ATG KHLTLANLLETLIVMPLDGX

300

MOUSe Rat

------T---T---------------------------G-----------C---------------------------TGG A A C ATC ACT GTT CAG TGG TAT GCT GGA GAG TTC CTT TGC AAA GTT CTC AGC TAT CTG WNITVQWYAGEFLCKVLSYL

360

Mouse

----------------------------------T-----------------------------------C-------AAG CTC TTC TCT ATG TAT GCC CCA GCC TTC ATG ATG GTG GTG ATT AGC CTG GAT CGC TCC KLFSMYAPAFMMVVISLDRS

420

M0U.W Rat

Rat

f

Rat

CTG

--------A---------------C-T-----A--A-----------C------------------A-----------TGCC GTC ACT CAG CCC TTA GCT GTC C A A AGC AAG AGC AAG CTT GAA CGG TCT ATG ACC L A,!L T Q P L A V Q S K S K L E 8 S M I

480

M0U.W Rat

------------------------------------------A-----------------------------------AGC CTG GCC TGG ATT CTC AGC ATT GTC TTT GCG GGA CCA CAG TTA TAT ATC TTC AGG ATG SLAWILSIVFAGPQLYIFRM

540

MOUSe Rat

--------------A-------------------C-A-----C-----------------------------------ATC TAC CTA GCC GAC GGC TCT GGG CCA GCA GTT TTC TCC CAA TGT GTG ACC CAC TGC AGC IYLADGSGPAVFSQCVTHC S

600

MOUSe Rat

------A---G-------------C-G---------------------------------G---------C-------TTT CCG CAA TGG TGG CAT GAA GCC TTC TAC AAC TTT TTC ACC TTC AGC TGC CTG TTC ATC FPQWWHZAFYNFFTFSCLFI

660

MOUSG2

Rat

------C---C-----------------------------------------------T---T-------G-------ATC CCT CTT CTC ATC ATG CTA ATC TGC AAT GCC AAA ATC ATC TTC GCC CTC A C A CGA GTC M L I C N A K I I F A L T R V I P L L I

720

MOUSe Rat

----------A-------------------------A---------G-------------------------------T CTT CAT CAG GAC CCA CGC AAA CTA CAG CTG ART C A A TCC AAG AAT AAT ATC CCA AGA GCA LHQDPRKLQLNQSKNNIPRA

780

MOUS2 Rat

--------------G-------------------C-------C---T-----------------G-------------CGG CTG AGA ACT CTA RAG ATG A C A GTG GCA TTT GCC ACC TCC TTT GTC ATC TGC TGG ACT RLRTLKMTVAFATSFVLCWT

840

MOUSe Rat

----------T-----------C---T-----------------------A-----------G---------------CCC TAC TAC GTC CTA GGA ATC TGG TAC TGG TTT GAT CCG GAA ATG TTA AAC AGG GTG TCA PYYVLGIWYWFDPEMLNRVS

900

MOUS.3

----------G-----------T-----------------------C-------------------------------C GAG CCA GTC AAT CAC TTC TTC TTT CTC TTT GCT TTT CTA A A C CCG TGC TTC GAC CCA CTT EPVNHFFFLFAFLNPCFDPL

960

---------------------------------G---ATA TAT GGG TAT TTC TCT TTG TAA TT L stop IY GY FS

986

MOUSk3

Rat MOUSe Rat

FiPure 1. cDNA and deduced amino acid sequence for the rGnRHR, and comparison to those of the mGnRHR. Numbers to the right indicate nucleotide residues beginning with 1 at the initiator ATG. Negative numbers indicate nucleotides 5’ to the start codon. Nucleotide differences between the mouse and rat GnRHRs are indicated above the rGnRHR nucleotide sequence. Amino acids which are different from the mGnRHR are underlined. 1648

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

NH2 190 00

B

0 I

Hydrophobicity

4

I

D

I

I

I

200 I

I

I

I

I

,

,

3 0

Hydrophilicity

-3 ;

I

( 200

Amino Acid Number FiPure 2, A, Model of the rGnRHR. Amino acid residues in black represent nonconserved amino acids between the rat and mouse GnRHRs; shaded amino acid residues are non-identical but conserved between the two species. Asterisks denote for protein potential glycosylation sites. Potential phosphorylation sites are indicated kinase C (arrowheads), casein kinase II (arrow), and protein kinase A (cross). B, Hydrophobicity plot of the rGnRHR, as determined by the method of Kyte and Doolittle (23).

Tissue Distribution determine

the distribution

of rGnRHR mRNA. A limited tissue screen was performed of expression of the rGnRHR mRNA

to

(Figure 3). The tissues

analyzed were chosen based on the results of previous GnRH binding studies (20-22). In aT3 cells, a predominant mRNA of approximately 4.5 Kb in size was detected by Northern blot analysis, consistent with previous reports (7). A smaller and less abundant mRNA, approximately 1.8 Kb in size, was also regularly detected under 1649

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 189, No. 3, 1992

A

12345

B

12345

w 20s -,

Cyclophilin

Figure 3. Northern blot analysis of mRNA from rat tissues and murine aT3 cells. All lanes contain 20 pg of total RNA. The positions of 28s and 18s RNA are indicated at the left of the blots. Arrowheads indicate the positions of the GnRHR mRNA signals. The lane designations are as follows: A, Rat ovary (Inne Z), rat liver (lane 2), intact adult female rat pituitary (lane 3), rat pituitary from ovariectomized rats (lane 4), and murine aT3 cells (lane 5); B, Rat placenta, 16 days gestation (lane Z), rat placenta, 21 days gestation (lane 2), rat testis (lane 3), rat ovary (lane 4), and murine aT3 cells (lane 5). Cyclophilin mRNA signals (used as a control to normalize for RNA loading to compare rGnRHR mRNA signal intensities in intact and ovariectomized rats) are indicated in A.

hybridization

and washing conditions

of high stringency.

between these two mRNAs is not known;

The nature and relationship

they may represent splicing variants of the

heteronuclear precursor RNA. In the rat pituitary, mRNAs of 4.5 and 1.8 Kb are also detected, the same sizes as those in the mouse gonadotrope cell line. The relative abundance of the two forms also seems to be similar between

the two species. In addition,

detected, consistent with an mRNA approximately

a larger, broad signal is

5.0-5.5 Kb in size. The nature of this

mRNA species is also unknown; it may represent heterogeneity in poly A(+) tail length. High affinity, specific GnRH binding has been reported in the rat ovary, testis, and placenta (20-22). We therefore tried to detect rGnRHR mRNA in these tissues. All three mRNAs detected in the rat pituitary were also detected in the rat ovary by Northern blot analysis, but were of lower signal intensity, suggesting that the rGnRHR gene is expressed in the ovary but at lower levels than in the pituitary. Low levels of signal intensity corresponding to the 4.5 Kb mRNA were also detected in rat testis RNA samples, suggesting that the rGnRHR gene is also expressed at low levels in this tissue. No signal was detected by Northern hybridization analysis of 20 pg of rat placental RNA derived from either 16 or 21 days gestation. This does not rule out the possibility that the GnRHR is expressed in rat placenta at levels below the limits of detection of Northern hybridization analysis, or that an alternate form of GnRHR mRNA is expressed in rat placenta, which is not easily detected with the rGnRHR probe used here. Further studies using ribonuclease protection analysis or PCR will be necessary to address these issues. No hybridization signal was detected in rat liver RNA, used as a negative control. 1650

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Regulation GnRHR

BIOCHEMICAL

3, 1992

mRNA

of rGnRHR

mRNA

AND BIOPHYSICAL

Levels

in the Rat Pituitarv.

levels in intact and 21-day post-OVX

increase in GnRHR

mRNA

RESEARCH COMMUNICATIONS

Comparison

female rats revealed

of

a 7-fold

levels after OVX (Figure 3). All three forms of GnRHR

mRNA were increased after OVX. These results are consistent with an increase in GnRH binding by the pituitary which has been reported previously (2), and suggest that this increase occurs at least partially at the pre-translational level. The mechanism of this increase in the GnRHR mRNA level, i.e., whether it occurs transcriptionally or posttranscriptionally,

remains to be determined.

Conclusion, cDNA

In this report, we describe the isolation

encoding

the rGnRHR

using

screening strategies. The availability facilitate

PCR amplification

of the rGnRHR

diverse studies of the mechanisms

delineation

of the ligand

binding

and sequence analysis of a and conventional

and mGnRHR

library

clones will greatly

of GnRH action. These studies include

site, allowing

the generation

of more potent

and

specific GnRH agonists and antagonists; detailed molecular analysis of the mechanisms of signal transduction and coupling to intracellular second messenger systems; analysis of the mechanisms the physiologic development

of the regulation

of receptor number and sensitivity;

roles of GnRH and the rGnRHR

in the regulation

and studies of of reproductive

and function.

Acknowledvments This work was supported, in part, by NIH Grant HD-19938 (to W. W. C.) and a Medical Research Council of Canada Clinician-Scientist Award (to U. B. K.).

References 1. Gharib, S. D., Wierman, M. E., Shupnik, M. A., and Chin, W.W. (1990) Endocr. Rev. 11: 177-199. 2. Clayton, R.N. (1989) J. Endocrinol. 120: 11-19. 3. Loumaye, E., and Catt, K. J. (1982) Science 215: 983-985. 4. Conn, P. M., and Crowley, W. F. (1991) N. Engl. J. Med. 324: 93-103. 5. Huckle, W.R., and Conn, P. M. (1988) Endocr. Rev. 9: 387-395. 6. Naor, Z. (1990) Endocr. Rev. 11: 326-353. 7. Tsutsumi, M., Zhou, W., Millar, R. I’., Mellon, P. L., Roberts, J. L., Flanagan, C. A., Dong, K., Gillo, B., and Sealfon, S. C. (1992) Mol. Endocrinol. 6: 1163-1169. 8. Reinhart, J., Mertz, L. M., and Catt, K. J. (1992) J. Biol. Chem. 267: 21281-21284. 9. Probst, W. C., Snyder, L. A., Schuster, D. I., Brosius, J., and Sealfon, S. C. (1992) DNA Cell. Biol. 11: l-20. 10. Windle, J. J., Weiner, R. I., and Mellon, P. L. (1990) Mol. Endocrinol. 4: 597-603. 11. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18: 5294-5299. 12. Kaiser, U. B., Lee, B. L., Carroll, R. S., Unabia, G., Chin, W. W., and Childs, G. V. (1992) Endocrinology 130: 3048-3056. 13. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74: 54635467. 1651

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14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning - A Laboratory Manual, ed. 2, pp. l-90 - l-104. Cold Spring Harbor Laboratory, Cold Spring Harbor. 15. Thomas, P.S. (1980) Proc. Natl. Acad. Sci. USA 77: 5201-5205. 16. Gharib, S. D., Bowers, S. M., Need, L. R., and Chin, W. W. (1986) J. Clin. Invest. 77: 582-589. 17. Danielson, P. E., Forss-Petter S., Brow, M. A., Calavetta, L., Douglass, J., Milner, R. J., and Sutcliff, J. G. (1988) DNA 7: 261-267. 18. Hubbard, S. C., and Ivatt, R. J. (1981) Annu. Rev. Biochem. 50: 555-583. 19. Kennelly, P. J., and Krebs, E. G. (1991) J. Biol. Chem. 266: 15555-15558. 20. Iwashita, M., and Catt, K. J. (1985) Endocrinology 117: 738-746. 21. Clayton, R. N., Katikineni, M., Chan, V., Dufau, M. L., and Catt, K. J. (1980) Proc. Natl. Acad. Sci. USA 77: 4459-4463. 22. Iwashita, M., Evans, M. J., and Catt, K. J. (1986) J. Clin. Endocrinol. Metab. 62: 127133. 23. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157: 105-132.

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