Cloning and sequencing of human LHhCG receptor cDNA

Vol.

172,

No.

November

BIOCHEMICAL

3, 1990

15,

and

Sequencing

of

Minegish*,

Kaoru

Mi

yamoto*‘,

Department

of School

%X Natlonal

hisa

September

tr ti ve G protein-coupled due extracel tion

Masao

isolated

and -

Su

ta,

Gunma 371

Gunma

lbuki

1

University Japan

Research Osaka

sequenced

chori sequence(699 segments

receptors. domai

ar

to

lnsti

565,

tute

Japan

1990

hormone ami no acid ansmembr ane I ui

Takakura,

Yoshi

Center, I

cDNA

lgarashi

and Gynecology, Maebashi,

tine,

rodai

17,

Yumi

1049-1054

a

encoding

n

(LH/hCG) contai sequence

residues) di spl ays The

n

cDNA

ogonadotropi

whi

ch

receptor contai

ns

the

human

consists N-linked

six

lu-

receptor. ni ng seven similarity of

The putato

335 glycosyla-

rest-

sites.

Whi I e previously the most

the

protein cloned

highly

segments

(91

LH

and udl

ng

I ati

ng

hormone.

is

94%

are

cycl structural

nism

of

coup1

ed

terized

they

act)

hormone-i receptors

ty

of

si

mi

on

medi

ated

common

subfami

correspondence

putative

of

been

of

glycoprotein

i vat i denti

f i ed

LH/hCG

common

should

G

ly

whose

be

n-coup1

addressed

G

mechaprote

are

th that

ed

that

n

char

ac-

transmembrane

wi

characteristics protei

of

seven

receptor

adeny-

common

members of

and

suggested a

fami

hormones

increase

,2)

A

Inc.

stimu-

n

reflects

1 on,

y,

Press,

follicle

ycoprotei

proteinscl

feature of

of

G

the vel

hormones

to

receptors

structural

I y

gl

receptors by

I with respect1

transmembrane ‘z. 1990 Academic

and

the

their

act

reveals a

the

family

ty

these

Comparison

of

overal receptor

respectively).

a

I ari

act

of

has the

% identical LH/hCG are

ty,

nduced

receptor(4,5,6)

whom

vi design

ns(3).

members

larl

87 ne

hormone(TSH) The

ase

by

domai

simi

members

that

the

and porci regions

thyroid-stimulating

observation I ate

85 and

rat conserved

and

hCG

i ncl

*To

COMMUNICATIONS

Receptor

Hasegawa,

Cardiovascular Fujisht

LH/hCG

Nakamura,

Yoshi

Obstetrics Medi

of

Human

Kazuto

and

have zi ng

RESEARCH

Pages

Takashi

We tel nj deduced

BIOPHYSICAL

1990

Cloning

Received

AND

receptors

TSH

and make

FSH them These

Vol.

172,

No.

3, 1990

receptors

of

the

pi

presence

ccl

Iul

tui a

been

not

elucidated We

the

the

for

the

similar regional

Mater

i al

s

and

0.

fi ed. cl ones clease pared was

%

The EcoRl Lambda This

SDS two

for done

and

on no

at selected

the

aci

ds

the

extent

% 65

SDS “C.

de

receptor is

de

le

sequence

the

of

receptor

is

counterpart. of

6 for

signifi-

identity

x

are

SSC

(0.15

15 min Several sequence

for and

stepwlse DNA

Sequence nation

termi

and

n(Fi

gl

deduced

noted

M

at clones

42

NaCl.

0.015

“C and were

0 I IdentlThese

analysis deleted templates

ng method

the

i ni

tl

having

cleavage

site

as

bi were

of

strands

x

exonupreof

DNA

(9)

3)

precedes

n.

A a

region

codon.

the

characteristl 1 ned

and

primary

first

ator

def

suggested protei

the The

.2).

analysjs(Fig

of

porcine(8)

PUC18 single-stranded

sequence a

I

clones

receptor(Fig.2)

zati

eoti

receptor

eoti

Whi

and

determlnatlon. chain

dered

acid

Hydropathy LH/hCG

ng

on

cons,

with

min were into

protei

no

peptide

ni

nucl

LH/hCG

nucl

in

0. IO

two

was ami

complete

human

complete

successively

dideoxy

sequenced

a

contal

cDNAs were prepared from human ovary polyadeends of the cDNA were blunted with 14 DNA adaptors were added. The cDNA was ligated gti0 and l,ackaged with Stratagene Gi gapack contains Ix10 Independent recombinants and was Ii brary(5x 105clones) was screened WI th n ch EcoRl fragment 2.5-ki lobase from the clone(7). Ni trocel I ulose fl I ters wer e

Discussi

complete

by

extra-

LH/hCG

receptor,

rat(7)

oned on

sequence the

by

sequence

and

in

for clones

subcl gesti

and

the

putative

structure

the

the

by

Methods

were JiJ di

We

its

receptor

1 and

Results

of

porcine(8)

for

LH/hCG

and washed Ci trate,pH7.0),

urn

ssc

cloning

differences

LH/hCG

hybridized M Sodi

though and

that

a

COMMUNICATIONS

characterized

n

onto

rat(7)

human to

Oligo(dT)-primed nyl ated RNAs. polymerase and to the vector Gold. The library amplified. translated32P-labeled rat

are

domai

grafted

reported,

report

cant

hormones

ated

Al

for

RESEARCH

yet.

cDNA

highly

is

cDNA

already

now

the

ycosyl

segments.

of

BIOPHYSICAL

n

gl

which

transmembrane

have

AND

glycoprotei

large,

locations

sequences

ami

tary

of

ar

seven

BIOCHEMICAL

by

methionl

Hei

model

extracellular of

1050

267

ami

of

jne(

with

possible

putative

was cs

Von

of

ne

This

alignment a

structure 1n fol

acids

lowed

a

SI

gnal

IO) rat,

for

porcl the

that

ne

organi-

domaln no

the

di

of

335

spl

ays

Vol.

172, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. I The cDNA and predicted amino acid sequence of human ovarian receptor: Position f I is assigned to the first nucleoLH/hCG tide of th e putative i ni ti ator codon, Numberi ng of the nucl eotides (above) and of the amino acid (underneath) is shown The N-linked glycosylation sites potenti al i n the extracel I ul ar domai n are underlined. a putative sites for protein kinase C phospholyl aton are indicated by dotted line. The segment which the shorter form of receptor does not contain is boxed

1051

Vol.

IIIH/hCGR rLH/hCGR PIH/l’CGR hTSHR iFSHR

172,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

KTFnRDFF~LLSKFCCCKRReELYR------------RXD~~~~c~~~~~~~*~~~~~~~~~~~~~~~~~*~~~~~~~~~~ ..P AS. ..a... .Y OPIPPR*ITH * .L. R .., 5. “0 : -----------i 0 YSTVM c x0 * 8 .o 0.4. ------------GORVPPKN 1~~~0IOVOXVTHEMRCG.RNMEOVYEL!EKSRLTPXKOGOISEEYMOTY~ IETSS*IHNFH* SHCSS*.PRYI SVYLVPLRHS ON 1 Yi E ” ’ ..’ YE”0 01 ~---------~~ E --... Fig.2 Detailed Only human represented sitated

comparison non TSH

identical and

between rat

by introduction

ing of the transmembrane the extracellular

amino

a

the

amino acids FSH receptor Alignment dot. of gaps

acids segments domain

is are

LH/hCG,

TSH and FSH receptor porcine LH/hCG receptors. rat, shown, identical resdues are of homologous regions has necesrepresented by dashes. The numberof are

indicated boxed. are denoted

on Conserved by

the filled

3.0000

0.0000

!6

-3.006

Fig.3 Hydropathy hydropathy residues. ments.

plot profile I to

364

of Vll

human the of correspond

699 730 635 764 b92

LH/hCG LH/hCG to

receptor. receptor, the putative

1052

right. cysteine circle.

The putative residues

in

T 699

A Kyte and Doolittle with a wlndow of I9 transmembrane seg-

Vol.

172,

No.

seven

possible

of

674

primary

structure.

gy

TSH at

cysteins

of

the

LH,FSH

and crucial

cellular

70%

for

the

with of

lular

kinases

play

nergi

c

a

the

change

ones

to

encodi

suggests

ng

a

role

the

of

and

it

logical

ly

active in

data

the is

as the

previous

one

extra-

than

90%

approximately

putative

phospholylati ati

i t

high i ntraon

on

by

i s

of

important

these

f i c adre-

t 0

sites

by

specl

decoupling

of

a

of

open

causes

IT n 0 w any

of

whether

the or

paper(

1053

as

frame, This

These

LH/hCG

forms

reading

observed.

splicing. porcine

monomer

large

also

alternative

known a

large

A in

for

I ),

was

truncated not

might

receptor.

protein of

found

specific

containing

shorter

with

stood,

described

clones

the

more

phosphoryl

agonist

LH/hCG

mechanism

compatible The

of

addition

rice

in

at

I n

bonds

and

sites

ns(l

and

respectively. are

G-protei

domain.

conserved

exhibits

receptor

Si

gly-

transmembrane

are

receptor

consensus

phosphorylation

functional In

FSH

the

lular

of

y

receptors.

threonines

in from

disulfide

domain

(Flg.1).

receptors

whether

and

three

role

of i ty

LH/hCG

and

C

ns

hormone

al

part

cystei

i ntegr

of

homolo-

potenti

and

d

mate1

45%

NH2-terminal

formation

porcine

with

and

extracel

these

spanning

serines

kinase

rice

aci

level

approxi

receptor

the

no

should

the

share

extracellular

the

TSH

domain

protein

putative

and

ns

putative in

glycoprotein

rat

ogy

proportion ccl

the

membrane

with

domai

ami

protein

Six

conformational

of

putative

homoi

present

Si

receptor,

domain

homology

are

protein.

TSH

mature

respectively. the

72

On

LH/hCG

in

a

tons).

ar

porcine

found

between

domain

I ui

receptor

are

junction

The

extracel

dal

COMMUNICATIONS

is

The

(75632

and

FSH

sites of

be

acids

rat

and

i on

Clusters the

no

with

with

cosyl

ami

RESEARCH

There

domain.

the

homology

BIOPHYSICAL

segments.

intracellular

nal

consist

85%

AND

transmembrane

COOH-termi

cl

BIOCHEMICAL

3, 1990

pattern

results

are

receptor.

the

receptor

LH/hCG an

01

is receptor

i gomer

not is

which

underphysl we

ohave

Vol.

172,

In tions by

No.

3, 1990

BIOCHEMICAL

man,

the

TSH

that

lead

to

autoantl

receptor

of

bodies

in

ovari

This for

as an

of

Acknowl

or

fai

wel

I ure

human

I

be

the

and in

COMMUNICATIONS

autoimmune

of

in the

improvement

the

r eac-

thyroid

idiopathic

gland myxoedema.

understandlng

in

the

of on

progress as

RESEARCH

target

disease

requires

LH/hCG

BIOPHYSICAL

hypostimulati

Grave’s Thus,

physiology

tion

can

hyper-

respectively(l3). i an

AND

di

isolation

of

agnosis

and

and

the

ovar-

management

character

za-

receptor.

edgments work Science

was

supported and Culture

by

a

grant from Japan(02670732)

of

the

Ministry

of

and

Educatron

Uehara

Memori

al

Foundation.

References 1.

Dufau.

2.

36,461-593 Ri bei

ro-Neto,

3.

Mol. Lefkowi

Endocrinol. tz,

4.

4993-4996 Parmentier, ard,

6.

Misrahi, Mantel, Commun.

F.,

7.

N.,

8.

Rosemblit, (1989) Loosfel

9. 10. 1 1 12. 13.

Science t, M.T.V.,

525-528 Sanger, Acad. Hei ji Benovi R.J. Minegishi. Chem. Reces-Smi Endocrinol

L.

482-491 Caron,

M.G.

Libert,

Braun, (1990) C.,

H.,

Vitamine

Birnbaumer,

Perret. J., G.,(l989)Science M., Loosfelt, A., and Miigrom. 166, 394-403 R., P.H. K.

I

K.J.(1978)

1, and

R.J.

Sprengel, Seeburg. McFarland,

Thi Jallal,

Catt,

M., C.,

Vassart, 5.

and

M.L.

F., Van

Field,

(1988)

J.

Maenhaut,

Nikolics. Endocrinol. R., K.,

494-499 ,

and

Bio.

C.,

M.

I

K., Phillips.

Chem.

S.,

Sal

Kohler,

esse, A.,

Milgrom,

E.

F.,

Nichlen,

S.,

and

Coulson,

A.R.

(1977)

14, M.G.. 83, M.L.(1987)

4683-4686 and 2797-2801

T., Kusuda, 17138-17143

262, th,

B., Rev.

9,

Mclachlan, 106-121

S.,

Res.

and S.M.,

1054

USA

Dufau, and

and M.,

Seeburg,

and

Caron, Sci.

Res.

D.L.,

B.,

Acids.

Ger-

and

GuiochonBiophys.

A., J.,

R.H., Acad.

263, A.,

J.E.

Jolivet, Garnier.

Sci USA, 74, 5463-5467 I G.V. (1986) Nucleic c, J.L., Strasser, Natl. (1986) Proc.

(1987)

Lefort,

D.L., M.,

B.

Segaloff, 525-530 H.S.,

Segaloff,

Atger, Guiochon-Mantel,

Hormones J,

Sande, J., Damont, 246, 1620-1622 H., Atger, M., Sar, E., (1990) Biochem.

T., Mol. Sprengel,

Nikolics. 245, Misrahi

and

R. I Sar

(1989)

Furmanai

P.H. Hal-Lau S.,

Science

245,

Proc.

Nat

Lefkowitz J. k.

Biol. J.

(1988)