263 Gene, 134 (1993) 263-265

Elsevier Science Publishers B.V.

GENE 07447

Cloning and sequencing of the cDNA encoding the murine mammary gland long-form prolactin receptor*

(Mus musculus; h phage library; RT-PCR; recombinant DNA)

Robert C. Moore and Takami Oka

Laboratory ofMolecular and Cellular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8, Room 319. 9000 Rockoille Pike, Bethesda, MD 20892, USA

Received by J. Piatigorsky: 8 April 1993; Revised/Accepted: 28 June 1993; Received at publishers: 23 July 1993

SUMMARY

The nucleotide sequence of a 1992-bp cDNA encoding the long form of the murine mammary prolactin receptor

(PRL-R) has been determined. The deduced 68-kDa protein has high sequence identity with long forms of prolactin

receptors from rat ovary and rabbit mammary gland.

INTRODUCTION

Prolactin is a peptide hormone which has multiple bio-

logical functions including the regulation of milk pro-

duction in the mammary gland. The effect of PRL is

mediated by the interaction of the hormone and its

plasma membrane receptor. Both short and long forms

of PRL-R cDNA have been identified that appear to be

the result of alternative splicing of transcripts. cDNAs

encoding three PRL-R short forms from mouse liver

(Davis and Linzer, 1989) and rat liver (Boutin et al., 1989)

have been cloned. Long form PRL-R cDNAs have been

isolated from rat (Shirota et al., 1990), rabbit (Edery et al.,

1989), bovine (Scott et al., 1992) and the human hepa-

toma cell line HepG2 (Boutin et al., 1989). Mammary

Correspondence to: Dr. T. Oka, Laboratory of Molecular and Cellular

Biology, National Institute of Diabetes, and Digestive and Kidney

Diseases, National Institutes of Health, Building 8, Room 319, 9000

Rockville Pike, Bethesda, MD 20892, USA. Tel. (l-301) 496-1404; Fax

(l-301) 402-0053; e-mail: moorer@ncifcrf.gov

*On request, the authors will supply detailed experimental evidence for

the conclusions reached in this Short Communication.

Abbreviations: aa, amino acid(s); bp, base pairs(s); kb, kilobase or

1000 bp; nt, nucleotide(s); PCR, polymerase chain reaction;

PRL, prolactin; PRL-R, PRL receptor(s); RT-PCR, reverse tran-

scriptase-PCR; TM. transmembrane.

glands of lactating mice produce the highest level of long

form PRL-R mRNA, compared to those of virgin and

pregnant mice (Nishikawa et al., 1993). Transfection ex-

periments have shown that the long form of PRL-R from

rabbit mammary gland is capable of transducing a signal

to milk protein genes (Lesueur et al., 1991) while the

role of the short form has yet to be determined.

EXPERIMENTAL AND DISCUSSION

(a) Isolation of mouse mammary gland long form PRL-R

cDNA

A mouse mammary gland library was prepared from

mRNA purified from a lactating C3H mouse. A cDNA

encoding the long form of PRL-R was isolated and its nt

sequence determined. A comparison of the deduced aa

sequences of the mouse mammary and rat ovary PRL-R

suggested that the cDNA clone lacked seven nt of the S-

end of the open reading frame. A DNA fragment contain-

ing part of the 5’-untranslated region and the first 802 bp

of the PRL-R coding region was amplified by RT-PCR

and used to construct a full length PRL-R cDNA. The

deduced mouse mammary PRL-R precursor consists of

a polypeptide of 608 aa. Analysis of this sequence by the

method of von Heijne (1986) predicts that the PRL-R

264

I”SII”“E~LISSPGDTLGSF‘GKAG~NV~K~SE~AGSGE GTGG~TGTGCMGMCGGGCUGCTTCCCTTCT~~~~~TCT~GGC~~CTCC~~G~~~TTGTCTATGCT~CCCC~~~TACGTG~~TT~C VAVGEGAKSFPSDKPWTSYPPLGEKGPIVYAKPPD~VEIH ~GTCMC~OLCGWIGTGCTATCATTACTCCC~G~~~~CUCCA~~~CCCTGGGGTTCCT~CCAGTMG~GTATGC~GGTATCTGGGGT~~~T 1:: K~NKDGVLSLLPKGRENHGTENPGVPETSKEVAKVSGVTLI 540 AA~AICAT~~TGGTGTTAGTGC~GACTCACGAGCCCA~CA~GCTTTGCTT~G~TCAGCCMGMGGTTCCACCATCGCTT~~~CCMTCTGAG~~TCTGGCCAGC 1798 RNILVLVPDSRAGNTALLEESAKKVPPSLE~R~~EKGLAS 580 TTTA~TG~~~T~~GWUCTGCAWCTCCMCTCCMCTGGG~GGCTG~TTACCTG~TCCTACGTGCTTCATG~CTCCTTT~CTGA~GCTA~CTTAT~CA~TT~CT~T~ 1918 F TATS S N C R L G L G R L D V CD P T C F NH S F H 1** 608

W\TTTCTCTTUCGTMCACTA~~GT~T~GTMTGTGGTCTGCTM~TGTTA~G~TGTG~TAT 1992

Fig. I. The nt sequence of the mouse mammary PRL-R cDNA and the deduced aa sequence. Sequence encoding predicted TM region is underlined.

Three asterisks indicate a stop codon. GenBank accession number is L13593. The mouse PRL-R sequence was obtained by: (a) isolation of a clone

(RM236) from a mouse mammary cDNA library constructed in kZAPl1 using a 32P-labeled PCR fragment encoding the receptor intracellular

domain; (b) sequencing RM236 after phagemid rescue using helper phage R408; (c) RT-PCR amplification of the missing 5’-end of the coding region

using mammary RNA and primers to the 5’-untranslated region of mouse liver PRL-R (Davis and Linzer, 1989) and RM236 coding sequence;

(d) exchange of restriction fragments to create RM252 and sequencing the complete nt sequence of clone RM252. Additional RT-PCR experiments

using long form specific primers confirmed the sequence and identity of the 5’-end of the cDNA. Methods: PRL-R cDNA was amplified in a final

volume of 100 pi in a I x PCR buffer (50 mM KCljlO mM TrisHCI (pH 8.3)/1.5 mM MgCl,) containing 0.2 mM each dNTPjlO0 ng each primer/phage

h cDNA/2.5 units of Taq polymerase. A 1217-bp PRL-R probe was synthesized by amplifying AZAPII library cDNA with PRL-R specific upstream

primer 8 (T-TATCTTGTCCAGACTCGCTGCAAGC) and downstream primer 12 (S-CACGTAGGATCCAGGTAATCCAGCC). The PCR program

was as follows: denaturation at 94-C for 1 min, annealing of primers at 60°C for 1 min and elongation at 72°C for 2 min. cDNA was synthesized

from mammary gland total RNA in a final volume of 20 ~1 in a I x PCR buffer containing 1 mM each dNTP/l unit of RNase inhibiter/5.2 pg

total RNA/random hexamers (2.5 pM112.5 units of Moloney Murine leukemia virus RT (Perkins Elmer Cetus). PRL-R cDNA was amplified

using upstream primer 24 (5’-GATCTAGAGAAGGGAGCCTCTGATCTATTGCCT) and downstream primer 25 (S-GATCTAGACAGG-

AACTGGTGGAAAGATGCAGGT) or downstream primer 68 (5’-ACTTCCAAGAATTCCACCAGCAAGT). Primers 24 and 25 contain a 5’-XbaI

site and primer 68 contains a single nt mutation (C-*T) to introduce an EcoRI site to facilitate cloning. PRL-R cDNA was amplified by PCR as

described above.

contains a 19-aa signal sequence. The mature receptor is

composed of 589 aa with a 210”aa extracellular domain,

a single 24-aa transmembrane (TM) region and a 355-aa

cytoplasmic domain (Fig. 1). The 3’-untranslated region

of the mouse mammary PRL-R cDNA sequence does not

have the polyadenylation ~onseusus SHp_li?Il~~

(AATAAA), as was the case with the rat ovary (Shirota

et al., 1990) and bovine (Scott et al., 1992) PRL-R cDNA

sequences.

(b) Sequence analysis

Long and short forms of PRL-R have highly conserved

extracellular and TM domains; i.e., the first 280 aa of the

PRL-R long form are identical to the mouse liver short

form. Among long forms, the mouse mammary receptor

shares 91% (of 610 aa) and 72% (of 616 aa) identity with

rat and rabbit receptors, respectively. The most conserved

region of the cytoplasm& domains of all forms of PRL-

R is adjacent to the TM region. All differences in peptide

sequence of the mature PRL-R that are due to the addi-

tion or deletion of aa are clustered within the central

region of the intracellular domain, from aa 384 to 524

(Fig. 2). This variable region of the receptor appears to

be dispensable because the PRL-R of the Nb2 rat lym-

phoma cell line, which lacks this region, is still capable

of stimulating milk protein gene expression (Ali et al.,

$8” N”“*IPPTPNPQ*l++xNNTPNCHTDTSRSTTWP*”LP”P Rt .***....vD.. *****GTN..F.V.RP..S...**.L*....“P......”........ P.B .**‘VTH.WD..TISLVDREI.YLGVNO...G...L”.Q*....NTN....N...~....T B0 .‘**LTCLQA..STSMGKI.YFLANGP..S...FPQ.*.SLYSP.YS..N.....E..L Hu THTWD.PCISHg**~'"GKI.YF.AGG..CS...*"..Q.S..NP..S..N.T...E..V

SO GHAG.TATS..QT~HA."**.A.KTIET.R.GK.TX*Q*RBSEGC.S'.PD.D.V..RP Hu G.AGAPATL.M(.CMA.g**.S.~IKSR..GX.T.+QREW..H.ETD"*.D.P.LLP

f$ QEKGPIVYAKPPDYVEIHKDGVLSLLPKQREN*HQTENPG 524 . ..S.T..V...................F......'N...K.. 526

Rb K..P.FISP..L............A....L..K..GD..GKA. 532 BO .D.T.LIS...tE.......SQ....A.F...N.R*P*****. 535 nu . ..T.FGS...L............A..........SGKPKK.. 538

Fig. 2. Comparison of variable regions in the intracellular domains of

long form PRL-R. Deduced aa 384-524 of mouse (Mu) mammary PRL-

R is compared to corresponding aa of rat (Rt) ovary (Shirota et al.,

1990), rabbit (Rb) mammary gland (Edery et al., 1989), bovine (Bo)

(Scott et al., 1992) and human (Hu) HepG2 cell (Boutin et al., 1989)

PRL-R. Dot indicates identical aa, asterisk indicates addition or dele-

tion of aa.

265

1991; 1992). The cDNA clone described here will be

useful in determining the domains through which the

mouse mammary long form PRL-R regulates the tran-

scription of milk protein genes.

(c) Conclusions

We have isolated and sequenced the mouse mammary

long form of the PRL-R cDNA. The mouse mammary

PRL-R is homologous to long forms of PRL-R from rat,

rabbit, bovine and human. The central region (aa

384-524) of the mouse mammary PRL-R intracellular

domain is the least conserved among the long forms of

PRL-R.

ACKNOWLEDGEMENTS

We would like to thank Drs. Lee Burns, Roland Owens

and Frank Tietze for critical reading of the manuscript.

We also thank Drs. Norio Nonomura and Seiji

Nishikawa for their helpful suggestions.

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