Multiple and tissue-specific promoter control of gonadal and non-gonadal prolactin receptor gene expression.

Prolactin receptors (PRLRs) are widely expressed, and multiple mRNA transcripts encoding PRLRs are present in prolactin target tissues. The molecular basis for the control of the PRLR gene expression is currently unknown. Analyses of the 5'-untranslated regions of PRLR mRNAs expressed in gonadal and non-gonadal tissues and their genomic organization revealed three alternative first exons designated as E11, E12, and E13. Each of these exons is alternatively spliced to a common noncoding exon (exon 2, nucleotides -115 to -56) that precedes the third exon containing the translation initiation codon. Alternative utilization of exons E11, E12, and E13, as well as alternative splicing of exon 2, generates multiple 5'-untranslated regions in PRLR transcripts. These alternative first exons (E11, E12, and E13) were found to be utilized in a tissue-specific manner in vivo. E11 is predominantly expressed in the ovary, E12 is specifically expressed in the liver, and E13 is expressed as a predominant form in the Leydig cell and as a minor form in the ovary and liver. Genomic 5'-flanking regions containing the three putative PRLR gene promoters (PI, PII, and PIII) that initiate the transcription of E11, E12, and E13, respectively, were identified. E11 was found to initiate from a single site at -549, E12 from multiple sites at -405, -461, and -506, and E13 from two major sites at -340 and -351. These findings indicate that multiple promoters control transcription of the PRLR gene and provide a molecular basis for the differential regulation of PRLR expression in diverse tissues.

Prolactin exerts diverse biological functions including lactation, reproduction, steroidogenesis, metabolism, behavior, immune regulation, growth, and water-salt balance (1) through specific prolactin receptors (PRLRs) 1 present in a wide range of target tissues (2,3). cDNAs encoding long and short forms of the prolactin receptor have been cloned from a variety of tissues of several species (3)(4)(5). Long and short forms of PRLRs mainly differ in the sequences and lengths of their cytoplasmic domains and have been classified as members of the cytokinegrowth hormone-prolactin receptor superfamily (3). Multiple PRLR mRNA species were identified in several tissues, corre-sponding to the long and short forms of the receptors. In the rat ovary, a 9.7-kb mRNA species was identified as coding for the long form of the receptor and was the most abundant transcript, while the 2.1-and 1.8-kb species encoding the short form of the receptor were less abundant (6). In the rat liver, the 1.8-kb mRNA coding for the short form receptor was the predominant species (7).
Besides its primary action on the mammary gland, prolactin has significant regulatory functions in the gonads (2). In the ovary, prolactin plays an essential role in the formation and maintenance of a functional corpus luteum by acting in concert with the gonadotropic hormones (2). In the testis, prolactin modulates Leydig cell functions by potentiation of luteinizing hormone-stimulated responses, partly through the control of luteinizing hormone receptor expression (2). Conversely, ovarian PRLRs and their mRNA levels were acutely up-and downregulated by administration of gonadotropins at different stages of ovarian development (6). In the male rat, luteinizing hormone treatment caused rapid and transient loss of Leydig cell prolactin receptors (8). These studies suggested that gonadotropins exert heterologous control of PRLR expression in the gonads. Furthermore, hepatic prolactin receptors were markedly induced by estrogen and during late pregnancy (4).
The diverse functions of prolactin and the wide distribution of its receptors suggested that expression of the PRLR is subject to complex regulation in different target tissues. In principle, the diverse actions of prolactin could be manifested by the expression of different receptor forms and signal transduction pathways and also by the differential control of PRLR gene transcription in individual target tissues. To investigate the mechanisms by which the expression of the PRLR is controlled, we have characterized the complex organization of the 5Ј-flanking regions of the gene and identified multiple and tissuespecific utilization of promoters in the gonads and liver.
Primer Extension and S1 Nuclease Protection-Sequences used for * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

RESULTS AND DISCUSSION
Heterogeneity and Tissue-specific Expression of PRLR mRNA 5Ј-UTR-Comparison of 5Ј-UTR sequences of reported PRLR cDNAs revealed sequence divergence 5Ј from nucleotide Ϫ60 (ATG ϩ1). Sequencing of 5Ј-RACE PCR products of the PRLR mRNAs from the rat ovary, Leydig cells, and liver verified the divergence of 5Ј-UTRs upstream from Ϫ60 and extended the 5Ј-UTR sequences to the 5Ј-ends (Fig. 1, left panel). Three distinct 5Ј-end mRNA sequences were identified and designated as E1 1 (442 nt), E1 2 (233 nt), and E1 3 (236 nt) (Fig. 1, right panel). Each of these sequences is followed by a region of either 55 nt (Ϫ115 to Ϫ60) or 23 nt (Ϫ115 to Ϫ93) with a 33-nt deletion at positions Ϫ92 to Ϫ60. In addition, an ovarian form with deletion at Ϫ115 to Ϫ60 was identified. All sequences downstream of Ϫ60 were identical and conformed to the reported PRLR cDNA forms.
Tissue-specific expression of the PRLR mRNA 5Ј-UTRs was revealed by Northern hybridization of PRLR mRNA and Southern hybridization of 5Ј-RACE PCR products using 5Ј-end sequence probes. Northern blots showed that E1 1 was expressed only in the ovary ( Fig. 2A, E1 1 ). However, very low levels of E1 1 were detected in Leydig cells but not in the liver by 5Ј-RACE PCR analyses. E1 2 was exclusively expressed in the liver and is the major form in this tissue ( Fig. 2A, E1 2 ). E1 3 is expressed in the three tissues as the predominant form in Leydig cells ( Fig.  2A, E1 3 and C) and as a minor form in the ovary and liver ( Fig.  2A, E1 3

and B).
In addition, it was observed in the ovary that the difference between the 2.1-and 1.8-kb mRNA species corresponding to the short form of the receptor (with brief cytoplasmic domain) could be accounted for by the presence in the 5Ј-UTR of E1 1 (442 nt) and E1 3 (236 nt), respectively (Fig. 2B). However, this 5Ј-UTR difference was not resolvable for the 9.7-kb species that encodes the long form of the receptor (with extended cytoplasmic domain). Since both E1 1 and E1 3 sequences were associated with PRLR mRNAs of both receptor forms, it is suggested that the generation of the long and short form of the receptor is independent of promoter specificity (see below) and may result from a posttranscriptionally regulated process. In the Leydig cell, only the E1 3 mRNA species was detected on Northern blots with major bands at 9.7 and 1.8 kb (Fig. 2C). In the liver, both E1 2 -and E1 3 -containing species were resolved as one broad 1.8-kb band due to the small size difference between E1 2 (290 - and common form, present in all three tissues. In parentheses are indicated mRNA species related to the specific 5Ј-end sequences in the gonads and liver (also see Fig. 2). Reverse primers used for 5Ј-RACE PCR analyses are indicated by arrowheads (#6 and #8, coding region and #4, noncoding region) and were used in two sets of independent 5Ј-RACE PCR analyses (#6 for the first strand cDNA synthesis and #8 as nested primer for the subsequent PCR, or #8 for the first strand cDNA synthesis and #4 for cDNA amplification). Positions at Ϫ115, Ϫ92, and Ϫ55 were alternative splicing sites (see also Fig. 3). Exon-intron junction was identified at Ϫ55 (sequences of 5Ј-UTRs diverged 5Ј from Ϫ60). On the right panel are shown the three classes of 5Ј-end mRNA sequences E1 1 , E1 2 , and E1 3 . 390 nt, see below) and E1 3 (236 nt) ( Fig. 2A, CRS, E1 2 , E1 3 ).
Heterogeneity of PRLR mRNA species can arise from differential transcripion initiation, alternative splicing of the coding region, and 3Ј alternative polyadenylation. Although the coding region of the long and short forms of the receptor partially accounted for the size difference between the mRNA transcripts encoding the two receptor forms (6, 7), it is evident from present data that differential transcriptional initiation also contributes to the mRNA heterogeneity (2.1 and 1.8 kb). In addition, the large size of the 9.7-kb species, which was more highly expressed in the ovary than in the testis ( Fig. 2A), can be accounted for by a long stretch (Ͼ7 kb) of 3Ј-UTR (based on the sizes of the coding region, 1.8 kb, and the 5Ј-UTRs, 0.35 and 0.55 kb).
Genomic Organization and Alternative Splicing of the PRLR mRNA 5Ј-UTR-The identification of three distinct 5Ј-end sequences was indicative of utilization of different promoters for transcription initiation of the PRLR gene. By employing the three 5Ј-UTR sequences (E1 1 , E1 2 , and E1 3 ) as probes, corresponding genomic clones were isolated from the rat genomic library (Fig. 3, middle). The gene region corresponding to the 5Ј-UTR spanned at least 60 kb (Fig. 3, top). Sequencing of gene fragments corresponding to E1 1 , E1 2 , and E1 3 confirmed the existence in the gene of the three 5Ј-end sequences identified by 5Ј-RACE PCR analyses. These sequences were designated as the alternative exon 1 of the PRLR gene. The first intron position was identified between the exon 1 (E1 1 , E1 2 , and E1 3 ) and nucleotide Ϫ115. Overlapping genomic clones containing both E1 1 and E1 2 were isolated, and E1 1 was localized ϳ10 kb upstream of E1 2 in the PRLR gene (Fig. 3, top). Since E1 3 genomic clones did not overlap with any other clones, their position in relation to E1 1 and E1 2 remains to be determined. The cloning of a genomic fragment corresponding to the oligomer probe 2 (Ϫ61 to Ϫ84) revealed the second exon at Ϫ115 to Ϫ56 and the second intron position at Ϫ55 (Fig. 3, bottom). An alternate splicing donor site was identified at Ϫ93 (TGAAG) of exon 2, which conforms to the consensus 5Ј-splicing site sequence (11) and accounts for the alternative splicing found in the PRLR mRNA 5Ј-UTRs (Fig. 1,  left and Fig. 3, top). Since the alternate donor sequence TGAAG at Ϫ93 is identical to the one at Ϫ56, the divergence of the PRLR mRNA 5Ј UTR occurs upstream from Ϫ60. Thus, the 5Ј-UTR of the PRLR gene is composed of at least three alternative first exons (E1 1 , E1 2 , and E1 3 ) and one common exon 2. The three FIG. 3. Genomic organization of PRLR 5-UTR and exon-intron boundaries. Top, the diagram of the genomic region corresponding to the 5Ј-UTR of the PRLR mRNA. E1 1 , E1 2 , and E1 3 are the three alternative first exons transcribed from three putative promoter regions PI, PII, and PIII, respectively (not in scale). The alternative splicing patterns are illustrated by lines and arrowheads connecting different exons. Gap regions between exons are indicated by //. Middle, the genomic clones with sizes (kb) and partial restriction enzyme mapping are indicated (E, EcoRI; X, XbaI; B, BamHI). Bottom shows sequences of the exon-intron boundaries and exon 2. Underlined TGAAGs at Ϫ93 and Ϫ56 are the two alternate 5Ј-donor recognition sites. a, the position of E1 3 in relation to E1 1 and E1 2 is arbitrary since no overlapping clones were isolated. b, this exon was deduced by the identification of the second intron position at Ϫ55 and another intron at ϩ54 (revealed from isolation of an exon of 133 base pairs located within the coding region at ϩ55 to ϩ187, not shown).

FIG. 4. Mapping of PRLR gene transcription initiation sites from the three putative promoter regions.
Primer extension was performed for PI (ovary mRNA), PII (liver mRNA), and PIII (ovary, Leydig cell, and liver mRNA). S1 nuclease protection assay was performed for PII (liver mRNA, lane 5). Yeast RNA was used for negative controls (lanes 2, 4, 6, 9, and 12). Sequence ladders were run along with the samples. Free probe at 362 base pairs was indicated (lanes 5 and 6). The specific extended or protected bands were determined within bandwidth error of Ϯ2 base pairs (arrows). The sizes are in base pairs from primer locations 166 (lane 1); 162, 218, 263 (lanes 3 and 5); and 107 and 118 (lanes 7, 8, 10, and 11) corresponding to transcription initiation sites (initiation codon ATG as ϩ1) at Ϫ549 (PI); Ϫ405, Ϫ461, Ϫ506 (PII); and Ϫ340 and Ϫ351 (PIII), respectively. The small arrow indicates a minor start site at Ϫ337 revealed only in the ovary. alternative first exons are transcribed from three putative promoter regions, and each of these is spliced to the second exon before joining to the third exon containing the translation initiation codon. The organization of the 5Ј-UTR in the gene has defined the alternate splicing variants of the PRLR mRNA 5Ј-UTR. However, the presence of additional alternative first exons and promoters of the PRLR gene in other expressing tissues not investigated in this study (i.e. mammary glands, prostate, adrenal, kidney, pancreas, and thymocytes) cannot be excluded. We conclude that multiple forms of PRLR mRNA 5Ј-UTRs resulted from the alternative utilization of three first exons and alternative splicing of the second exon.
Mapping of Transcriptional Initiation Sites and Sequences of the Three Putative Promoter Regions of the PRLR Gene-A major transcription initiation site for E1 1 at Ϫ549 was found in the ovary by primer extension analysis, conforming to the 5Ј-end derived from the 5Ј-RACE PCR product (Fig. 4, lane 1). Multiple transcription initiation sites for E1 2 were identified at Ϫ405, Ϫ461, and Ϫ506 in the liver by both primer extension (Fig. 4, lane 3) and S1 nuclease protection analyses (Fig. 4, lane 5). Two major transcription initiation sites for E1 3 were identified at Ϫ340 and Ϫ351 in the ovary, Leydig cells, and the liver by primer extension analyses, consistent with the 5Ј-ends derived from 5Ј-RACE PCR analyses (Fig. 4, lanes  7, 8, and 10). In addition, a minor transcriptional initiation site for E1 3 at Ϫ337 was observed in the ovary (Fig. 4, lane 11) only after prolonged exposure of the gel.
Analyses of the three putative promoter region sequences (PI, PII, and PIII) have revealed consensus sequences for several transcription factors, which may be important for the basal as well as hormonally regulated promoter activities. Although no canonical TATAA element was observed within an expected distance from the transcription initiation sites, TATA-like sequences (AATAA) were found at Ϫ559 in PI and at Ϫ408 and Ϫ478/Ϫ474 in PII. In addition, a CCAAT element and a C/EBP site were found in PI (Ϫ623 and Ϫ636, respectively), and SP1 sequences were observed in PI (Ϫ449) and PIII (Ϫ262, Ϫ273). Also AP-1 and AP-2 sites along with several other consensus elements were observed in these promoter regions (Fig. 5).
Multiple and tissue-specific promoters have been reported for several other genes (12-17). Multiple and differential promoter control of the PRLR gene is consistent with the numerous functions of prolactin in diverse tissues in which the PRLR expression may require being differentially regulated. The finding of three unique 5Ј-end exons in the 5Ј-UTR, which do not appear individually to associate with specific receptor forms, raises an intriguing question of whether the difference present in the PRLR mRNA 5Ј-UTR may play a role in regulation of the PRLR gene expression posttranscriptionally. It has been shown that the 5Ј-UTR was associated with the mRNA stability as well as the translatability in other genes (18,19). Furthermore, the deletion of partial or entire exon 2 in some of the mRNA forms further diversifies the PRLR 5Ј-UTR. Interestingly, the sequence deleted at Ϫ93 to Ϫ60 can potentially form a stem loop structure, and therefore its presence or absence may be significant in regulating the PRLR mRNA stability and/or translatability.
In summary, three alternative first exons and corresponding putative promoter regions, PI, PII, and PIII, of the PRLR gene that are utilized in a tissue-specific manner in vivo were identified in gonadal and non-gonadal tissues. PI and PII function as major promoters in the ovary and in the liver, respectively, while PIII is the dominant promoter in Leydig cells and minor promoter in the ovary and liver. The differential control of these multiple promoters may provide the molecular basis of tissue-specific regulation of the PRLR expression in diverse prolactin target cells.