(Received for publication, September 15, 1995; and in revised form, January 18, 1996)
From the
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 E1, E1
, and E1
. 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 E1
, E1
, and
E1
, as well as alternative splicing of exon 2, generates
multiple 5`-untranslated regions in PRLR transcripts. These alternative
first exons (E1
, E1
, and E1
) were
found to be utilized in a tissue-specific manner in vivo.
E1
is predominantly expressed in the ovary, E1
is specifically expressed in the liver, and E1
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 E1
, E1
, and
E1
, respectively, were identified. E1
was found
to initiate from a single site at -549, E1
from
multiple sites at -405, -461, and -506, and E1
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) ()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
cytokine-growth hormone-prolactin receptor superfamily(3) .
Multiple PRLR mRNA species were identified in several tissues,
corresponding 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 down-regulated 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 5`-flanking regions of the gene and identified multiple and tissue-specific utilization of promoters in the gonads and liver.
Figure 1:
Multiple forms of PRLR mRNA 5`-UTRs and
their unique 5`-end sequences derived from 5`-RACE PCR analyses. Left panel, diagram depicting multiple forms of 5`-UTRs of the
PRLR mRNA; ovarian form, predominantly in the ovary; liver form,
specifically in the liver; and common form, present in all three
tissues. In parentheses are indicated mRNA species related to
the specific 5`-end sequences in the gonads and liver (also see Fig. 2). Reverse primers used for 5`-RACE PCR analyses are
indicated by arrowheads (#6 and #8, coding
region and #4, noncoding region) and were used in two sets of
independent 5`-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 -115, -92, and -55
were alternative splicing sites (see also Fig. 3). Exon-intron
junction was identified at -55 (sequences of 5`-UTRs diverged 5`
from -60). On the right panel are shown the three
classes of 5`-end mRNA sequences E1, E1
, and
E1
.
Figure 2:
Northern blot analyses of tissue-specific
expression of PRLR mRNA 5`-UTR. Panel A, hybridization to
blots of poly(A) RNA from ovary, Leydig cells, and
liver using the common region sequence (CRS, -55 to
+390) and the three 5`-UTR sequences (E1
,
E1
, and E1
) as probes. Panel B (ovarian mRNA) and panel C (Leydig cell mRNA) illustrate
the profile of multiple PRLR mRNA species revealed by hybridization to
different probes (SCD, short unique C-terminal domain of 320
base pairs in the short form PRLR). Sizes of mRNA species are indicated
in kb.
Figure 3:
Genomic organization of PRLR 5`-UTR and
exon-intron boundaries. Top, the diagram of the genomic region
corresponding to the 5`-UTR of the PRLR mRNA. E1,
E1
, and E1
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 -93 and -56 are the two
alternate 5`-donor recognition sites. a, the position of
E1
in relation to E1
and E1
is
arbitrary since no overlapping clones were isolated. b, this
exon was deduced by the identification of the second intron position at
-55 and another intron at +54 (revealed from isolation of an
exon of 133 base pairs located within the coding region at +55 to
+187, not shown).
Tissue-specific expression of the PRLR mRNA 5`-UTRs was revealed by
Northern hybridization of PRLR mRNA and Southern hybridization of
5`-RACE PCR products using 5`-end sequence probes. Northern blots
showed that E1 was expressed only in the ovary (Fig. 2A, E1
). However, very low levels of
E1
were detected in Leydig cells but not in the liver by
5`-RACE PCR analyses. E1
was exclusively expressed in the
liver and is the major form in this tissue (Fig. 2A,
E1
). E1
is expressed in the three tissues as
the predominant form in Leydig cells (Fig. 2A, E1
and C) and as a minor form in the ovary and liver (Fig. 2A, E1
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 5`-UTR of E1 (442 nt) and E1
(236 nt), respectively (Fig. 2B). However, this
5`-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
and E1
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
mRNA species was detected on Northern blots with major
bands at 9.7 and 1.8 kb (Fig. 2C). In the liver, both
E1
- and E1
-containing species were resolved as
one broad 1.8-kb band due to the small size difference between E1
(290-390 nt, see below) and E1
(236 nt) (Fig. 2A, CRS, E1
, E1
).
Heterogeneity of PRLR mRNA species can arise from differential transcripion initiation, alternative splicing of the coding region, and 3` 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 (>7 kb) of 3`-UTR (based on the sizes of the coding region, 1.8 kb, and the 5`-UTRs, 0.35 and 0.55 kb).
Figure 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 ±2 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 +1) at -549 (PI); -405, -461, -506 (PII); and -340 and -351 (PIII), respectively. The small arrow indicates a minor start site at -337 revealed only in the ovary.
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 -559 in PI and at -408 and -478/-474 in PII. In addition, a CCAAT element and a C/EBP site were found in PI (-623 and -636, respectively), and SP1 sequences were observed in PI(-449) and PIII(-262, -273). Also AP-1 and AP-2 sites along with several other consensus elements were observed in these promoter regions (Fig. 5).
Figure 5: Nucleotide sequences of the three 5`-flanking regions with their respective partial exon 1. PI (-1566 to -124), PII (-1264 to -181), and PIII (-1427 to -179) are shown. Primers used for primer extension analyses are marked by dashed overlines, transcriptional initiation sites are indicated by arrowheads, 5`-ends of the mRNA 5`-UTR derived from the 5`-RACE PCR are marked with a dot under the nucleotide, TATA-like sequences are boxed, and consensus elements for transcription factor binding sites are underlined.
Multiple and tissue-specific promoters have been reported for several other genes(12, 13, 14, 15, 16, 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 5`-end exons in the 5`-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 5`-UTR may play a role in regulation of the PRLR gene expression posttranscriptionally. It has been shown that the 5`-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 5`-UTR. Interestingly, the sequence deleted at -93 to -60 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34897[GenBank], U34898[GenBank], U34899[GenBank], and U34900[GenBank].