Vol.. 189, No. 1, 1992 November

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30, 1992

CLONING,

Pages

SEQUENCING,

Sham S. Kakar,

Department

Received

of

AND REPRESSION OF HUMAN GONADOTROPIN RELEASING (GnRH) RECEPTOR'

Lois

C. Musgrove, Daniel C. Devor, and Jimmy D. Neil1

Physiology

September

and Biophysics, Birmingham,

30,

Jeffrey

University AL 35294

of

289-295

HORMONE

C. Sellers

Alabama

at

Birmingham,

1992

Summary: Gonadotropin releasing hormone is a hypothalamic decapeptide that stimulates the release of gonadotropic hormones from the anterior pituitary gland. Therapeutically, the human pituitary GnRH receptor is the target of agonists used in the suppression of prostate cancer. Here we report the isolation of a cDNA representing this receptor. It encodes a protein with a transmembrane topology similar with that of other G protein-coupled, 7transmembrane receptors. Binding studies of the cloned receptor demonstrate high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot and reverse transcriptase/PCR analysis revealed that its mRNA is expressed in pituitary, ovary, testis, breast, and prostate but not in liver and spleen. Availability of a human GnRH receptor cDNA should permit the design of improved analogs for therapeutic applications. 0 1992 AcademicPress. Inc.

Gonadotropin is

the

by

hypothalamic

releasing

key neural

anterior

lobe

There,

hormones

the (2).

identified (3),

other

brain

Most known rat shown

(2). it

' Sequence Libraries

(4)

data under

site

of is

receptors

tissues

of

where

their

studies

from this Accession

the

process

of the

of

of GnRH in

that

synthesized

carried

rat

the

to

member

pituitary

of

the

for

as the are

not

in molecular

article have been No. LO3380.

deposited

gland

G-protein (3),

definitively

have

(5),

with

the

coupled,

adrenal studies

been cortex in the

investigators

have 60 kD),

EMBL/GenBank 0006-291X/92

289

is

have

(64 kD vs.

the

the

established.

come from size

(LH

of

gonadotropic

GnRH also

gonads

human GnRH receptor

receptor

hormones functions

and secretes

Receptors such

functions

gonadotropic the

G&H-receptors (2).

the

is

and

and hormonal

of GnRH receptors rat

It

manner,

gametogenic

action

the

decapeptide

(1).

of the hypothalamo-hypophysealportal

an established

of

from

a hypothalamic

a pulsatile

the

expresses

receptor

is

secretion

regulate

characteristics

In limited to differ

in

glandbyway

that

family in

and

cell

This

Ca'+-dependent

(GnRH) reproductive

GnRH stimulates

in turn, The primary

gonadotrope,

the

secreted

of the pituitary

circulation. (1).

of

neurons,

and FSH) which, gonads

hormone

regulator

in

Data $4.00

Copyright 0 1992 by Academic Pre.vs, Inc. All rights of reproduction in any fbrm rrserrri.

Vol.

189, No. 1, 1992

binding

affinity

and divalent

BIOCHEMICAL for

concentrations

are

GnRH (6)

cancer pituitary cloned,

approved

pituitary

towards

for

to It

treatment

gland long

to

monovalent

acting

supraphysiological GnRH agonists

GnRH receptors,

secretion.

is

on this

of precocious

basis

leading that

puberty,

(7)

to profound

analogs

of GnRH

endometriosis,

and

(7).

a cDNA clone cell

the

or continuously

in humans

sequenced

of

desensitizationof

in gonadotropin

Recently, anterior

exposure of

currently

we have

and sensitivity

some GnRH antagonists,

down regulationand

decreases prostate

RESEARCH COMMWNICATIQNS

cations.

Paradoxically, causes

AND BIOPHYSICAL

line

for (8).

a GnRH receptor Using

and expressed

has

been

isolated

the mouse GnRH receptor a GnRH receptor

from

from

mouse

cDNA as a probe, the human pituitary

gland. Materials

and Methods

cDNA cloning. The probe for screening the human pituitary cDNA library was mouse anterior pituitary GnRH receptor cDNA representing its open reading frame (8) prepared by reverse transcriptase/PCR (9, 10) as follows. Total RNAwas prepared from mouse anterior pituitary gonadotrope cell line (aT3-1) by the guanidinium/CsCl method (ll), and poly (A+)-RNA was purified by oligo(dT)cellulose chromatography. First strand cDNA was prepared using oligo(dT) primer and AMV reverse transcriptase following the protocol provided by the supplier (Promega). The primers used for PCR were sense 5'-ATGGCTAACAATGCATCTTG-3' and antisense 5'-CTACAAAGAGAAATACCCAT-3'(8). A lambda gtl0 cDNA library (12) prepared fromhuman pituitary poly (A+)-RNA primed with oligo(dT) (kindly donated by Dr. Olivier Civelli, Oregon Health Sciences University, Portland) was screened with 32P-labeled mouse GnRH receptor cDNA. In brief, approximately 5 x lo5 plaques from the library were plated and screened by plaque hybridization (13). Positive clones were purified to homogeneity. Inserts from the positive clones were excised with EcoRl and subcloned into the pcDNA-I vector. Nucleotide sequencing of three clones was performed completely onboth strands using the Sequenase 2.0 kit (US Biochemical) and synthetic primers. The sequence analysis presented was performed using the Wisconsin GCG program on a VAX computer. Transfection of COS-7 cells with human GnRH receDtor cDNA for bindinn assays. COS-7 cells grown on 60 mm Petri dishes were transfected with 5.0 ug of CsClpurified plasmid DNA (pcDNA-I) bearing the human GnRH receptor cDNA using 60 ~1 of lipofectin as described previously (9). After 60 to 72 hr, the cells were washed with PBS, scraped-off the dishes, and homogenized in buffer (10 mM TrisHCl, pH 7.4) to prepare the cell membranes for GnRH receptor radioassay as described by Wormald et al. (5) for human pituitary. The radioligand was "'1 [D-Ala61 des-Glyl*-GnRH. Nonspecific binding was determined in the presence of 10-h [D-Ala6]des-Glyl' -GnRH (GnRH-A, a GnRH agonist). Calcium measurements. COS-7 cells were transfected described above, collected after 65 hr, and attached with poly-L-lysine. Calcium measurements in these Furaas previously described (9).

with GnRH receptor to glass coverslips cells were performed

cDNA as coated using

Tissue exoression of human GnRB receotor mNf?A. Normal human tissues were obtained from The Tissue Procurement Facility, Comprehensive Cancer Center, University of Alabama at Birmingham. Such tissues are collected at the time of autopsy (3-12 hr after death) or biopsy and are immediately frozen and stored in liquid nitrogen. Total RNA from human pituitary,ovary, testis, breast, 290

Vol.

BIOCHEMICAL

189, No. 1, 1992

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

prostate, liver, and spleen was prepared as described earlier. Twenty pg of RNA from each tissue was separated on a 1.0% agarose gel. Northern blot analysis was performed under high stringency conditions using random hexamer primed 32Plabeled full length human GnRH receptor (1.5 kb) as previously described (14). For reverse transcriptase/PCR analysis of GnRH receptor mRNA, we prepared first strand cDNA using 2 rg of the total RNA in a 20 ~1 reaction volume as described earlier. A five pl aliquot of this solution was then used in a polymerase chain The primers used were sense 5'reaction (PCR) using the GeneAmp kit. 5'-CCTAGGACATAGTAGGGGCTTGAAGCTCTGTCCTGGGA-3' (overline, Fig. 1) and antisense 3'(underline, Fig. 1). The reaction conditions were 1.5 min at 95'C, 1.5 min at 54'C, and 2 min at 72'C for 30 cycles in a Perkin-Elmer Cetus DNA thermal cycler. Twenty ~1 of the 100 ~1 reaction mixture was then electrophoresed through a 1.2% agarose gel and stained with ethidium bromide.

Results Ten cDNA clones from

the human

were

subcloned

Three

of

pituitary

the pcDNA-I

sequenced

nucleotide

sequence

end.

to

cDNA

occurred

protein

(relative

not

(8) ;

there

(8).

Hydropathic

potential

hydrophobic for

glycosylation residues

the

phosphorylation which

also

four 2).

was reported

for

among 7-transmembrane, receptor's

GnRH receptor 1251[D-Ala6]des-Gly'o high

affinity

(Fig.

3):

the

(16,

is

amino

acids

acid

level

2).

The

modification,

cysteine

residues,

domain

are

The

receptor

lacks

the

mouse

GnRH-receptor

coupled

that

that

3'this

oligo(dT)

a 328 amino

acid

75% identity

with

open reading

frame

two

receptors

seven

stretches

protein

contains

five

the

include

(8);

and for

threonine regulatory

cytoplasmic this

(15),

several

two N-linked

serine

candidates

a C-terminal

receptors

of

identified which

and;

that

its

between

protein

(Fig.

long exhibits

GnRH

at the

encodes

with

The

composed

we assume

frame

were

of the

is

poly(A+)-tail

sufficiently sequence

hence

sequence cDNA

signal,

predicted

G-protein

propensity

identical.

and 85% identity

the

cytoplasmic (Fig.

were

an apparent

but

post-translational

sites; in

at of

amino

sequences acid

clones

expression. and

1.

The nucleotide

analysis

and eukaryotic

The

isolated

4 of these

frames

The open reading

cDNA overall

from

amino

Fig.

cDNA were

reading

a polyadenylation

region.

89% identity

sites

in

and contains

Mr-37,730).

is

open

predicted

shown

of

inserts

sequencing their

a poly(A+)-tail

in this

the mouse GnRH receptor

highly

are

absence

is

priming

of

strands;

and corresponding

the

poly(A+)-region

for

complete

1550 nucleotides

Due

The EcoRI

vector

contained on both

(hGnRHR)

approximately

mouse GnRH receptor

cDNA library.

4 clones

completely receptor

thathybridizedwith

into

the

and Discussion

is

domain

unprecedented

and may relate

to this

to desensitize. radioassays

performed

-G&H,

demonstrate

17).

The

[D-Ala6]des-Gly"-GnRH

IC,,

that values

(GnRH-A),

on

transfected

COS-7

the cDNA encodes for

various

0.6x10-9M;

cells

a GnRH receptor

ligands GnRH,

were

as

using with follows

[Ac-D-pCl-

5x10-‘M;

Phe1~2,D-Trp3,D-Arg6D-Ala1o]GnRH(GnRH-Antl),lxlO~~;[D-pGlu1,D-Phe2,D-Trp3~6]GnRH (GnRH-Ant et&.

2),

4x10-'M,

(5) performed

and;

[D-Phe2,6,Pro3]GnRH

GnRH receptor

radioassays 291

(GnRH-Ant

3),

on human pituitary

50~10~~.

Wormald

cell

membranes

Vol.

189, No. 1, 1992

-55 1 21 41 61 81 101 121 141 161 181 201 221 241 261 281 301 321

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

GTTGTGCTGTCAGTGCACCAGAGACACAAGGCTTGAAGCTCTGTCCTGGGAAAAT

-1

ATGGCAAACAGTGCCTCTCCTGAACAGAATCAG~TC~TCACTG~CAGCCATC~C~CAGC MANSASPEONONHCSAINNS ATCCCACTGATGCAGGGCAACCTCCCCACTCTGACCTTGTCT~~G~TC~GA~TG~CG IPLMQGNLPTLTLSGKIRVT GTTACTTTCTTCCTTTTTCTGCTCTCTGCGACCTTTAATGCTT VTFFLFLLSATFNASFLLKL CAGAAGTGGACACAGAAGAUGAGAAAGGGAAAAAGCTCTCAAGAATGAAGCTGCTCTTA QKWTQKKEKGKKLSRMKLLL AAACATCTGACCTTAGCCAAC~GTTGGAGACTCTGATTG KHLTLANLLETLIVMPLDGM TGGAACATTACAGTCCAATGGTATGCTGGAGAGTTA~CTGC~GTT~CAGTTATCTA WNITVOWYAGELLCKVLSYL AAGCTTTTCTCCATGTATGCCCCAGCCTTCATGATGGTGGTGATCAGC~TG~AC~GC~CC KLFSMYAPAFMMVVISLDRS CTGGCTATCACGAGGCCCCTAGCTTTGAAAAGCAACAGCAGTCGGACAGTCCATGGTT LAITRPLALKSNSKVGQ GGCCTGGCCTGGATCCTCAGTAGTGTCTTTGCAGGACCACAGTTATACAT~TCAGGATG GLAWILSSVFAGPQLYI ATTCATCTAGCAGACAGCTCTGGACAGACAAAAGTTTTCTCACACTGC IHLADSSGQTKVFSQCVTHC AGTTTTTCACAATGGTGGCATCAAGCATTTTATTTTTGCTGCCTCTTC SFSQWWHQAFYNFFTFSCLF ATCATCCCTCTTTTCATCATG~GATCTGCAATGCAAAATCATCTTCACCCTGACACGG IIPLFIMLICNAKIIFTLTR GTCCTTCATCAGGACCCCCACGAACTACAACTGAATCAGTGA V L H Q DPHELQ L N Q S KNNIPR GCACGGCTGAAGACT~AGACGGTTGCATTTGCCCTGTCTGCTGG ARLKTLKMTVAFATSFTVCW ACTSCCTACTATGTCCTAGGAATTTTGGTATCCAGGTTG TPYYVLGIWYWFDPEMLNRL TCAGACCCAGTAAATCACTTCTTCTTTCTCTTTGCCTTTTCCCATGCTTTGATCCA SDPVNHFFFLFAFLNPCFDP CTTATCTATGGATATTTTTCTCTGTGATTGATAGACTACAC~G~GTCATATG~G~G LIYGYFSL#

60 120 180 240 300 360 420 480 S

M

V

F

R

M

540 600 660 720 780 840 900 960 1020

GGTAAGGTAATGAATCTCTCCATCTGGGAATGATTAACAC~TGTTGGAGCATGTTTAC ATACAAACAAAGTAGGATTTACACTTAAGTTATCATTCTTCTCAGTCTTCAGA GCCTCAATTATTAAGGAAAAGCTCTTCAGGAAAAATACTAAAATATTTCTCTTCCTCATA AGCTTCTAAATTAATCTCTGCCTTTTCTGACCTCATATAAATGTAGGTTTCTTA TCACTTTCTCTTTGCATAATAATGTACTACT~TATTT~TACCTTCAGCCT~GGCAC~ GGATGCC-CAAAGGTGAGAAACCACAACACAGGTCTAAACTCAGCATGCTTTGG TGAGTTTTTCTCCAAAAGGGGCATATTAGCAATTAGAGTTAGAGTTGTATGCTATAT~TACATAG AGCACAGAGCCCTTTGCCCATAATATCAACTTTCCCTCCTTTCCCTCCTATAGTT~ AA

1080 1140 1200 1260 1320 1380 1440 1500 1502

sequence of the human GnRR receptor cDNA and deduced amino FIG. 1 Nucleotide A The nucleotide sequence is listed above and acid sequence of the protein. numbered on the right while the amino acid sequence is listed below and numbered The overline (sense) and underline (antisense) designate the on the left. sequences of synthetic oligonucleotides used for the polymerase chain reactions shown in Fig. 5.

and

reported

similar

values

our

values

hormone

(TRH)

with

releasing

Thus, the 3). characteristics shown cloned

GnRH receptor functionally in

for

clonedhuman cDNA for

(8).

for

5

GnRH

and

4

and

nM,

the

GnFM receptor, measurement

of

Ant

2

which

are

Finally,

(AII)

were

a protein

the

anterior

increases

in

Ca2+ also

when RNA transcribed

To determine

for

virtually with

highly

thyrotropin inactive

the

(Fig.

pharmacological

GnRH receptor.

from

intracellular

nM

respectively.

encodes

human to

4.2

II

isolated

in cells

mouse GnEW receptor, oocytes

the

of couple

increases

nM

and angiotensin

expected

to

Xenonus receptor

of

cDNA we have

The dependent

4.8

of

whether we used (Ca2+)i 292

pituitary

have

been

from Ca2+ serves

COS-7 cells levels

of

intracellular

using

the

rats

has

demonstrated

been GnRH-

Ca2+ (2). with

cDNA was injected as the

transfected video-based

second with

the into

messenger the GnRH

fluorescence

Vol.

189,

No.

BIOCHEMICAL

1, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Extracellular

Intracellular of the human GnRH receptor. The AFIG. 2 Proposed seven transmembrane topography shaded area represents the cell membrane. Also indicated are the canonical sites of N-linked glycosylation (Y), potential sites for phosphorylation by protein kinase C (*), and cysteine residues in the extracellular loops (v). ratio

imaging

of

About

12% of

the

exhibited

a mean

the calcium transfected 2.0-fold

indicator

dye,

COS-7 increase

cells in

the

Fura-2, responded Fura-

as described to

lo-'M

fluorescence

previously

(9).

G&H-A

and

they

ratio

(Fig.

4).

80 g

FIG. 3. Unlabelled receptor radioassay human GnRH receptor assays derived from abbreviations of the

ligand displacement curves in an 1251-[D-Ala6]-des-Gly10-GnRH performed on membranes from COS-7 cells transfectedwith cDNA. The results shown are the mean values derived independent transfections. See text for definition ligands.

293

from

the 2-4

of the

Vol.

BIOCHEMICAL

189, No. 1, 1992

0.20.

~

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

GnRH

o.ooJ

,

2

0

4

,

(

,

6

8

IO

TIME (min.)

FIG. 4. GnRH-A-stimulated increase in intracellular Ca'+ levels in two COS-7 cells transfected with the human GnRH receptor cDNA. In 2 independent transfection assays, 20 of 165 COS-7 cells (12.1%) responded to GnEH-A (lo-'M); none of 60 cells responded to GnEH-A when transfected with pcDNA-I plasmids carrying the human GnEH receptor cDNA in the antisense orientation. None of the cloned

COS-7 cells

in

the

receptor

(hGnRNR)

intracellular

also

analysis

length

revealed

whether

GnRH mRNA is

sensitive

breast,

reverse

corresponding

blot

a 5.0

testis,

to

cancer

Although was highest expressed.

bearing

to

the GnRH receptor

cDNA

Thus,

GnRH

GnRN-A.

functionally

coupled

cell

hybridizations kb EcoRI

kb mRNA in

expressed

size ovary,

(Fig.

in pituitary

compared

Pharmacological

insert

from

the

human

to

increases

in

were

determined

in

than

of

the

not

to other

and

pituitary,

GnRH receptor breast, in

liver tissues

clone).

in

This

other

tissues

(800

and prostate and spleen (Fig. been

characterization

test

we used

expression

cDNA has

functional

not

hexamer

As an additional

As shown in Fig.

semi-quantitative,

GnRN receptor

a random

the XgtlO

but

shown).

other

testis,

5) but

is only

a human

as a probe

technique.

expected

line

using pituitary

not

in tissues

transcriptase/PCR the

human

(data

our RT-PCR assay summary,

of the human GnRN receptor

prostate)

in human pituitary,

In

be

hGnRHR cDNA (1.5

(ovary,

breast

to

and size

Northern

full

visible

a plasmid

responded

appears

distribution

stringency

primed,

with

direction

Ca2+.

Tissue high

transfected

opposite

the more

5, PCR products bp)

are

clearly

and in the MCF-7 (data

not

shown).

of GnRH receptor

5). cloned,

sequenced, of

the

and

receptor

Human GnEH receptor mBHA expression in various tissues and a breast LFIG. 5 tumor cell line. Ethidium bromide stained DNA is shown which was generated from tissue or cell mENA using reverse transcriptase/polymerase chain reactions. Abbreviations: Pit, anterior pituitary gland: Ov, ovary; Bst, breast; Pros, prostate gland; Test, testis; MCF-7, breast tumor cell line. 294

of

Vol.

189,

support

1, 1992

the

identical this

No.

conclusion

with

receptor

cancer, agonists

BIOCHEMICAL

and will

the

native

cDNA will

that

receptor

BIOPHYSICAL

cloned the

human

is

highly

study

of

its

of improved

COMMUNICATIONS

similar The availability

human pituitary.

to the development for

RESEARCH

receptor

in the

facilitate

contribute

and antagonists

the

AND

role

in

reproduction

if

not of and

GnRH receptor-specific

therapy.

Acknowledgments We wish to thank Dr. Pamela L. Mellon, University of California, San Diego, for the uT3-1 cell line, Dr. Olivier Civelli, Oregon Health Sciences University, Portland, for the human pituitary cDNA library, Dr. William E. Grizzell, Tissue Procurement Center, Comprehensive Cancer Center, University of Alabama at Birmingham, for normal human tissues, Dr. Jeffrey A. Engler for synthesis of oligonucleotide primers (supported by NC1 Grant CA 13148 to the UAB Comprehensive Cancer Center), Dr. Elliot Lefkowitz for assistance with sequence analysis, the UAB Center for AIDS Research (P30 AI 27767) for use of the VAX computer, and Dr. Raymond A. Frizzell for the use of his facilities in the measurements of (Ca'+),.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Fink, G. (1988) In The Physiology of Reproduction (E. Knobil and J.D. Neill, Eds.). pp. 1349-1377. Raven Press, New York. Huckle, W.R. and Conn P.M. (1988) Endocr. Rev, 9, 387-395. Pieper, D.R., Richards, J.S., andMarshall, J.C. (1981) Endocrinology 108, 1148-1155. Jennes, L., Dalati, B., and Conn, P.M. (1988) Brain Res. 452, 156-164. Wormald, P.J., Eidne, K.A., and Millar, R.P. (1985) J. Clin. Endocrinol. Metab. 61, 1190-1194. Belchetz, P.E., Plant, T.M., Nakai, Y., Keogh, E.J., andKnobi1, E. (1978) Science 202, 631-633. Barbieri, R.L. (1992) Trends Endocrinol. Metab. 3, 30-34. Tsutsumi, M., Zhou, W., Miller, R.P., Mellon, P.L., Roberts, J.L., Flanagan, C.A., Dong, K., Gillo, B. and Sealfon, S. (1992) Molec. Endocrinol. 6, 1163-1169. Kakar, S.S., Sellers, J.C., Devor, D.C., Musgrove, L.C., and Neill, J.D. (1992) Biochem. Biophys. Res. Commun. 183, 1090-1096. Kakar, S.S., Riel, K.K., and Neill, J.D. (1992) Biochem. Biophys. Res., Commun. 185, 688-692. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Grandy, D.K., Marchionni, M.A., Makam, H., Stofko, R.E., Alfano, M., Frothingham, L., Fischer, J.B., Burke-Howie, K.J., Bunzow, J.R., Server, A.C., and Civelli 0. (1989) Proc. Natl. Acad. Sci. USA 86, 9762-9766. Sambrook, D., Fritsch, E.F., and Maniatis T: (1989) Molecular Cloning: A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory, New York. Kakar, S.S., Wei, N., Mulchahey, J.J., LeBoeuf, R.D., and Neill, J.D. (1992) Neuroendocrinology (in press). Attwood, T.K., Eliopoulos, E.E., and Findlay, J.B.C. (1991) Gene 98, 153159. Clayton, R.N. and Catt, K.J. (1980) Endocrinology 106, 1154-1159. Loumaye, E., Naor, Z., and Catt, K.J. (1982) Endocrinology 111, 730-736.

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