Vol.

175,

March

No.

29,

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

Pages

1991

Cloning

and

Takashi

Sequencing

Department

of School

Received

of

Minegish*,

Kazuto

Yoshi

29,

FSH

Receptor

Nakamura.

lbuki

and

Obstetrics Medicine.

of

Janaury

to

Human

Yumi

Masao

Takakura,

lgarashi

and Gynecology, Maebashi,

Gunme 371 I

Gunma

1991

stmulating

hormones izing

whi

of

on

their

ed

by

G

and

the fol 1 i cle emi no acid setransmembrane protein-coupled extracellular sites. the previously regions are 0 1991

whose

of

members of

receptors presence

of

Academic

locations transmembrane

whom

correspondence

the

which

the

is

segments.

should

ated

grafted

domai onto

Al

be

1125

though

been

n a

the

va-

identistructural

Moreover are

I

these

characterized of

structure complete

putati

at-

these acti

common

hormones

ycosyl

of

hormone-induced

by

act

medi

structures

has

n-

simi-

they

activity

domains(3).

gl

I utei

that

receptors

characterized

n

The

of

protein-coupled

large,

gl ycoprotei

hormone(TSH).

cyclase

mechanism

glycoprotein a

of

observation

that

transmembrane

pituitary

ng

adenylate

common G

ati

and

suggested

are

seven of

cellular

a

member

choriogonadotropin(hCG1.

increase

.2)

family

mul

hormones to

a

is

d-sti

human

glycoprotein

reflect A

(FSH)

thyroi

udes

proteins(l

feature

*To

i ncl

receptors

on.

fied

hormone

(LH) the

receptors

seven

ch

hormone

larity

the

University Japan

Inc.

Follicle

ti

1125-1130

cDNA

We have isol ated and sequenced a cDNA encoding stimul ati ng hormone (FSH) receptor. The deduced contai ni ng seven putative quence(678 residues) segments which displays sequence si mi lari ty to G receptors. The receptor consists of 359 residue domain which contains four N-linked glycosylation Whi le the protein is 89 X identical overal I with cloned rat FSH receptor , the most highly conserved the putative transmembrane segments (95% similarity). Press,

COMMUNICATIONS

by ve

extra-

containing nucleoti

de

0006-291X/91

$1.50

addressed.

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

175,

No.

BIOCHEMICAL

3, 1991

sequences

of

the

reported,

the

cDNA

cDNA

for

for

rat

the

AND

BIOPHYSICAL

FSH

receptor(4)

human

FSH

and

complete

RESEARCH

COMMUNICATIONS

have

receptor

been

is

not

al

ready

elucidated

yet. We the

now

report

cDNA

highly regi

for lar

one1

cloning

the

simi

Mater

the

human to

FSH

the

i als

and

receptor.

Whi

counterpart

differences

in

nucl

of

the

le

rat

extent

of

eoti

de

sequence

the

of

receptor

is

receptor,

significant

identity

are

noted.

Methods

Oligo(dT)-primed nyl ated RNAs. pol ymerase and to the vector Gold. The library

cDNAs were prepared from human ovary polyadeends of the oDNA were blunted wi th T4 DNA adaptors were added. The cDNA was ligated gtl0 end ackaged wi th Stratagene Gi gapack contains 1x10 I dependent recombinants, and was 5 was screened with nick This Ii brary(5x 10 clones) :;;y:532 P _ I abel ed 2.D-ki I obase(Kb) fragment from the rat FSH receptor cl ens(4). Nitrocellulose filters were hybridized and washed successi vel y in 6 x SSC (0. 15 M NaCl, 0.015 M Sodium 0.1 % SDS for 15 min at 42°C and 0.1 x SSC, 0.1 % Ci trate,pH7.0). SDS for IO mi n at 65°C. Several clones were identified, and one clone was selected for sequence analysis. These clones were stranded DNA templates were subcloned into PUC18 and single Sequencing of strands of DNA prepared for sequence determination. was done by the dideoxy chain termi nation method (5).

Results

and

Figure cDNA

Discussion

1 and

PI rst

The EcoRl Lambda

shows

amino

the acid in

This

was

characteristics defined

by

Von

signal

A a

region

a

ami

of FSH

peptide

with

The

the

acid

initiator

sequence a

cloned

receptor.

considered

no

the

having

cleavage

the

site

as

jne(6).

suggested

protein.

the

was

by

analysis(Fig.2)

tor(Fig.3)

transmembrane

Hei

for

sequence

followed a

sequence

deduced

the

of

Hydropathy

precedes

sequence

methlonine

codon.

2,327-nucleotides

and a

putative

possible extracel

of segments.

254

ami

no

There

alignment

with

model

for

lular

domain

acids is 1126

that a

65

the

rat organization of

of

359

displays amino

r ecep-

FSH

ami seven

acid

no

the acids

possible

COOH-terminal

Vol.

175,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ATG ,“,t

GCC ~,a

CTG ,..u

CTC L.v

CT0 Leu

OTC “al

TCT Ssr

TTG L.”

CTG La”

GCA ALa

TTC Ph.

CT0 La”

AGC S.r

,,G L.u

GGC G,y

TCA S.r

GGA GIY

TO, CY.

CAT His

CA, HI,

COG ,.rp

ATC I,,

TO, cyr

CI\C “is

,GC cyr

TC, ser

r\*c /\rn

100 drp

OTT v,,,

T,, ~,,a

CTC L.u

TGC CYS

CAbi Gin

GAG 01”

AGC S.,

LAG Lrr

GTG “al

PICA Thr

GAG 01”

IT, II,

CC, Pro

TCT s.r

GAC.CTC ASP L.u

CC0 P,.

AGO A,D

A.&T Aso

GCC AI.

A,, I,.

GAA 0,~

CTG L.U

A.60 A,)

T,, P,,,

OTC “a,

C,C L.U

ACC Thr

AA0 Lo,

c,, L,U

CGA ~rp

OTC “.I

TTT Ph.

GCC AI.

AD, S.r

G&G 01”

CT0 L.”

,CA S.r

OTC “.I

,AC TYI

ACT Th,

CT0 L.”

ACA ,hr

GC, A,.

ATC II.

&CC Thr

G&A G,”

AGA Arm

,GG ,,I

CA, HI,

ICC Thr

ATC II.

AC0 Thr

CA, “I,

GCC A,,

&TO M.t

CAG 0,”

CTG I..”

GAC A,P

TGC Cys

AA0 LYS

GTG “a,

CAG 0,”

C,C Lnu

CGC Am

CAT His

GCT. PII*

GCC AI.

AGT S.r

OTC “al

ATG M.t

GTG “a,

A,G M.t

GGC 0,~

TOG ,r.n

AT, ,I.

T,G L.”

f,, Ph.

GCT Air

TTT Phm

GCA Ala

GCT Ala

GCC Ala

CTC La”

TTT Ph.

CCC Pro

ATC II.

TTT Ph.

GGC 01~

ATC I,.

AGC S-r

AGC S.r

TAC TY’

ATG M.t

AAG L)(I

GTG “al

AGC Ssr

ATC II.

TGC CYZ

CT0 C.”

CCC Pro

ATG “.t

GA, *s.

ATT ,I.

GAC )I,*

AGC S.r

CC, Pro

,,G C,”

TCA S.7

CA0 G,”

CTG L.”

TAT ,I,

GTC Y.,

ATG Mat

,CC Sar

CTC La”

CTT L.”

GTG “.I

CTC LS”

AAT AL0

GTC “.I

CTG L.aU

GCC Ala

MG LY.

CGC AirI

ATO Idat

GCC AIS

ATG Met

CTC L.”

ITC II.

,,C Ph.

ACT Tilr

GAC Asp

,TC Ph.

CTC L.”

TGC CY.

ATG Hat

GCA ,&,a

CCC Pro

A,, II.

TCT Ser

TTC Ph.

TTT Ph.

GCC *Ia

ATT II.

TCT S.r

GCC *Ia

TCC Ssr

CTC L-v

AAG Lrr

GTG “al

CCC Pro

Fig.1 The cDNA and predicted amino acid sequence of human ovarian FSH receptor: Position t I Is assigned to the first nucleotlde of Numbering of the nuoleoti des the putative initiator codon. The poten(above) end of the amino acid (underneath) is shown. in the extracellular domain are tlal N-linked glycosylation sites under1 i ned.

i ntracel ami

no

I ul acids

the

extracsl

rat

FSH

respeoti

ar

domai (76465 I ul

receptor vely.

The

n. dal

ar

Four

42X potential

protein

On

tons).

domains and

mature the

should

level

of

approximately

share homology

wi th glyoosylation 1127

consist primary 86%

human

676

structure, homology

LH/hCG sites

of

wi th

receptor(7) are

found

in

Vol.

175,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

i

-3.000 Fig.2 Detailed comparison non Identical amino receptor are shown, Alignment of homologous gaps represented by

indicated on the boxed. Conserved are denoted by fi

the

putative

present

in

regions

dashes.

lul

and ns

receptor(4,8,9,10,11),

lar

domain

The with

of

LH/hCG nines

and are

receptor.

found

in

by

specific

decoupling

important sites

of to

causes

know any

Moreover,

when

recently

described

this

fragment

funoti we

compared cloned

is

a

part

human of

the

a

role

might

human

96%

be lu-

and

the

agonist

in

threophospspecif-

G-proteins(l21,

it one

of

is

these

reoeptor. of

fragment(g), FSH

human

Si rice

on FSH

homology with

serines

in

from

sequences DNA

ts

domain.

of

1128

bi

of

phosphorylati

change the

TSH

extracel

homology

proportion

the onal

large

exhi 70%

receptors

whether

and

bonds the

domain

play

adrenergic

protein.

LH

disulfide

intracellular kinases

the

the

receptors.

high

putative

FSH,

of

are

between of

In

approximately A

cysteins

junction

domain

integrity

spanning and

sre domain

of

the

of

hormone

receptor

at

conserved

onal

membrane

TSH

and

formation

glyooprotein

FSH

horylation ic

conformati

putative rat

are

segments

extracellular

transmembrane

the the

the

Clusters

part

ar

cystei

for

transmembrane in

domain.

NH8 -terminal

the

these

crucial

FSH and LH/hCO receptor: Only rat FSH receptor and human LH/hCO residues are represented by a dot. has necessitated introduction of The numbering of the amino acids is

right. The putetive cystei ne residues I led ci role.

extracel

rice

the of

extracellular

putative SI

between acids ldentlcel

our

FSH we

receptor.

receptor observed

and that

Vol.

175,

No.

3,

1991

Fig.3 Hydropathy hydropathy

BIOCHEMICAL

plot profi

of I e

I

residues.

of

toMI

AND

human FSH the LH/hCO correspond

BIOPHYSICAL

RESEARCH

receptor.

A

receptor. to

Kyte

and

with

the

COMMUNICATIONS

a

Dooli

wl ndow

putative

ttie

of

19

transmembrane

segments.

In ti

ons

by

man.

the

that

lead

autoanti

TSH to

bodies

receptor hyperin

respectively(l3). i an of

or

Grave’s Thus,

physiology ovarian

can

as failure

wel

I

be

requires

target

of

hypostimulation

of

disease progress

as

the

and in

improvement the

the in

isolation 1129

in

autoimmune the

thyroid

idiopathic

and

of and

the

gland myxoedema.

understanding diagnosis

reac-

ovar-

management

characteriza-

Vol.

175,

tion

No.

of

LH/hCG that

3, 1991

human receptor

further

Acknowl

BIOCHEMICAL

gonadotropin and

AND

BIOPHYSICAL

receptors. FSH

receptor

understanding

for

In in

these

man

RESEARCH

this have

diseases

COMMUNICATIONS

regard, been wi

since cloned,

I I

be

both we

hope

achieved.

edgments

This work for Science Foundation. ructi ng

was

the

supported and Culture We also thank I i brary.

by

a

of

grant from the Japan(02670732) Dr. Kaoru Mi yamoto

Ministry and for

of Uehara help

Education Memorial i n cons

References I I

2. 3. 4. 5. 6. 7.

8.

9.

10.

I I *

12. 13.

M.L. and Catt, K. J. (1978) Vi taml ns and Hormones Dufau, 36,461-593 Reichert,Jr.L.E. and Dettatreyamurty, B.(l969) Bi ol .Reprod. 40.13-26 Lefkowi tz, R.J. and Caron, M.G. (1988) J. Bio. Chem. 263, 4993-4996 Sprengel, R., Braun, T., Nikollcs, K., Segaloff, D.L., and Seeburg, P.H. (1990) Mol. Endoorinol. 525-630 Sanger, F., Nichlen, S., and Coulson. A.R. (1977) Proc. Nat. Acad. Sci USA, 74, 5463-5467 Hei ji, G.V. (1986) Nucleic Acids. Res. 14, 4683-46864 Minegishi, T., Nakamura, K., Takakura, Y., Mlyamoto, K., Hasegawa, Y.. lbuki, Y. and lgerashi, M. (1990) Bioohem. Biophys. Res. Commun. 172, 1049-1054 McFarland, K. C., Sprengel, R., Phillips, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L., Seeburg. P.H. (1989) Science 245, 494-499 Loosfel t. H., Misrahi , M., Atger, M. I Sal esse, R. I Hai-Lau Thi , M.T.V., Jolivet, A., Guiochon-Mantel, A., Sar, S., JaIlal, B., Garnier, J.. and Milgrom, E. (1989) Science 245, 525-528 Parmentier, M., Libert, F., Maenhaut, C., Lefort, A., Gerard, C., Perret, J., Van Sande, J., Damont, J.E. and Vassart, G., (1989)Science 246, 1620-1622 Misrahi, M.. Loosfelt. H.. Atger. M., Sar, S., Guiochonand Milgrom, E., (1990) Biochem. Biophys. Res. Mantel I A., Commun. 166, 394-403 Benovic, J.L.. Strasser, R.H., Caron, M.G. I and Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801 Furmanai k, J. (1988) Reces-Smith. B.. Molachlan, S.M., and Endocrinol Rev. 9, 106-121

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