Prolactin is a protein hormone of the anterior pituitary gland that was originally named for its ability to promote lactation in response to the suckling stimulus of hungry young mammals. We now know that prolactin is not as simple as originally described. Indeed, chemically, prolactin appears in a multiplicity of posttranslational forms ranging from size variants to chemical modifications such as phosphorylation or glycosylation. It is not only synthesized in the pituitary gland, as originally described, but also within the central nervous system, the immune system, the uterus and its associated tissues of conception, and even the mammary gland itself. Moreover, its biological actions are not limited solely to reproduction because it has been shown to control a variety of behaviors and even play a role in homeostasis. Prolactin-releasing stimuli not only include the nursing stimulus, but light, audition, olfaction, and stress can serve a stimulatory role. Finally, although it is well known that dopamine of hypothalamic origin provides inhibitory control over the secretion of prolactin, other factors within the brain, pituitary gland, and peripheral organs have been shown to inhibit or stimulate prolactin secretion as well. It is the purpose of this review to provide a comprehensive survey of our current understanding of prolactin's function and its regulation and to expose some of the controversies still existing.
Prolactin is a polypeptide hormone that is synthesized in and secreted from specialized cells of the anterior pituitary gland, the lactotrophs. The hormone was given its name based on the fact that an extract of bovine pituitary gland would cause growth of the crop sac and stimulate the elaboration of crop milk in pigeons or promote lactation in rabbits (1477). However, we now appreciate that prolactin has over 300 separate biological activities (184) not represented by its name. Indeed, not only does prolactin subserve multiple roles in reproduction other than lactation, but it also plays multiple homeostatic roles in the organism. Furthermore, we are now aware that synthesis and secretion of prolactin is not restricted to the anterior pituitary gland, but other organs and tissues in the body have this capability. Indeed, the multiple roles and sources of prolactin had led Bern and Nicoll (154) to suggest renaming it “omnipotin” or “versatilin.”
In this review we integrate the burgeoning information on prolactin's structure (sect. ii), synthesis and release from varying sources (sect. iii), the intracellular mechanism of its action (sect. iv), its major biological functions (sect.v), and the patterns (sect. vi) and regulation of its secretion (sect. vii).
II. PROLACTIN CHEMISTRY AND MOLECULAR BIOLOGY
A. Prolactin: Gene, Primary Structure, and Species Specificity
Based on its genetic, structural, binding and functional properties, prolactin belongs to the prolactin/growth hormone/placental lactogen family [group I of the helix bundle protein hormones (195, 791)]. Genes encoding prolactin, growth hormone, and placental lactogen evolved from a common ancestral gene by gene duplication (1311). The divergence of the prolactin and growth hormone lineages occurred ∼400 million years ago (357, 358). In the human genome, a single gene, found on chromosome 6, encodes prolactin (1363). The prolactin gene is 10 kb in size and is composed of 5 exons and 4 introns (357, 1772). Transcription of the prolactin gene is regulated by two independent promoter regions. The proximal 5,000-bp region directs pituitary-specific expression (160), while a more upstream promoter region is responsible for extrapituitary expression (159). The human prolactin cDNA is 914 nucleotides long and contains a 681-nucleotide open reading frame encoding the prolactin prohormone of 227 amino acids. The signal peptide contains 28 amino acids; thus the mature human prolactin is composed of 199 amino acids (1640).
The prolactin molecule is arranged in a single chain of amino acids with three intramolecular disulfide bonds between six cysteine residues (Cys4-Cys11, Cys58-Cys174, and Cys191-Cys199 in humans) (357). The sequence homology can vary from the striking 97% among primates to as low as 56% between primates and rodents (1640). In rats (358) and mice (968), pituitary prolactin consists of 197 amino acids, whereas in sheep (1036), pigs (1035), cattle (1851), and humans (1624) it consists of 199 amino acids with a molecular mass of ∼23,000 Da.
B. Secondary and Tertiary Structure of Prolactin
Studies on the secondary structure of prolactin have shown that 50% of the amino acid chain is arranged in α-helices, while the rest of it forms loops (169). Although it was predicted earlier (1311), there are still no direct data about the three-dimensional structure of prolactin. The tertiary structure of prolactin was predicted by homology modeling approach (635), based on the structural similarities between prolactin and other helix bundle proteins, especially growth hormone (2, 438). According to the current three-dimensional model, prolactin contains four long α-helices arranged in antiparallel fashion (2, 438).
C. Prolactin Variants
Although the major form of prolactin found in the pituitary gland is 23 kDa, variants of prolactin have been characterized in many mammals, including humans. Prolactin variants can be results of alternative splicing of the primary transcript, proteolytic cleavage and other posttranslational modifications of the amino acid chain.
1. Alternative splicing
Alternative splicing of prolactin mRNA has been proposed as one source of the variants (1639, 1640). Indeed, evidence suggestive of the existence of an alternatively spliced prolactin variant of 137 amino acids has been described in the anterior pituitary (501, 1882). In addition, alternative splicing involving retention of introns is also possible. However, alternative splicing is not considered a major source of prolactin variants.
2. Proteolytic cleavage
Of the cleaved forms that have been characterized, 14-, 16-, and 22-kDa prolactin variants have been most widely studied. The 14-kDa NH2-terminal fragment is a posttranslational product of the prolactin gene that is processed in the hypothalamus and shares biological activity with the 16-kDa fragment (335,1765). Both seem to possess a unique biological activity, which will be described later. The 16-kDa fragment [prolactin-(1—148)] was first described in rat pituitary extracts (1207) and has subsequently been found in mouse (1642) and human (1643) pituitary glands as well as in human plasma (1643). The 16-kDa prolactin is a product of kallikrein enzymatic activity. Kallikrein is an estrogen-induced, trypsin-like serine protease that is found in the Golgi cisternae and secretory granules of lactotrophs (1433). This enzyme will cleave rat prolactin in a thiol-dependent manner. Thiol alters the conformation of prolactin such that kallikrein recognizes it as a substrate. Treatment of native prolactin with carboxypeptidase-B results in a 22-kDa prolactin fragment [prolactin-(1—173)]. Surprisingly, this synthetic fragment can be detected in pituitary extracts by Western blot using an antiserum produced specifically against the 22-kDa prolactin fragment (45). It seems that the production and release of these proteolytic fragments from the pituitary gland is specific to female rats and sensitive to inhibition by dopamine (45). Although these and other fragments have been found in pituitary gland and serum, more work is required to determine their physiological significance since the possibility remains that they may be preparative artifacts (1640).
3. Other posttranslational modifications
Besides proteolytic cleavage, the majority of prolactin variants can be the result of other posttranslational processing of the mature molecule in the anterior pituitary gland or the plasma. These include dimerization and polymerization, phosphorylation, glycosylation, sulfation, and deamidation (1702).
a) dimerization and polymerization: macroprolactins. Dimerization and polymerization of prolactin or aggregation with binding proteins, such as immunoglobulins, by covalent and noncovalent bonds may result in high-molecular-weight forms. In general, the high-molecular-weight forms have reduced biological activity (1640). The role of prolactin-IgG macromolecular complexes in the detection and differential diagnosis of different prolactinemias is targeted primarily in clinical studies (299).
b) phosphorylation. Phosphorylation of prolactin occurs within the secretory vesicle of lactotrophs just before exocytosis and involves esterification of hydroxyl groups of serine and threonine residues (670). Phosphorylated prolactin isoforms have been isolated from bovine (224) and murine (1337) pituitary glands. Phosphorylated isoforms of prolactin may constitute as much as 80% of total pituitary prolactin in cattle (938). Although phosphoprolactin has been shown to be secreted in vitro, it is not known if it is secreted into the plasma in vivo. The significance of phosphorylated and nonphosphorylated prolactins has been reviewed in detail (736). Phosphorylated prolactin has much lower biological activity than nonphosphorylated prolactin (1859). However, phosphorylated prolactin may subserve a unique role as an autocrine regulator of prolactin secretion since it suppresses the release of nonphosphorylated prolactin from GH3 cells (768). Phosphorylation of prolactin as well as the relative ratio of phosphorylated to nonphosphorylated isoforms seems to be regulated throughout the estrous cycle (769), although the physiological relevance of this finding is not yet appreciated. However, novel data indicate that phosphorylated prolactin acts as an antagonist to the signal transduction pathways (362) and proliferative activities initiated by unmodified prolactin on Nb2 lymphoma cells (315). Further investigation is needed to determine the significance of phosphorylated prolactin in primary cells and tissues.
c) glycosylation. Glycosylated prolactin has been found in the pituitary glands of a wide variety of mammalian, amphibian, and avian species (1640). The degree of glycosylation varies from 1 to 60% among species and may also vary between reproductive states within species (1640). The linkage of the carbohydrate moiety may be either through nitrogen (N-glycosylation) or oxygen (O-glycosylation). The carbohydrate residues of the oligosaccharide chain may contain varying ratios of sialic acid, fucose, mannose, and galactose that differ considerably between species, physiological, and pathological states (1640). Like other prolactin variants, glycosylation also lowers biological activity (1127,1641) as well as receptor binding and immunologic reactivity of prolactins (740). Glycosylation also alters the metabolic clearance rate of prolactin (1641). Taken together, glycosylation of prolactin may play a role either in regulation of the biological activity or clearance of the molecule.
III. SITES OF SYNTHESIS AND SECRETION OF PROLACTIN
A. Anterior Pituitary Gland
1. Morphology of lactotrophs
The cells of the anterior pituitary gland which synthesize and secrete prolactin were initially described by light microscopy using conventional staining techniques (753). These cells, designated lactotrophs or mammotrophs, comprise 20–50% of the cellular population of the anterior pituitary gland depending on the sex and physiological status of the animal. Lactotrophs were subsequently identified unequivocally by immunocytochemistry in the anterior pituitary gland of the mouse (109), rat (110, 1287), and human (111,725, 1387) using species-specific prolactin antibodies. Ontogenetically, lactotrophs descend from the Pit-1-dependent lineage of pituitary cells, together with somatotrophs and thyrotrophs (348, 643, 1382,1599).
The morphology and distribution of lactotrophs have been best described in the rat (1768), where prolactin-containing cells are sparsely distributed in the lateroventral portion of the anterior lobe and are present as a band adjacent to the intermediate lobe (1287). Their shapes are heterogeneous, appearing as either polyhedral or angular but at times rounded or oval (429). With the use of either velocity sedimentation at unit gravity (1650) or discontinuous Percoll gradients (1813) to separate cell populations, it has been shown that lactotrophs vary based on their secretory granule size and content (1650) as well as on the amount of prolactin and prolactin mRNA present (1813).
2. Functional heterogeneity of lactotrophs
Aside from morphological heterogeneity, lactotrophs display functional heterogeneity as well. Development of the reverse hemolytic plaque assay (572, 576, 1300) led to a more precise description of functional heterogeneity in lactotrophs (1090). Although prolactin is largely found and secreted from a distinct cell type in the pituitary gland, the lactotroph, both prolactin and growth hormone can also be secreted from the intermediate cell population called mammosomatotrophs (572, 574, 576,1300). These bifunctional cells, which predominate in the pituitary of neonatal rats (770), differentiate into lactotrophs in the presence of estrogen (191). Mammosomatotrophs also differentiate into lactotrophs in pups in the presence of a maternal signal that appears in early lactation (1427) and is delivered to the pups through the mother's milk (1429).
There also appears to be functional heterogeneity among lactotrophs with regard to their regional distribution within the anterior lobe (1246) as well as to the nature of their responsiveness to secretagogues (188); that is, lactotrophs from the outer zone of the anterior lobe respond greater to thyrotrophin releasing hormone (TRH) than those of the inner zone, adjacent to the intermediate lobe of the pituitary gland (188). On the other hand, dopamine-responsive lactotrophs (84) are more abundant in the inner than the outer zone of the anterior pituitary. Surprisingly, functional heterogeneity is also reflected in the discordance between prolactin gene transcription and prolactin release in some lactotroph populations (296,1562). Taken together, it is clear that lactotrophs are not homogeneous in their morphology, hormonal phenotype, distribution, or function.
The first observation that prolactin is produced in the brain was by Fuxe et al. (594) who found prolactin immunoreactivity in hypothalamic axon terminals. Prolactin immunoreactivity was subsequently found in the telencephalon in the cerebral cortex, hippocampus, amygdala, septum (433), caudate putamen (502, 737), brain stem (433,737), cerebellum (1589), spinal cord (737, 1630), choroid plexi, and the circumventricular organs (1741).
Prolactin immunoreactivity is found within numerous hypothalamic areas in a variety of mammals (29, 677,678, 737, 1321,1630, 1741). Within the rat hypothalamus, prolactin immunoreactivity is detectable in the dorsomedial, ventromedial (676), supraoptic, and paraventricular (735) nuclei. Several approaches have been taken to prove that prolactin found in the hypothalamus is synthesized locally, independent of prolactin synthesis in the pituitary gland. Indeed, hypophysectomy has no effect on the amount of immunoreactive prolactin in the male hypothalamus and only diminishes but does not abolish the quantity of immunoreactive prolactin in the female rat hypothalamus (433).
With the use of conventional peptide mapping (434) and sequencing of a polymerase chain reaction (PCR) product of hypothalamic cDNA from intact and hypophysectomized rats (1882), it has been established that the primary structure of prolactin of hypothalamic and pituitary origin is identical. Thus it seems that the prolactin gene expressed in the rat hypothalamus is identical to the prolactin gene of the anterior pituitary (501,1882).
Although the role of prolactin of hypothalamic origin in the central nervous system (CNS) is not apparent, it should be noted that the hypothalamus contains the appropriate proteolytic enzymes to cleave 23-kDa prolactin into 16- and 14-kDa fragments (435). We do not know if prolactin of neural origin exerts its central effect as a neurotransmitter, neuromodulator, or a central cytokine regulating vascular growth and/or glial functions. To ascribe a role for prolactin of neural origin is troublesome, in part, because it is difficult to differentiate between the effects of prolactin of pituitary versus hypothalamic origin in the CNS. One cause of these difficulties is that pituitary prolactin from the circulation bypasses the blood-brain barrier and enters the CNS through the choroid plexi of the brain ventricles. Coincidentally, choroid plexi have a very high density of prolactin receptors (prolactin-Rs) as demonstrated by autoradiography (1113, 1853, 1854), immunocytochemistry (1495), standard receptor binding assays (1242), reverse transcriptase PCR, and ribonuclease protection assay (590). Interestingly, prolactin enhances the expression of its own receptors in the choroid plexus (1113). Aside from passage from the blood to the cerebrospinal fluid by way of the choroid plexus, pituitary prolactin may also reach the brain by retrograde blood flow from the anterior pituitary to the hypothalamus (1192, 1351). Therefore, because the actions of prolactin in the CNS can be due to the hormone of pituitary or hypothalamic origin, in this review we refer to its effects in the CNS without attributing a source.
2. Regulation of hypothalamic prolactin synthesis
Some well-established stimulators of pituitary prolactin secretion also affect hypothalamic prolactin production. For example, ovarian steroids modulate hypothalamic synthesis and release of prolactin (436, 437). Approximately 33% of the prolactin immunoreactive neurons in the medial basal hypothalamus can be labeled with [3H]estradiol (436), suggesting that these neurons have estrogen receptors. Ovariectomy lowers hypothalamic prolactin content, whereas estrogen replacement elevates it (436, 437). Of the known hypophysiotrophic factors, angiotensin II stimulates release of prolactin from hypothalamic fragments (437), and intracerebroventricular injection of vasoactive intestinal peptide will increase the amount of hypothalamic prolactin mRNA (212). However, other established stimulators of pituitary prolactin secretion such as TRH are without effect (437). Obviously, much more work is needed to establish the control of hypothalamic prolactin synthesis and release.
C. Placenta, Amnion, Decidua, and Uterus
The placenta, in addition to its bidirectional fetomaternal metabolic transport functions, has a wide array of endocrine functions as well. Among its many secretory products are a family of placental lactogens found in the rat (354, 393,537, 744, 1487-1489,1491, 1651), mouse (1605,1896), hamster (874,1662-1664), cow (46, 1612), pig (568), and human (728). The rat placenta produces a bewildering array of prolactin-like molecules that bear structural similarity to pituitary prolactin (1058,1652). These placental lactogens (PL) or prolactin-like proteins (PLP) have been variously identified as PL-I, PL-II, PL-Im (mosaic), PL-Iv (variant) (349, 1487, 1490), or PLP-A, -B, -C, -D, -E, -F, and -G (356, 392,851). In addition, the placenta contains a lactogen known as proliferin (PLF) (1056) and proliferin-related protein (PRP) (1057).
The decidua, on the other hand, produces a prolactin-like molecule, that is indistinguishable from pituitary prolactin in human (35, 342, 1475,1707), but is somewhat dissimilar in rat (688). A novel member of this family is prolactin-like protein J (1752), which is produced by the decidua during early pregnancy. Each of these prolactin-like molecules can bind to the prolactin-R (755, 860), and their secretion is regulated by local decidual (638,689, 729-731, 1745,1746), but not hypothalamic (637) prolactin-releasing factors (PRF). Progesterone has also been identified as a potent stimulator of decidual prolactin production (1143). In addition to stimulatory factors, a substance with inhibitory activity is found in decidual conditioned media (731). This substance decreases basal decidual prolactin release and competes with the decidual PRF (731). Recently, the N5 endometrial stromal cell line, which expresses the prolactin gene driven by the extrapituitary promoter, has been identified as a possible model system to study decidual prolactin gene expression (210). Ample evidence indicates that decidual prolactin diffuses into the amniotic fluid (1473,1474, 1476, 1501). Although the function of amniotic prolactin is uncertain, it has been suggested that it may serve an osmoregulatory (1781), maturational (864), or immune (732) role in the embryo/fetus.
Finally, the nonpregnant uterus has been shown to be a source of prolactin as well. Indeed, a decidual-like prolactin, indistinguishable from pituitary prolactin (611), has been identified in the myometrium of nonpregnant rats (1855). Interestingly, although progesterone stimulates the production of decidual prolactin, it appears to be a potent inhibitor of myometrial prolactin production (611). The physiological role for myometrial prolactin has yet to be identified.
D. Mammary Gland and Milk
Prolactin can be detected in epithelial cells of the lactating mammary gland (1326) as well as in the milk itself (680). There is little doubt that a portion of the prolactin found in the milk originates in the pituitary gland and reaches the mammary gland through the circulation. Thus some of the prolactin found in milk is taken up rather than produced by the mammary epithelial cells. Indeed, a significant amount of radiolabeled prolactin introduced into the circulation appears in milk (685, 1253). Apparently, prolactin reaches the milk by first crossing the mammary epithelial cell basement membrane, attaches to a specific prolactin binding protein within the mammary epithelial cell, and is ultimately transported by exocytosis through the apical membrane into the alveolar lumen (1352,1583).
In addition to uptake of prolactin from the blood, the mammary epithelial cells of lactating animals are capable of synthesizing prolactin. The presence of prolactin mRNA (992,1682) as well as synthesis of immunoreactive prolactin by mammary epithelial cells of lactating rats has been described (1063, 1064). It is possible that de novo synthesis of mammary prolactin requires a systemic trophic factor since the amount of both prolactin mRNA and immunoreactive prolactin declines over 24–48 h in mammary gland explants (992). The mammary gland may also act as a posttranslational processing site for prolactin. In both human (499) and rat (888,889) milk, the number of prolactin variants far exceeds that found in serum. Indeed, the mammary gland is the site of formation of the important 16-kDa variant of prolactin mentioned previously (332). Although prolactin, produced locally by mammary epithelial cells promotes proliferation, the 16-kDa cleaved prolactin variant inhibits local angiogenesis, which makes this proteolytic step a possible target of breast cancer research (636).
The physiological role for milk-borne prolactin has only been described in the rat, which is born immature relative to many other mammals. Indeed, during a brief window of neonatal life, the gastrointestinal tract lacks the ability to digest protein and likewise possesses the ability to absorb intact protein. This is particularly important since the rat pituitary gland is relatively quiescent during this period. Approximately 20% of the prolactin ingested in milk passes to the neonatal circulation (686). It has been shown that milk prolactin participates in the maturation of the neuroendocrine (1596, 1629) and immune (687, 702) systems.
E. The Immune System
A great deal of evidence suggests that lymphocytes can be a source of prolactin as well (599, 882,1214, 1516). Indeed, immune-competent cells from thymus and spleen as well as peripheral lymphocytes contain prolactin mRNA and release a bioactive prolactin that is similar to pituitary prolactin (445, 612,613, 1214-1216, 1523). Not only is an immunoreactive 22-kDa prolactin found in murine (1214) and human (1886) immune-competent cells, but size variants of prolactin have been described as well (1215, 1398, 1523,1592).
Although the control of pituitary prolactin secretion differs from that of lymphocytic origin, there is abundant evidence that lymphocytes contain dopamine receptors that may be involved in the regulation of lymphocytic prolactin production/release (432). Pharmacological characterization of lymphocytic dopamine receptors suggests that rather than the classical D2 type receptors found on lactotrophs, both the D4 and D5predominate on lymphocytes (186, 187,1361, 1470, 1545,1824). Moreover, mRNA for the D1, D3, and D5 receptors have been identified in rat lymphocytes (283).
The question remains of the role for pituitary and lymphocytic prolactin in the immune response. It is interesting to note that pituitary prolactin gene expression (1601), bioassayable serum prolactin (1601), immunoassayable serum prolactin (1505), and lymphocyte number (1505,1601) are elevated during acute skin allograft rejection in mice. Administration of bromocryptine, a D2 receptor agonist, or antilymphocytic serum diminishes circulating levels of prolactin in grafted animals and prolongs graft survival (1294, 1505). Because bromocryptine has little direct effect on lymphocytic prolactin secretion (1294), such data suggest that pituitary prolactin may modulate the elaboration of lymphocytic prolactin and that suppression of pituitary prolactin is thus a requirement for graft survival (1131). Indeed, such a role for prolactin in transplant rejection warrants further investigation.
F. Prolactin-Secreting Cell Lines
To study the synthesis, processing and secretion of prolactin at the cellular and molecular level, cell lines derived from pituitary tumors have been developed. The first cell line was a mammosomatotroph (MtT/W5) isolated from a radiation-induced pituitary tumor produced in a Wistar-Furth rat (1724, 1907). Because these cell lines secreted mostly growth hormone, they were designated as GH cells. It was subsequently found that some of the subclones were pluripotent and heterogeneous (1721,1722). For example, GH3 cells may release growth hormone only (somatotrophs), prolactin only (mammotrophs), both hormones (mammosomatotrophs), or neither hormone (189,192). Similarly, the GH1 and GH4C1 cell lines produce both prolactin and growth hormone but in varying ratios (1721).
The most obvious advantage of using cell lines rather than primary pituitary lactotrophs is that clonal cells are usually immortal, can be easily stored, and thus provide a perpetual supply of cells without sacrificing animals and purifying primary pituitary cultures. To critically use these cells, one should recognize their dissimilarity to primary cultures of pituitary cells. For example, unlike pituitary cells, the vast majority of the prolactin synthesized by GH cell lines is rapidly released and not stored (1722); thus there is no intracellular degradation of prolactin (381). Moreover, GH cells lack functional dopamine receptors, and thus they are resistant to the prolactin-inhibiting actions of dopamine (538). This can be viewed as either an advantage or a disadvantage. Because cell lines lack the complete receptor repertoire of a normal pituitary cell, one must be careful when drawing conclusions that apply to normal lactotrophs on the basis of data collected from cell lines. On the other hand, with knowledge of the defect borne by cell lines, one can study the role of an absent phenotype in control of cellular function. For example, one can transfect GH4C1 cells with a dopamine receptor gene, thus isolating and examining the role of that particular dopamine receptor subtype in lactotroph function (22,244, 249, 491, 666,1784, 1807, 1924).
Not all clonal lactotroph lines deviate as markedly from primary cells. For example, the MMQ cell line derived from the estrogen-induced rat pituitary tumor 7315a secretes prolactin exclusively, expresses functional dopamine D2 receptors (881), and behaves in a manner similar to (but not the same as) that of normal lactotrophs (567,699).
IV. PROLACTIN RECEPTORS
A. Prolactin Receptor: Gene, Splicing Variants, and Isoforms
The prolactin-R is a single membrane-bound protein that belongs to class 1 of the cytokine receptor superfamily (131, 132, 926,997). Just like their respective ligands, prolactin and growth hormone receptors share several structural and functional features despite their low (30%) sequence homology (632,633). Each contains an extracellular, transmembrane, and intracellular domain (1899). The gene encoding the human prolactin-R is located on chromosome 5 and contains at least 10 exons (131, 132). Transcriptional regulation of the prolactin-R gene is accomplished by three different, tissue-specific promoter regions. Promoter I is specific for the gonads, promoter II for the liver, and promoter III is “generic,” present in both gonadal and nongonadal tissues (812). Numerous prolactin-R isoforms have been described in different tissues (24, 386, 1031). These isoforms are results of transcription starting at alternative initiation sites of the different prolactin-R promoters as well as alternative splicing of noncoding and coding exon transcripts (809, 812). Although the isoforms vary in the length and composition of their cytoplasmic domains, their extracellular domains are identical (184,926, 1031). The three major prolactin-R isoforms described in rats are the short (291 amino acids), intermediate (393 amino acids), and long (591 amino acids) forms (184). In mice, one long and three short forms have been described (338, 386). In addition to the membrane-bound receptors, soluble prolactin-binding proteins were also described in mammary epithelial cells (158) and milk (1430). These soluble forms contain 206 NH2-terminal amino acids of the extracellular domain of the prolactin-R (159). The soluble prolactin binding proteins are also products of the same prolactin-R gene, but it is still uncertain whether they are results of alternative splicing of the primary transcript or products of proteolytic cleavage of the mature receptor (or both) (184).
B. Activation of Prolactin-R and the Associated Signal Transduction Pathways
1. Prolactin-R domains and receptor activation
a) extracellular domain: ligand-induced dimerization. The extracellular domain of all rat and human prolactin-R isoforms consists of 210 amino acids (196,197) and shows sequence similarities with other cytokine receptors (cytokine receptor homology domain, CRH) (1872). The extracellular domain can be further divided into NH2-terminal D1 and membrane-proximal D2 subdomains (926, 1872). Both D1 and D2 show analogies with the fibronectin type III molecule, which drives the receptor-ligand interactions in the majority of cytokine receptors (1872). The most conserved features of the extracellular domain are two pairs of disulfide bonds (between Cys12-Cys22 and Cys51-Cys62) in the D1 domain and a “WS motif” (Tpr-Ser-x-Trp-Ser) in the D2 domain (1872). The disulfide bonds and the WS motif are essential for the proper folding and trafficking of the receptor, although they are not responsible for binding the ligand itself (632). Activation of the prolactin-R involves ligand-induced sequential receptor dimerization (184) (Fig. 1). Each prolactin molecule contains two binding sites (site 1 involves helices 1 and 4, while site 2 encompasses helices 1 and 3). First, prolactin's binding site 1 interacts with a prolactin-R molecule (634). The formation of this initial hormone-receptor complex is the prerequisite for the interaction of binding site 2 on the same prolactin molecule with a second prolactin-R (184). Disruptive mutation of prolactin binding site 2 is detrimental to prolactin-R activation, which can be initiated only when a trimeric complex (2 receptors, 1 hormone) is formed (184, 634).