Purification, Cloning, and Expression of the Prolactin Receptor'

BIOLOGY

OF

REPRODUCTION

40,

27-3

Purification, PAUL

A. KELLY,2’3

MARIKO

2 (1989)

Cloning,

JEAN-MARIE

SHIROTA,3

and Expression BOUTIN,3

MARC

EDERY,4

CHRISTINE ISABELLE

Laboratory

of the Prolactin

Receptor’

JOLICOEUR,3

HIROAKI

DUSANTER-FOURT,4

of Molecular

and

OKAMURA,3 JEAN

DJIANE4

Endocrinology3

McGill University Royal Victoria Hospital Montreal,

Quebec

Canada

H3A and d ‘Endocrinologie

Unite Institut

National

1A1 Mol#{233}culaire4

de la Recherche

CRJJ,

78350

Agronomique

Jouy-en-Josas

France ABSTRACT The

rat

liver

prolactin

receptor

has

been

purified

to

homogeneity,

and

partial

amino

aicd

sequences

have

been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 receptor show regions of suggesting

that

and 598 amino strong sequence

the prolactin

and growth

acids, respectively). identity with the

hormone

receptors

INTRODUCTION The

anterior

primarily

pituitary

hormone

responsible

mammary number

for

the

gland and lactation. of other reproductive

in nonhuman

species.

In the

prolactin

(PRL)

development However, functions,

of

gland,

monoclonal receptor

a

and itself

the

prolac-

addition

to

the

with

specific,

classical

effects

in

as well as other by the interaction high-affinity

the

mammary

located

plasma membrane and widely distributed of tissues (Posner et al., 1974a, Djiane Different biochemical approaches immunoprecipitation, or immunoblot

on

2

Royal 1A1,

Victoria Canada.

Hospital,

687

Pine

West,

al.,

1984,

in different

tissues.

Prolactin

the

in a number et al., 1977). (cross-linking, analysis with

Ave.

et

ancestor.

have shown that molecular weight

1985,

1987;

levels In rat

pregnancy plays

-

(Kelly by

one

et

al.,

role

1984;

regulated

of the

tissues

with

receptor levels vary et al., 1975), increase

et al.,

estrogens

a major

Kelly

bonds to al., 1983;

are differentially liver,

prolactin binding, estrous cycle (Kelly

stimulated

the prolactin of 40,000

not linked by disulfide subunits (Haeuptle et

1984). receptor

edly

1974), (Posner

in

the

own receptor, inducing both updepending on the concentration

and

and et

are a!.,

mark1974b).

regulation

of its

down-regulation, and duration

of

exposure to prolactin (Posner et al., 1975; Manni a!., 1978; Djiane et al., 1979). As is true for growth hormone (GH), no means signal transduction has been There are no clear effects adenosine 3’, 5’-monophosphate guanosine 5’ -monophosphate pholipids, phosphorylation,

work was supported in part by grants from the Medical ReCouncil of Canada and the National Cancer Institute of Canada. Reprint requests: Dr. Paul A. Kelly, Laboratory of Molecular En-

docrinology, Quebec H3A

apparently to other

Sakai et al., Prolactin

during

‘This search

or

a common

antibodies) a relative

has

the highest during the

effects of prolacof the hormone

receptors

from

is

Katoh

tin stimulates the expression of milk protein genes by increasing both gene transcription and mRNA halflife (Guyette et a!., 1979; Houdebine et al., 1985). In gland, reproductive tin are initiated

originate

is

it also has especially

mammary

Both the short and long forms of the prolactin human and rabbit growth hormone receptors,

Montreal,

27

identified for prolactin. of prolactin on cyclic (cAMP), cyclic (cGMP), inositol phosCa2, or ion channels

et of

28

KELLY

(Matusik bine

and

et

a!.,

ceptor

Rosen, 1985).

appears

1980;

Kelly

Neither to

be

et a!.,

the a

GH

tyrosine

1984;

nor

pletely

Houde-

the

PRL

kinase.

A

ET AL.

re-

When

better

disappears

carried is

out

a partially

run

on

understanding involved in

of prolactin receptor structure, regions hormone binding, signal transduction,

Western

and tors

homology shed some

antibody, (Fig. 1B).

possible should

which In

prolactin the present

induces report,

with light

other hormone on the mechanism

its various actions. we describe the

zation, purification, cloning, and expression of the prolactin its structure with PRL receptors lated from hepatoma that

have

(Leung

et al.,

been

characteri-

amino acid sequence receptor and compare we have recently iso-

the rabbit mammary as well as with rabbit

ceptors

recepby

recently

gland and and human cloned

and

human GH re-

sequenced

1987).

CHARACTERIZATION

AND

PURIFICATION

The

PRL

receptor

weight (Mr4O,OOO) the characterization [‘25]I-oPRL to specific band with the hormone-receptor

in rat

liver

is a small

protein. Figure of the receptor hepatic membrane

an Mr of 61,000 corresponding complex is seen, which

A

to corn-

analyzed

using

by

a monoclonal

of the receptor is 41,000 amounts of PRL receptor were

solubilized, estrogen-treated preparations (Katoh et 6 mg of partially purified

and was

to homogeneity after reduction

the protein electroeluted.

band

homogeneous

PRL

analysis.

identified. From three oligonucleotide

B

female rat al., 1987). receptor

in

the

by polyacrylaand alkyla38,000-43,000

receptor

was

reduced

and

Ten the

readable

sequences

first few sequences probes were prepared

were obtained, (Boutin

1988).

Initial library

10

receptor

M

CLONING

MrX

and

1).

(-j

sequence

et al.,

A

of PRL

gel

analysis

is

(Fig.

alkylated and digested exhaustively with trypsin. The resulting peptides were purified on reverse-phase high-performance liquid chromatography (HPLC). Amino acid sequences were obtained by gas-phase

molecular

1 demonstrates by cross-linking receptors.

polyacrylamide

the apparent Preparative

reaction oPRL

preparation

receptor were purified mide gel electrophoresis

The PRL RECEPTOR

purified

binding

of excess

prepared by immunoaffinity-chromatography E2 1, a monoclonal antibody specific to the rat receptor. Preparative amounts 100 i.tg) of

tion, region

OF THE

a

the

presence

immunoblot

purified from liver membrane Approximately were using PRL

when

in the

RECEPTOR

screening with

of

a

cDNA

polysome-enriched

oligonucleotide

Xgtll

PRL-2A

12 5 ‘GT -3

MrXIO

3

detected

TGAT

positive

TCCCA

66-41

31-

bp) to

TC-3’

phages,

contained the peptide probe, plus

clones

El

the

entire used to the regions

corresponding to two flanking tryptic fragments. Insert E2 was shorter and was included within the sequence of El. A longer cDNA clone (F3) was subsequently isolated from a library prepared using size-selected clone El

mRNA as an RNA

screened). A restriction Figure 2. The hepatic FIG. 1. Prolactin receptor characterization: sodium dodecylsulfatepolyacrylamide gel electrophoresis. A) Cross-linked E’2511-ovine prolactin (oPRL) to bound membrane (microsomal) PRL receptor from the rat liver in the absence (Lane 1) and presence (Lane 2) of an excess of unlabelled 0PRL. The PRL-receptor complex migrates with an Mr of 61,000. B) Partially purified receptor revealed by Western immunoblot analysis using a monoclonal antibody (E21) against the rat liver PRL receptor (Lane 3) (Boutin et al., 1988).

TC

recombinant

and E2. Insert El (93 sequence corresponding prepare the oligonucleotide

97-

43-

two

AA

messenger 1635 bp

from probe

estrogen-treated (2.5 X l0

map of clone PRL receptor

-2.2 kb in length are included in the

(see insert

liver with recombinants F3 is shown in is encoded by a

Fig. 4), of which F3, containing a

single open reading frame that corresponds to a protein of 310 amino acids (Boutin et al., 1988). The initial methionine is followed by a 19hydrophobic amino acid stretch indicative of a signal

THE

PROLACTIN

TT

.1 II

I.

500

0

1000

3’

1500

FIG. box tion

(PRL)

2. Prolactin

indicates sites are

receptor

F3

El cDNA

restriction

the predicted coding region of clone indicated, as well as the localization

map.

The

F3. Unique of El (Boutin

blot analysis glycosylation,

purified receptor trifluoromethanesulfonic an Mr fragments

______________________ -

29

Western for by

-

5’

RECEPTOR

hatched restricet al.,

1988).

of

sequence

attempts

to

(Gascuel obtain

unsuccessful, which 20 may be present receptor

thus

the

and

Danchin,

N-terminal

suggests that as a pyro-Glu

corresponds

protein

with

a theoretical

between and the

the predicted Mr Mr of the hepatic

to Mr

sequence

Two were

the GIn at position residue. The mature a

of

1986).

291-

33,368.

amino

acid

Differences

of the F3-encoded protein PRL receptor detected by

34,000 previously

were

located

from

F3

to

the

strongly (230-25

to the separating region

plasmic

region,

and

the

ASN

an

tissue tissues

RNA

234

-r--

acids region

region of the extracellular short

C-terminal

to probe

BLOT

ANALYSIS

derived

from

of mRNA This figure

PRL PRL cyto-

El

were

Large amounts used to clearly

of Poly demonstrate

(A)’

of receptor mRNA relative abundance

the

cDNA

El,

was analyzed in 17 shows that mRNAs present

that previously had been shown receptors and were absent in all other

only

in tissues

to contain PRL tissues examined.

mRNA this fact

(100 zg) and confirm

were the

in negative tissues. of the mRNAs correlated

well with specific binding (Posner et al., 1974a; Kelly

of PRL in these tissues et a!., 1984). Indeed, the

strongest

in

signal

was

found

the

liver,

and a weak signal expressing the PRL

followed

by

was present receptor,

in in-

cluding the mammary gland. Moreover, the level of expression was hormone-dependent in liver, since the signal was weak or absent in hypophysectomized and normal males, higher in normal females, and markedly (Posner ple

increased et al., 1974b,

As is the case for forms of mRNAs

only one found species in the FIG. 3. Schematic representation of the rat liver prolactin (PRL) receptor. The localization of the three Asn-glycosylation sites and the five Cys residues in the extracellular domain are indicated.

other

amino this

it probably is involved in signal 3 schematically demonstrates rat liver prolactin PRL receptor.

probe

distribution (Fig. 4).

ovary, and prostate, all other tissues

211

In addione

illustrated.

hybridizing

(203)

sequence

of the five Cys residues (amino acid 31, and 203) and the three potential Asn sites (amino acids 54, 99, and 127) are

absence The

-CYS

tryptic sequence

The location 41, 70, 81, glycosylation

(41)

(127)-

peptide,

with with

Figure of the

(31)

ASN ( 99)-

signal

very

All

protein

mismatches.

transmembrane the N-terminal,

from

F or a band

transduction. the structure

the rat

(70) (81)

the any

hydrophobic region of 24 3) was observed. Presumably

With

-CYS -CYS

in

accounted of highly

shown). by protein

within

without

hydrophobic

binding

1

ASN ( 54)-

be

endoclycosidase acid results

NORTHERN

-CYS -CYS

might treatment

(data not identified

deduced

corresponds receptor, peptide

with

analysis tion

(41,000) since

band form

was was

seen the

in estrogen-treated 1975; Djiane et a!., other exist.

in the prostate

hormone The 2.2

liver and

female 1979).

receptors, kb band

rats

multiwas the

and was clearly a major ovary. A slightly larger

in the adrenal. Another major mRNA 4 kb band, which is the predominant

KELLY

30

ET AL.

.2 o

52

M

-

...,

)

)_

-

.2

.

N

52

.4

5252

...‘

0

53

*1:

A

#{149}t.’.

-

‘

V.

-

-2.8

.

-.

-2.1 -1.7

.

-1.3

-0.6

FIG. 4. Northern blot analysis of prolactin receptor mRNA expression. Northern blot of 17 rat mentary to clone El (93 bp). Poly (Aye mRNA (100 big) was used in each lane. The film was exposed for 36 h with an intensifying screen to detect low-abundance mRNA5 in testis, kidney, adrenal, and son, all lanes have been mounted on the same figure (Boutin et al., 1988).

form

in the

were testis,

also the

kidney seen signal

and

mammary

were seen at 2.2 and mRNA was detected et a!.,

gland.

Larger

in the ovary and the adrenal. was very weak, although faint 4 kb. only

In addition, in this tissue

forms

in

In the bands

These curves ity confirming

CHO

cells

tissues analyzed with an RNA probe complefor 16 h. The same blot was autoradiographed mammary gland. For convenience of compari-

stably

transfected

demonstrate

with

classical

that

clone

pKCR2/F3.

hormone

F3

encoded

specificthe

PRL

a 0.6 kb (Boutin

1988).

120 A

EXPRESSION

A

To the

determine

whether

synthesis

of

heterologous

and vector

clone

functional

cell

ent vectors Bluescript

the

system,

F3

F3

would

rat

PRL

was

inserted

direct

receptor

expressed in various with its transcription

in

into

0

a

differ-

cell types. promoter

0

The T3

0

was used to synthesize F3 mRNA, which was microinjected into Xenopus oocytes. The expression of the PRL receptor at the cell surface was shown by significant

specific

of

[‘25I]-PRL

(data

the

transiently

and

PRL

stably

containing and Transient

receptor

was

expressed

in mammalian

the promoter polyadenylation transfactions

cells

by

conferring

G4l8.

Similar

shows

the

and competition

stable curve

resistance results

were

transfections. of

[‘25I]-oPRL

to

the

rPRL

.-u

hPL oGH

0

region, origin of signal of the SV4O were performed in

neo

pSV2 analog

#{149}-. hGH a’c

INSULIN

using

plasmid

transient

a-c oPRL

z

A-a

both

kidney cells using the pECE/F3 stable CHO cell lines were established of the pKCR/F3 construct with the

both

60

not

COS-7 monkey construct, and by cotransfection mycin

80

40

shown). In addition, vectors replication, genome.

binding

100

0 .01

HORMONE

neo-

observed

for

Figure binding

20

5

.1

1

10

IN INCUBATION

100

1000

(ng/tube)

FIG. 5. Expression of rat liver prolactin (PRL) receptor/F3 CHO cells stably transfected with pKCR2/F3. Values are a percentage of specific binding calculated in the absence beled hormone (Boutin et al., 1988).

.:DNA in expressed as of any unla-

THE

receptor.

Transformation

Scatchard

analysis

extracts with an liver

of both affinity

PRL

of the

PRL

binding

demonstrates COS similar

receptor

that

data

by

membrane

7 and CHO cells to that reported

(Posner

PROLACTIN

bound PRL for the rat

et al. (1974b;

Kelly

et a!.

1983). REGULATION

OF PRL

RECEPTOR

EXPRESSION

GENE

Sex steroids are major regulators expression in rat liver. Using a probe

of PRL encoding

receptor the rat

PRL

receptor

mRNA

receptor

levels

in

(F3), female

response

to

we rat

studied liver

estrogen

PRL during

and

androgen

ontogeny

and

treatment.

Steady

PRL mRNA

analysis. receptor levels

stimulated

From

prepuberty

numbers increased

PRL

to

increased 8-fold, 3-fold. Estrogen

receptor

numbers

6-fold,

levels were only increased 3-fold. androgen administration, the number tors in rats previously 3-fold, but mRNA These

results

pression or

with only

estrogen declined

that

PRL

receptor

liver

is regulated

post-transcriptional

tional

level

level

(unpublished

at the as well

form of the rat mammary

which

multiple

single ever,

decreased 1.5-fold.

(Fig. 4). mechanism GH

receptors

moto

et

1987)

receptors

al.

transferin, respectively. milk,

cerebral

clone

F3

used

as a prove,

splicing is the the different

thus

of

a part

receptors. Howkinase activity nor

of

the

structural

IGF-II/M6P primarily

and prolactin fluid,

short

PRL

arrangement

et a!. 1984),

act

of the

LDL

(Yama-

(Morgan

et

as transporters

mannose 6-phosphate, has been detected and

semen.

This

al. for in sug-

transcriptional

as at the

PRL RECEPTOR

transla-

RABBiT

Gli RECEPTOR RUMAI4

HUMAN/RABBIT

uvu

MAIPATOMA

STRUCTURE 1_

With

been

site in the cytoplasmic growth factor receptors.

segment

spinal

have

ex-

RAT

RECEPTOR

are

of the

1984),and cholesterol In fact,

RNA for

class tyrosine

(Schneider that

both The

appear.

cytoplasmic

results).

OF PRL

that

is reminiscent

of

in the

in which identified.

mRNAs

Alternative responsible

receptor

transferin

species been

of receptor

a potential phosphorylation domain, as is true for many short

form

identified

is probably also present and in other tissues in

and

PRL

second

been

first have

membrane-spanning they do not possess

uvu COMPARISON

has

receptor gland,

forms

of the

this

(PRL-R2)

longer in the

Both

GH receptors in this of both the rabbit and

domain

making it the the receptor

identified probable

and

PRL

Interestingly,

receptor

rat ovary, forms of

of the

mRNA

gene

receptors. PRL

receptor

whereas treatment but

the

The

response to of PRL recep-

treated levels

suggest

in rat

In

between the cytoplasmic

human

in

adulthood,

31

identity extended

forms

state mRNA levels were determined by Northern blot analysis and densitometric analysis. Messenger RNA levels were compared to receptor numbers assessed by Scatchard

RECEPTOR

cDNAs

1_

1

encoding

the prolactin receptor from rabbit mammary gland and human hepatoma have just been isolated. In addition, cDNAs encoding the PRL receptor in rat mammary gland, ovary, fied recently. A second appears rabbit

to be the predominant and human (Fig. 4). The

a 592-amino acid prolactin receptor ever, a much longer PRL receptor (598 structure second size to

and kidney form of

have been identithe PRL receptor

species in both rabbit PRL receptor

the is

211

#{149}..

211

291

protein highly similar to the rat (Edery et al., 1989), with, howcytoplasmic domain. The human amino acids) has a similar overall

to the rabbit mammary receptor. The form of the PRL receptor is much closer in the rabbit and human GH receptor, which has

recently been cloned and sequenced (Leung et a!., 1987). In addition to the highly conserved sequence just past the transmembrane segment (Boutin et a!., 1988),

211

there

are 3 additional

regions

of strong

sequence

592 FIG. 6. Schematic comparison rat liver, rabbit mammary gland, rabbit or human growth hormone

820 of the prolactin (PRL) receptor and human hepatoma compared (GH) receptor.

from to the

KELLY

32 gests

that

and

testis-where

in

the

localized-that their normal

mammary

these function

transport proteins, one compartment investigate

The the

localized

GH

mains Whether

await

than

regions

and

PRL

suggests

form

where

membrane transfer

more

in the

important alone

been

may, in addition transduction, act

of sequence

regions,

mutagenesis

one

plexus, have

the hormone It will be interesting

receptors some

these

some other ble for the

receptors of signal

in tissues

choriod

receptors

translocating to another.

if more

tor is present occurs.

gland,

prolactin

to as

from to

of the

PRL

recep-

than

one

action

identity cytoplasmic

between do-

functional

role.

or in combination

with

of

component the hormonal

studies

in functional

(s)

are responsimessage must systems.

ACKNOWLEDGMENTS These studies were supported in part by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. We gratefully acknowledge the technical support of Sally Raguet, Joseph zachwieja and Rejean Melan#{231}on. We also thank Nirmala Brunel for typing the manuscript.

REFERENCES JM, Jolicoeur C, Okamura H, Gagnon J, Edery M, Shirota M, Banville D, Dusanter-Fourt I, Djiane J, Kelly PA, 1988. Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family. Cell 53:69-77 Djiane J, Clauser H, Kelly PA, 1979. Rapid down-regulation of prolactin receptors in mammary gland and liver. Biochem Biophys Res Commun 90:1371 -78 Djiane J, Durand P. Kelly PA, 1977. Evolution of prolactin receptors in rabbit mammary gland during pregnancy and lactation. Endocrinology 100:1348-56 Edery M, Jolicoeur C, Levi-Meyrueis C, Dusanter-Fourt I, P#{233}tridou B, Boutin

Boutin J-M, Lesueur L, Kelly PA, Djiane J, 1989. Identification and sequence analysis of a second form of prolactin receptor by molecular cloning of complementary DNA from rabbit mainmary gland. Proc Nati Acad Sci (USA): (In press) Gascuel 0, Danchin A, 1986. Protein export in prokaryotes and eukaryotes: indications of a difference in the mechanism of exportation. J Mol Evol 24:130-42 Guyette WA, Matusik RJ, Rosen JM, 1979. Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell 17:1013-23 Haeuptle MT. Aubert ML, Djiane J, Kraehenbuhl JP, 1983. Binding

ET AL. sites for lactogenic and somatogenic hormones from rabbit mammary gland and liver. J Biol Chem 258:305-14 Houdebine LM, Djiane J, Dusanter-Fourt I, Martel P, Kelly PA, Devinoy E, Servely JL, 1985. Hormonal action controlling mammary activity. J Dairy Sci 68:489-500 Katoh M, Djiane J, Kelly PA, 1985. Prolactin binding components in rabbit mammary gland: characterization by partial purification and affinity labelling. Endocrinology 116:2612-20 Katoh M, Djiane J, Leblanc G, Kelly PA, 1984. Characterization of antisera to a partially purified prolactin receptor: effect on binding in different target tissues. Mol Cell Endocr 34:191 -200 Katoh M, Raguet S. Zachwieja J, Djiane J, Kelly PA, 1987. Hepatic prolactin receptors in the rat: characterization using monoclonal antireceptor antibody. Endocrinology 120:739-49 Kelly PA, Djiane J, Katoh M, Ferland LH, Houdebine LM, Teyssot B, Dusanter-Fourt I, 1984. The interaction of prolactin with its receptors in target tissues and its mechanism of action. Recent Prog Horm Res 40:379-436 Kelly PA, Djiane J, Leblanc G, 1983. Preferential establishment of a slowly dissociable component in plasma membrane compared to intracellular prolactin receptors. Proc Soc Exp Biol Med 172: 219-24 Kelly PA, Posner BI, Friesen HG, 1975. Effects of hypophysectomy, ovariectomy, and cycloheximide on specific binding sites for lactogenic hormones in rat liver. Endocrinology 97:1408-15 Kelly PA, Posner BI, Tsushima T, Friesen HG, 1974. Studies of insulin, growth, hormone, and prolactin binding: onrogenesis, effects of sex and pregnancy. Endocrinology 96:532-39 Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters Mi, Wood WI, 1987. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature (Lond) 330:537-43 Manni A, Chambers MJ, Pearson OH, 1978. Prolactin induces its own receptors in rat liver. Endocrinology 103:2168-71 Matusik RJ, Rosen JM, 1980. Prolactin regulation of casein gene expression: possible mediators. Endocrinology 106:252-59 Morgan DO, Edman JC, Standring FN, Fried VA, Smith MC, Roth RA, Rutter Wi, 1987. Insulin-like growth factor II receptor as a multifunctional binding protein. Nature (Lond) 329:301-07 Posner

BI, Kelly PA, Friesen HG, 1974b. Induction of a lactogenic receptor in rat liver: influence of estrogen and the pituitary. Proc NatI Acad Sci USA 71:2407-10 Posner B!, Kelly PA, Friesen HG, 1975. Prolactin receptors in rat liver: possible induction by prolactin. Science 188:57-59 Posner BI, Kelly PA, Shiu RPC, Friesen HG, 1974a. Studies of insulin growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 96:521-31 Sakai S. Katoh M, Berthon P, Kelly PA, 1984. Characterization of prolactin receptors in porcine mammary gland. Biochem J 224: 911-22 Schneider C, Owen MJ, Banville D, Williams JG, 1984. Primary structure of human transferrin receptor deduced from the mRNA sequence. Nature (Lond) 311:675-78 Yamamoto T, Davis CG, Brown MS. Schneider WJ, Casey ML, Goldstein JL, Russell DW, 1984. The human LDL receptor: a systeinerich protein with multiple alu sequences in its mRNA. Cell 39: 27-38