Functional Characterization of the Intermediate Isoform of the Human Prolactin Receptor

Abstract

Prolactin-dependent signaling occurs as the result of ligand-induced dimerization of the prolactin receptor (PRLr). While three PRLr isoforms have been characterized in the rat, studies have suggested the existence of several human isoforms in breast carcinoma species and normal tissues. Reverse transcription polymerase chain reaction was performed on mRNA isolated from the breast carcinoma cell line T47D, revealing two predominant receptor isoforms: the previously described long PRLr and a novel human intermediate PRLr. The nucleotide sequence of the intermediate isoform was found to be identical to the long isoform except for a 573-base pair deletion occurring at a consensus splice site, resulting in a frameshift and truncated intracytoplasmic domain. Scatchard analysis of the intermediate PRLr revealed an affinity for PRL comparable with the long PRLr. While Ba/F3 transfectants expressing the long PRLr proliferated in response to PRL, intermediate PRLr transfectants exhibited modest incorporation of [3H]thymidine. Significantly, however, both the long and intermediate PRLr were equivalent in their inhibition of apoptosis of the Ba/F3 transfectants after PRL treatment. The activation of proximal signaling molecules also differed between isoforms. Upon ligand binding, Jak2 and Fyn were activated in CHO-K1 cells transiently transfected with the long PRLr. In contrast, the intermediate PRLr transfectants showed equivalent levels of Jak2 activation but only minimal activation of Fyn. Last, Northern analysis revealed variable tissue expression of intermediate PRLr transcript that differed from that of the long PRLr. Taken together, differences in signaling and tissue expression suggest that the human intermediate PRLr differs from the long PRLr in physiological function. Prolactin-dependent signaling occurs as the result of ligand-induced dimerization of the prolactin receptor (PRLr). While three PRLr isoforms have been characterized in the rat, studies have suggested the existence of several human isoforms in breast carcinoma species and normal tissues. Reverse transcription polymerase chain reaction was performed on mRNA isolated from the breast carcinoma cell line T47D, revealing two predominant receptor isoforms: the previously described long PRLr and a novel human intermediate PRLr. The nucleotide sequence of the intermediate isoform was found to be identical to the long isoform except for a 573-base pair deletion occurring at a consensus splice site, resulting in a frameshift and truncated intracytoplasmic domain. Scatchard analysis of the intermediate PRLr revealed an affinity for PRL comparable with the long PRLr. While Ba/F3 transfectants expressing the long PRLr proliferated in response to PRL, intermediate PRLr transfectants exhibited modest incorporation of [3H]thymidine. Significantly, however, both the long and intermediate PRLr were equivalent in their inhibition of apoptosis of the Ba/F3 transfectants after PRL treatment. The activation of proximal signaling molecules also differed between isoforms. Upon ligand binding, Jak2 and Fyn were activated in CHO-K1 cells transiently transfected with the long PRLr. In contrast, the intermediate PRLr transfectants showed equivalent levels of Jak2 activation but only minimal activation of Fyn. Last, Northern analysis revealed variable tissue expression of intermediate PRLr transcript that differed from that of the long PRLr. Taken together, differences in signaling and tissue expression suggest that the human intermediate PRLr differs from the long PRLr in physiological function. prolactin prolactin receptor polymerase chain reaction interleukin Chinese hamster ovary phosphate-buffered saline The neuroendocrine hormone prolactin (PRL)1 exhibits high homology to growth hormone and is also related to the peptide hormones of the interleukin family (1Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1879) Google Scholar, 2Bazan J.F. Immunol. Today. 1990; 11: 350-354Abstract Full Text PDF PubMed Scopus (511) Google Scholar). PRL has been implicated in the proliferation and differentiation of lobular units as well as the initiation and maintenance of lactation (3Horseman N.D. Zhao W. Montecino-Rodriguiez E. Tanaka M. Nakashima K. Engle S.J. Smith F. Markoff E. Dorshkind K. EMBO J. 1997; 16: 6926-6935Crossref PubMed Scopus (501) Google Scholar, 4Riddle O. Bates R.W. 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Montone K.T. Powell C.M. Tomaszewski J.E. Clevenger C.V. Endocrinology. 1997; 138: 5555-5560Crossref PubMed Scopus (175) Google Scholar) has been identified. PRL exerts its effects at the molecular level by inducing the homodimerization of the prolactin receptor (PRLr). A member of the cytokine receptor family, the PRLr lacks intrinsic enzymatic activity, thus requiring the activation of associated kinases and other signaling factors for ligand-driven transduction. Two protein-tyrosine kinases found in association with the PRLr are p59 fyn (11Clevenger C.V. Medaglia M.V. Mol. Endocrinol. 1994; 8: 674-681Crossref PubMed Google Scholar) and p120 jak2 (12Rui H. Lebrun J.-J. Kirken R.A. Kelly P.A. Farrar W.L. Endocrinology. 1994; 135: 1299-1306Crossref PubMed Scopus (116) Google Scholar, 13Lebrun J.-J. Ali S. Sofer L. Ullrich A. Kelly P.A. J. Biol. Chem. 1994; 269: 14021-14026Abstract Full Text PDF PubMed Google Scholar, 14DaSilva L. Howard O.M.Z. Rui H. Kirken R.A. Farrar W.L. J. Biol. Chem. 1994; 269: 18267-18270Abstract Full Text PDF PubMed Google Scholar, 15Rui H. Kirken R.A. Farrar W.L. J. Biol. Chem. 1994; 269: 5364-5368Abstract Full Text PDF PubMed Google Scholar). Through Jak2, PRL stimulation activates Stat family members in lymphocytes (16Gilmour K.C. Reich N.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6850-6854Crossref PubMed Scopus (55) Google Scholar) and breast tissues (17Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (715) Google Scholar, 18Groner B. Altiok S. Meier V. Mol. Cell. Endocrinol. 1994; 100: 109-114Crossref PubMed Scopus (44) Google Scholar), resulting in the initiation of transcription for interferon regulatory factor-1 and β-casein gene products, respectively. PRLr dimerization also induces the GRB2/SOS/Ras/Raf/MEK/MAPK signaling cascade, ultimately activating several transcription factors necessary for cell cycle progression including Myc, Jun, and T cell factor (19Seth A. Gonzalez F.A. Gupta S. 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Paquereau L. Le Cam A. Clark J. Nielsen J.H. Djiane J. Postel-Vinay M.-C. Edery M. Kelly P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 957-961Crossref PubMed Scopus (90) Google Scholar, 23Sato N. Sakamaki K. Terada N. Arai K.-I. Miyajima A. EMBO J. 1993; 12: 4181-4189Crossref PubMed Scopus (330) Google Scholar, 24Venkitaraman A.R. Cowling R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12083-12087Crossref PubMed Scopus (80) Google Scholar). The Box 2 domain is hydrophobic and acidic, and its signaling function is largely uncharacterized. Several isoforms of the PRLr have been identified in both mammals (25Kelly P.A. Djiane J. Postel-Vinay M.C. Edery M. Endocr. Rev. 1991; 12: 235-251Crossref PubMed Scopus (660) Google Scholar, 26Goffin V. Kelly P.A. Journal of Mammary Gland Biology and Neoplasia. 1996; 2: 7-17Crossref Scopus (103) Google Scholar, 27Postel-Vinay M.-C. Horm. Res. 1996; 45: 178-181Crossref PubMed Scopus (25) Google Scholar, 28Anthony R.V. Smith G.W. Duong A. Pratt S.L. 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Endocrinol. 1990; 4: 1136-1143Crossref PubMed Scopus (221) Google Scholar), long form (80–85 kDa) (36Boutin J.-M. Jolicoeur C. Okamura H. Gagnon J. Edery M. Shirota M. Banville D. Dusanter-Fourt I. Djiane J. Kelly P.A. Cell. 1988; 53: 69-77Abstract Full Text PDF PubMed Scopus (452) Google Scholar), and a mutant intermediate form found on the PRL-dependent rat T cell lymphoma line Nb2 (65 kDa) (Fig. 3 B) (36Boutin J.-M. Jolicoeur C. Okamura H. Gagnon J. Edery M. Shirota M. Banville D. Dusanter-Fourt I. Djiane J. Kelly P.A. Cell. 1988; 53: 69-77Abstract Full Text PDF PubMed Scopus (452) Google Scholar, 37Ali S. Pellegrini I. Kelly P.A. J. Biol. Chem. 1991; 266: 20110-20117Abstract Full Text PDF PubMed Google Scholar). In humans, the only PRLr isoform characterized thus far is the long form cloned from the liver (Fig. 3 A) (38Boutin J.M. Edery M. Shirota M. Jolicoeur C. Lesueur L. Ali S. Gould D. Djiane J. Kelly P.A. Mol. Endocrinol. 1989; 3: 1455-1461Crossref PubMed Scopus (238) Google Scholar). Previous studies have, however, provided evidence that other human PRLr isoforms may be expressed in human tissues (30Nagano M. Chastre E. Choquet A. Bara J. Gespach C. Kelly P.A. Am. J. Physiol. 1995; 268: G431-G442PubMed Google Scholar, 39Clevenger C.V. Chang W.P. Ngo W. Pasha T.L.M. Montone K.T. Tomaszewski J.E. Am. J. Pathol. 1995; 146: 1-11PubMed Google Scholar). In this study, we identify a novel isoform of the human PRLr cloned from the human breast cancer cell line T47D. The isoform is analyzed for 1) in vivo surface expression and its ability to bind ligand, 2) induction of cell proliferation and cell survival in response to ligand, 3) the ability to activate associated kinases, and 4) the relative levels of its corresponding mRNA in normal human tissues. T47D cells, an estrogen receptor/PRLr-positive human breast cancer cell line, were used for mRNA isolation. Whole RNA was purified from 107 washed cells using Trizol reagent (Life Technologies, Inc.) as described previously (39Clevenger C.V. Chang W.P. Ngo W. Pasha T.L.M. Montone K.T. Tomaszewski J.E. Am. J. Pathol. 1995; 146: 1-11PubMed Google Scholar). Messenger RNA was then purified from the whole RNA preparation with oligo(dT)-cellulose (Invitrogen, San Diego, CA). 5 μg of T47D mRNA was used for first strand synthesis of cDNA using the Superscript II RT cDNA kit (Life Technologies, Inc.). Negative controls consisted of reactions containing no T47D mRNA or no reverse transcriptase. A positive control reaction consisted of chloramphenicol acetyltransferase mRNA template. For polymerase chain reaction, 2 μl of the corresponding cDNA reactions were added to a 50-μl reaction containing 5-μl 10× PCR buffer, 3 μl of 25 mmMgCl2, 1 μl of 10 mm dNTP mix, 5 units ofTaq polymerase (Life Technologies, Inc.), and primers for amplification. As the positive control reaction, primers A (5′-GACATGGAAGCCATCACAGAC-3′) and B (5′-CGACCGTTCAGCTGGATATTA-3′) were used to amplify a fragment of the chloramphenicol acetyltransferase gene from control cDNA. The PRLr gene amplification reaction contained primers PRLR-F3 (5′-ATGAAGGAAAATGTGGCA-3′) and PRLR-1 (5′-TCAGTGAAAGGAGTGTGT-3′), which correspond to the 5′- and 3′-ends of the human long PRLr open reading frame. The primary cycle of the reaction consisted of 94 °C for 2 min, 42 °C for 1 min, 72 °C for 3 min, and 94 °C for 2 min, which was followed by 30 cycles of 94 °C for 30 s, 47 °C for 30 s, and 72 °C for 2 min. It was then extended at 72 °C for 3 min. Isolated PCR fragments were subcloned into the TA vector pCR 2.1 (Invitrogen, San Diego, CA) and analyzed by dideoxynucleotide sequencing. For eukaryotic expression of the intermediate isoform, the gene was reamplified by PCR with primers PRLR-Kl (5′-CGAATTCCACCATGAAGGAAAATGTGGCA-3′) and PRLR-599′ (5′-GCGCTCGAGTCAGTGAAAGGAGTGTGTAAA-3′), which contain a 5′EcoRI restriction site and Kozak initiation sequence and a 3′ XhoI restriction site, respectively. Alternative 3′ primers were also utilized to remove the tertiary stop codon from the open reading frames of the isoforms, allowing the addition of a carboxyl-terminal V5 epitope tag when ligated into vector pEF1-V5/HisA (Invitrogen). The intermediate isoform was reamplified with primers PRLR-Kl and PRLR-INT′ (5′-GCGCTCGAGGGAGTCCCGGGCTTC-3′), while the long isoform was reamplified with PRLR-Kl and PRLR-LONG′ (5′-CGCTCGAGGTGAAAGGAGTGTGTAAA-3′). The DNA fragments were digested with EcoRI and XhoI and ligated into the corresponding restriction sites of pcDNA3 and pEF1-V5/HisA. The clones were subsequently checked for amplification errors by dideoxynucleotide sequencing. T47D cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The mouse interleukin 3 (IL-3)-dependent pro-B cell line Ba/F3 was maintained in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in the presence of 1 ng/ml IL-3 (PeproTech, Rocky Hill, NJ). Chinese hamster ovary (CHO-K1) cells were maintained in Ham's F-12 medium (Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Ba/F3 cells (107) were transfected with 50 μg of intermediate or long isoform cDNA clones in pcDNA3 by exposure to a single voltage pulse (0.6 kV, 25 microfarads for 0.1 s) in a Gene Pulser electroporator (Bio-Rad). Stable clones were obtained by limiting dilution by selection in 750 μg/ml G418. CHO cells (2 × 105) were transiently transfected with 2 μg of intermediate or long isoform cDNA clones in pEF1-V5/HisA in conjunction with 2 μg of human Jak2 (gift of Dr. Roy Duhe) or murine Fyn (gift of Dr. Paul Stein) cDNA in pEF1-V5/HisA using Fugene 6 (Roche Molecular Biochemicals) as instructed. Total RNA from cells was isolated from T47D cells by extraction with Trizol reagent (Life Technologies) as described previously (39Clevenger C.V. Chang W.P. Ngo W. Pasha T.L.M. Montone K.T. Tomaszewski J.E. Am. J. Pathol. 1995; 146: 1-11PubMed Google Scholar). 10 μg of total RNA was denatured and subjected to electrophoresis on a 1% agarose formaldehyde gel and transferred onto a nylon membrane. A cDNA probe generated from bp 73–702 of the extracellular domain of the human PRLr long isoform was labeled with [α-32P]dCTP in the presence of random hexamers using the Oligo Labeling Kit (Amersham Pharmacia Biotech). The probe was hybridized to the membrane at a final concentration of 100 ng/ml at 68 °C for 1 h in Express-hyb solution (CLONTECH, Palo Alto, CA) as per the manufacturer's instructions. The blot was then washed three times in 2× SSC, 0.05% SDS at room temperature followed by two 20 min washes in 0.1× SSC, 0.1% SDS at 50 °C, followed by autoradiography. A master tissue blot of human total mRNA (CLONTECH) was probed with cDNAs specific for either the intermediate or long PRLr isoforms. Equal loading of mRNAs was confirmed by the quantitation of eight distinct housekeeping genes. The cDNA probe specific for the long isoform was composed of nucleotides 1037–1347 of the long form open reading frame (38Boutin J.M. Edery M. Shirota M. Jolicoeur C. Lesueur L. Ali S. Gould D. Djiane J. Kelly P.A. Mol. Endocrinol. 1989; 3: 1455-1461Crossref PubMed Scopus (238) Google Scholar). This entire region is deleted in the intermediate isoform open reading frame. The probe for the intermediate isoform spans the 573-bp deletion due to alternative splicing. This corresponds to nucleotides 910–1054 of the intermediate isoform open reading frame (or 910–1580 of the long isoform open reading frame (38Boutin J.M. Edery M. Shirota M. Jolicoeur C. Lesueur L. Ali S. Gould D. Djiane J. Kelly P.A. Mol. Endocrinol. 1989; 3: 1455-1461Crossref PubMed Scopus (238) Google Scholar)). Hybridization conditions were performed as instructed byCLONTECH. Under these conditions, no cross-hybridization was observed between isoforms (data not shown). The blot was exposed to x-ray film for 4 days, and signal intensities were obtained using ImageQuaNT densitometry software (Molecular Dynamics, Inc., Sunnyvale, CA). CHO cell transfectants were lysed in Laemmli buffer containing SDS and 2-mercaptoethanol (5Clevenger C.V. Russell D.H. Appasamy P.M. Prystowsky M.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6460-6464Crossref PubMed Scopus (213) Google Scholar). Lysates were electrophoresed through an 8% SDS-polyacrylamide gel and transferred to nitrocellulose. Nonspecific binding was blocked with 5% milk in PBS/Tween 20. Antigen was labeled with 1 μg of horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen) per ml. Antigen-antibody complexes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). 106 Ba/F3 transfectants were harvested and washed with PBS at 4 °C. Cells were then stained with a 1:100 dilution of rabbit anti-PRLr antiserum developed by our laboratory and characterized elsewhere (40Leav I. Merk F.B. Lee K.F. Loda M. Mandokt M. McNeal J.E. Ho S.-M. Am. J. Pathol. 1999; 154: 863-870Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) for 1 h at 4 °C. After washing three times with PBS, bovine serum albumin, 0.1% sodium azide, the cells were incubated for 30 min with a 1:2000 dilution of fluorescein 5-isothiocyanate-conjugated goat anti-rabbit secondary antibody. Cells were washed three times with PBS/bovine serum albumin/sodium azide, fixed with PBS, 4% paraformaldehyde for 15 min and resuspended in PBS/bovine serum albumin/sodium azide. Cellular immunofluoresence was examined using a Zeiss Axioskop2 Inc., and an This antiserum was specific for PRLr the addition of peptide was previously shown to PRLr (40Leav I. Merk F.B. Lee K.F. Loda M. Mandokt M. McNeal J.E. Ho S.-M. Am. J. Pathol. 1999; 154: 863-870Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). binding were as described previously C.M. 1996; PubMed Google Scholar). Ba/F3 cells were incubated with of human in a total of 100 μl of 0.1% sodium azide. Nonspecific binding was by with PRL at a concentration 100 times that of labeled Cells were incubated at 4 °C for 2 h with and through an 10% were from the and was on a 5 × long and intermediate isoform Ba/F3 transfectants were in medium of RPMI 1640 medium supplemented with sodium and in the presence of μg/ml human PRL or murine After cells were with of at °C for 4 of was by the of PRLr isoform Ba/F3 cells transfected with the PRLr intermediate isoform, the long isoform or control vector were at in 2 of RPMI 1640 with or 10 ng/ml Cells were harvested a and the of both and cells were by The of was by the of × After PRL stimulation 2 × CHO cells transfected with PRLr isoforms in conjunction with Jak2 or Fyn cDNAs expressed in vector were lysed and as described previously (11Clevenger C.V. Medaglia M.V. Mol. Endocrinol. 1994; 8: 674-681Crossref PubMed Google Scholar) using 3 μl Inc., CA) or Antigen-antibody complexes were isolated by the addition of 50 μl of A After three washes with buffer, were washed with buffer mm 100 mm and 100 The were then in 30 μl of buffer mm 10 mm and 10 of After 20 at 30 °C, the reactions were by the addition of 2× Laemmli buffer with and the reaction were analyzed by 10% SDS-polyacrylamide gel electrophoresis followed by autoradiography. three isoforms of the PRLr have been in the rat S. Edery M. Pellegrini I. J. Djiane J. Kelly P.A. Endocr. Proc. 1991; Scholar), only has been found expressed in human tissues. from our laboratory (39Clevenger C.V. Chang W.P. Ngo W. Pasha T.L.M. Montone K.T. Tomaszewski J.E. Am. J. Pathol. 1995; 146: 1-11PubMed Google Scholar) and (30Nagano M. Chastre E. Choquet A. Bara J. Gespach C. Kelly P.A. Am. J. Physiol. 1995; 268: G431-G442PubMed Google Scholar) found evidence the existence of at human identify and these PRLr isoforms, reverse transcription PCR was performed on cDNA generated from the breast cancer cell line T47D using to the 5′- and 3′-ends of the human long PRLr. electrophoresis of the cDNA revealed DNA fragments of and (Fig. control reactions to amplify DNA that the identified were generated from cDNA and not the result of DNA (Fig. 1 and The three fragments were and and their DNA were The was found to be the long PRLr isoform previously described (38Boutin J.M. Edery M. Shirota M. Jolicoeur C. Lesueur L. Ali S. Gould D. Djiane J. Kelly P.A. Mol. Endocrinol. 1989; 3: 1455-1461Crossref PubMed Scopus (238) Google Scholar). The fragment was an mRNA splice for a truncated extracellular domain B. and C. V. in In contrast, the sequence revealed an mRNA splice for an isoform with a deletion in the domain (Fig. 2 This DNA sequence is most the result of an RNA a consensus splice site was at the between and (39Clevenger C.V. Chang W.P. Ngo W. Pasha T.L.M. Montone K.T. Tomaszewski J.E. Am. J. Pathol. 1995; 146: 1-11PubMed Google Scholar). The open reading frame is to the long isoform to pair a deletion of nucleotides pair to (Fig. 2 While this isoform homology to the long form of pair the deletion a in the reading the sequence and a stop codon after the splice (Fig. 2 on the of the splice to the gene deletion in the rat Nb2 intermediate isoform (Fig. S. Pellegrini I. Kelly P.A. J. Biol. Chem. 1991; 266: 20110-20117Abstract Full Text PDF PubMed Google Scholar), this PRLr was the human intermediate sequence of the human intermediate PRLr open reading frame of the intermediate The sequence from the long PRLr isoform, the the and the the Box 1 and Box 2 frameshift region of the intermediate PRLr nucleotide of the long the of a pair deletion and frameshift within the intermediate of the human intermediate PRLr isoform with human and rat isoforms. extracellular Box 1 Box 2 variable of a in reading from the primary sequence of the long the physiological of the intermediate isoform in PRLr a eukaryotic expression vector containing the intermediate PRLr cDNA was transfected transiently into CHO cells and into the murine pro-B cell line as vector and the long PRLr isoform were also used to a DNA probe to the extracellular domain to both isoforms, Northern analysis of the transfectants showed mRNA of the molecular for both long and intermediate clones (data not shown). The intermediate isoform was also analyzed for its ability to be The cDNAs for both forms were subcloned into the vector which the addition of a V5 epitope tag to the carboxyl-terminal of both PRLr of from CHO cells transiently transfected with the revealed of the molecular previously for the long form kDa) A. S. Okamura H. Kelly P.A. Endocrinology. 1992; PubMed Scopus Google Scholar) and 50 for the intermediate isoform (Fig. The molecular for the intermediate PRLr is of the extracellular domain in a to that found on the long PRLr for the in the and observed of both transfectants with in a in molecular (data not shown). the expression of the intermediate PRLr on the cell Ba/F3 transfectants were stained with anti-PRLr antiserum and examined for surface of the receptor (Fig. long and intermediate PRLr transfectants showed high levels of a cell surface with vector (Fig. 1 and 2 while with serum only in of the transfectants (Fig. The rat intermediate PRLr has been shown to bind ligand with a affinity the rat long form S. Pellegrini I. Kelly P.A. J. Biol. Chem. 1991; 266: 20110-20117Abstract Full Text PDF PubMed Google Scholar). the ligand binding of the human long and intermediate Scatchard analysis was performed on Ba/F3 After for binding of PRL, and levels of surface expression were observed for both the long PRLr a of while the intermediate