I. INTRODUCTION

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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

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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.

Studies on the secondary structure of prolactin have shown that 50% of the amino acid chain is arranged in -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 -helices arranged in antiparallel fashion (2, 438).

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.

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).

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

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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).

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.

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.

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.

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.

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 D₅ predominate 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.

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

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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).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Mechanism of prolactin receptor activation. Activation of prolactin-R involves ligand-induced sequential receptor dimerization (184) driven by the prolactin molecule containing two binding sites. First, prolactin's binding site 1 interacts with a prolactin-R molecule (step 1). The extracellular (EC) domain of all prolactin-R isoforms consists of NH2-terminal D1 and membrane-proximal D2 subdomains (926), both of which show analogies with the fibronectin type III molecule driving the receptor-ligand interactions in cytokine receptors (1872). The formation of the initial hormone-receptor complex induces the interaction of binding site 2 on the same prolactin molecule with a second prolactin-R (184) (step 2). Although the intracellular (IC) domains of prolactin-R isoforms differ in length and composition, there are two conserved regions, termed box 1 and box 2 (1260). Both the presence of the proline-rich box 1 (1014) and strict homodimeric stoichiometry of prolactin-R dimers (550) are necessary for the activation of the tyrosine kinase termed Janus kinase2 (Jak2), constituitively associated with the IC domain of the prolactin-R (1013). Jak2 kinases transphosphorylate each other (550) (step 2) and phosphorylate (P) the Tyr residues (Y) of the prolactin-R itself (step 3) (1514). Although phosphorylation of Jak2 is a key event in the activation of all prolactin-R isoforms, Tyr phosphorylation of the receptor itself does not occur upon activation of the short form of the prolactin-R, despite the presence of four Tyr residues in its intracellular domain (660).

B) INTRACELLULAR DOMAIN: ACTIVATION OF JAK2 AND RECEPTOR PHOSPHORYLATION. I) Transmembrane and intracellular domains. The role of the 24-amino acid-long transmembrane domain in the activation of prolactin-R is unknown (184). The intracellular domain, however, is a key player in the initiation of the signal transduction mechanisms associated with the prolactin-R (184). The intracellular domains are different in length and composition among the various prolactin-R isoforms and show little sequence similarities with other cytokine receptors (184). However, there are two relatively conserved regions termed box 1 and box 2 (1260). Box 1 (Fig. 1) is a membrane-proximal, proline-rich motif necessary for the consensus folding of the molecule recognized by the transducing molecules (184). Box 2 is less conserved and is missing in the short isoform of the prolactin receptor (632, 926).

II) Activation of Jak2. Although the intracellular domain of the prolactin-R is devoid of any intrinsic enzymatic activity, ligand-mediated activation of prolactin-R results in tyrosine phosphorylation of numerous cellular proteins (1479), including the receptor itself (926, 1267). The membrane proximal region of the intracellular domain is constitutively (i.e., not induced by ligand binding) associated with a tyrosine kinase termed Janus kinase 2 (Jak2) (266, 834, 1013). Phosphorylation of Jak2 occurs within 1 min after prolactin binding, suggesting a major upstream role for Jak2 (1014)(Fig. 1). Experimental data suggest two major prerequisites for Jak2 activation: 1) presence of the proline-rich box 1 motif in the intracellular domain of the prolactin-R (1014) and 2) homodimeric stoichiometry of the ligand-induced prolactin-R dimers (307, 549, 550). Although the association of Jak2 with prolactin-R has been undoubtedly proven (266, 1013, 1515), the exact structure of their association is not known. Although box 1 of the intracellular domain adopts the typical SH3 (src kinase homology domain 3) folding (1464), no matching SH3 region is found in the sequence of Jak2, implying either the presence of an adapter protein or a mechanism different from the well-known SH3-SH3 binding (1357a). Activation of Jak2 occurs by transphosphorylation upon receptor dimerization, which brings two Jak2 molecules close to each other (550). Experiments with chimeric receptors suggest that mere juxtaposition of box 1 regions does not guarantee Jak2 activation (306). Exact homology of the rest of the intracellular domain is also required, suggesting the significance of the COOH-terminal residues (550).

III) Phosphorylation of the prolactin-R. Jak2 kinases transphosphorylate each other and are involved in the phosphorylation of Tyr residues of the prolactin-R itself (1514) (Fig. 1). Phosphotyrosines are of interest since they are potential binding/docking sites for transducer molecules containing SH2 domains. Although phosphorylation of Jak2 occurs in all active prolactin-R isoforms, Tyr phosphorylation of the receptor itself does not occur upon activation of the short form of the prolactin-R, despite the presence of four Tyr residues in its intracellular domain (660). Certain cellular functions, like proliferation, mediated by the short form of the prolactin-R, can take place without prolactin-R phosphorylation (1906). The long form of the prolactin-R also contains numerous Tyr residues, many of which are phosphorylated upon prolactin-R activation (1412).

A) STAT PROTEINS. The signal transducer and activator of transcription (STAT) protein family has been shown to be a major transducer in cytokine receptor signaling (834). The STAT family currently consists of eight members. Four of them, STAT1, STAT3, and especially STAT5a and STAT5b, have been identified as transducer molecules of the prolactin-R (631, 852). STAT contain five conserved features: a DNA-binding domain, an SH3-like domain, an SH2-like domain, and an NH₂- and a COOH-terminal transactivating domain (552). According to the consensus model of STAT activation (184, 552), a phosphorylated Tyr residue of the activated cytokine receptor interacts with the SH2 domain of STAT (Fig. 2). Then STAT, while docked at the receptor, is phosphorylated by the receptor-associated Jak kinase. The phosphorylated STAT dissociates from the receptor and hetero- or homodimerizes through its phosphotyrosine residues with the SH2 domain of another phosphorylated STAT molecule (184) (Fig. 2). Finally, the STAT dimer translocates to the nucleus and activates a STAT DNA-binding motif in the promoter of a target gene (184, 291). The consensus DNA motif recognized by STAT1, STAT3, and STAT5 homo- or heterodimers is termed GAS (-interferon activated sequence) (791) (Fig. 2). GAS consists of a palindromic sequence: TTCxxxGAA (791). Numerous promoters contain the GAS consensus motif, and multiple cytokines have been shown to activate these promoters in vitro (548, 658). It has been proposed that STAT interact with other signal transducers (e.g., glucocorticoid receptor) to initiate a cell- and cytokine-specific response (1687, 1688).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Signal transduction pathways initiated by activation of the prolactin (PRL) receptor. Jak/STAT pathway: members of the signal transducer and activator of transcription (STAT) protein family (834), STAT1, STAT3, STAT5a, and STAT5b are the central transducer molecules of the signal transduction pathways initiated by prolactin-R (PRL-R) activation (631, 852). STAT contain a DNA-binding domain, an SH3-like domain, an SH2-like domain, and an NH2- and a COOH-terminal transactivating domain (552). A phosphorylated Tyr residue (Y) of the activated long prolactin-R isoform interacts with the SH2 domain of a STAT. STAT, while docked at the receptor, is phosphorylated by the receptor-associated Jak kinase. Then, phosphorylated STAT dissociates from the receptor and hetero- or homodimerizes through its phosphotyrosine residues with the SH2 domain of another phosphorylated STAT molecule. Finally, the STAT dimer translocates to the nucleus and activates a STAT DNA-binding motif in the promoter of a target gene (184), termed GAS (-interferon activated sequence) (791). The tyrosine residues of the short form of prolactin receptor are not phosphorylated by Jak2, but the phosphotyrosine of Jak2 can serve as docking site for Stat1 (184). MAPK cascade: activation of the prolactin-R also activates the mitogen-activated protein kinase (MAPK) cascade (1417), which is involved in the activation of a wide range of transcription factors/immediate early genes by phosphorylation. Phosphotyrosine residues of the activated long prolactin-R isoform serve as docking sites for adapter proteins (Shc/Grb2/SOS) connecting the receptor to the Ras/Raf/MAPK cascade (382). Novel data indicate communication between the Jak/STAT and MAPK pathways (698). Ion channels: box 1 of the intracellular domain of prolactin-R is also involved in the activation of a tyrosine kinase-dependent, calcium-sensitive K+ channels through Jak2 (1435). The COOH terminal of prolactin-R's intracellular domain is involved in the production of the intracellular messengers [inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6)] that open voltage-independent Ca2+ channels (1452, 1659). Src kinases: prolactin also induces the activation of members of the Src kinase family, c-src (150, 1658) and Fyn (20), which are involved in the Tyr phosphorylation of phosphatidylinositol 3-kinase (PI3K) (152, 1453). Downregulation: Jak/STAT pathways can be inhibited by SOCS (suppressors of cytokine signaling) which inhibit Jak kinases (503, 762, 1289, 1312, 1411, 1672) or CIS (cytokine-inducible SH2-containing protein), which compete with STAT for docking sites on prolactin receptor (1144, 1914).

Of the STAT1, STAT3, and STAT5 proteins, STAT5 (earlier known as mammary gland factor, MGF) is recognized as the most important transducer of the long and intermediate isoforms of the prolactin-R (1060). STAT5 has two isoforms, STAT5a and STAT5b, encoded by two different genes, with 95% sequence homology and differences only in the COOH-terminal domain. Both isoforms possess a Tyr-694, which is phosphorylated by Jak2 (659). In addition to Tyr phosphorylation, activation of STAT involves serine/threonine phosphorylation as well. The major difference between STAT5a and -b isoforms lies in their serine/threonine phosphorylation sites (133). Protein kinase C (PKC)- and casein kinase II have been proposed as serine/threonine kinases activating STAT5 (133). Novel data indicate that STAT5 may fulfill inhibitory roles in regulation of gene transcription (1088).

B) OTHER SIGNALING PATHWAYS. I) Ras/Raf/MAP kinase pathway. Although Jak/STAT are the most important pathways initiated by activation of the prolactin-R, a number of reports implicate activation of the mitogen-activated protein (MAP) kinase cascade as well (242, 345, 383, 384, 518, 1307, 1323, 1417). Phosphotyrosine residues of the prolactin-R can serve as docking sites for adapter proteins (Shc/Grb2/SOS) connecting the receptor to the Ras/Raf/MAPK cascade (291, 382) (Fig. 2). Although initially the Jak/Stat and MAPK pathways were regarded as independent or parallel pathways, there are data suggesting that these pathways are interconnected (698).

II) Other kinases: c-src and Fyn. Several recent reports indicate prolactin-induced activation of members of the Src kinase family, c-src (150, 267, 1658) and Fyn (31a) (Fig. 2). Recently, prolactin-induced rapid Tyr phosphorylation of insulin receptor substrate-1 (IRS-1) and a subunit of the phosphatidylinositol (PI) 3'-kinase (103, 152, 1453) have been described. Both IRS-1 and PI 3'-kinase seem to be associated with the prolactin-R complex. It has been proposed that prolactin-induced activation of PI 3'-kinase is mediated by Fyn (31a) (Fig. 2).

III) Intracellular ion concentration. At least two events and two regions of the prolactin-R are involved in prolactin-induced ionic changes. Box 1 of the intracellular domain of the prolactin-R is involved in the activation of tyrosine kinase-dependent K⁺ channels by Jak2 (1435), whereas the COOH terminal of the intracellular domain is involved in the production of the intracellular messengers {inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P₄] and inositol hexakisphosphate (InsP₆)} that open voltage-independent Ca²⁺ channels (351, 1452, 1659) (Fig. 2).

C) DOWNREGULATION OF PROLACTIN-R SIGNAL: TYR PHOSPHATASES AND INHIBITOR PROTEINS. Because activation of prolactin-R results in Tyr phosphorylation of multiple signal molecules, it is expected that inactivation of signaling pathways involves Tyr phosphatases (184). Experimental data indicate that SH2 containing Tyr phosphatases SHP-1 and SHP-2 play less of a role in downregulation of prolactin signaling than in GH or other cytokines (23, 147, 490, 1411, 1445, 1770).

A newly revealed facet of cytokine receptor signaling is identification of SH2-containing protein families inhibiting the Jak/STAT pathways. These protein families are referred to as cytokine-inducible SH2-containing protein (CIS) (1042, 1144, 1914) and suppressors of cytokine signaling (SOCS) (503, 762, 1289, 1312, 1672). Their main mechanism of action in prolactin receptor signaling has been recently characterized (1411). The data indicate that prolactin induces acute and transient expression of SOCS-1 and SOCS-3 (1411). SOCS-1 and SOCS-3 switch off the prolactin receptor-mediated signaling by inhibiting the catalytic activity of Jak2 and activation of STAT proteins (1411). The CIS and SOCS-2 genes respond with prolonged activity to prolactin administration, and SOCS-2 seems to restore the cells' sensitivity to prolactin receptor stimulation probably by suppressing SOC-1's inhibitory effect (1411).

C.  Distribution of Prolactin-R

1.  Subcellular distribution: surface targeting, internalization, and nuclear translocation of prolactin-R

For proper surface targeting, glycosylation of the asparagyl residues (Asn³⁵, Asn⁸⁰, Asn¹⁰⁸) of the extracellular domain of the prolactin-R is crucial, although not an absolute requirement for prolactin-R activation (256). Although prolactin-R is mainly a cell-surface receptor, deglycosylated forms of prolactin-R can accumulate in the Golgi apparatus (256). Nitric oxide activates N-acetylglucosamine transferase, which is responsible for glycosylation of these intracellular receptors and promotes migration of these newly glycosylated receptors to the cell surface (183).

Earlier, endocytosis of prolactin and prolactin-R had been shown in several cell types (149, 447, 877). Surprisingly, even translocation of prolactin (1451) and prolactin-R to the nucleus has been demonstrated in different cell types (344, 1028, 1450). Nuclear translocation of prolactin-R can be accompanied by nuclear actions like stimulation of PKC (241, 343, 1449). Because activation of "classical" cytokine signaling pathways (Jak/STAT, MAP kinase) (1404) do not require nuclear translocation of prolactin-R, the mechanism and in vivo importance of prolactin-R internalization and nuclear actions still remain to be determined.

It is not surprising that prolactin-R and its message are found in the mammary gland and the ovary, two of the best-characterized sites of prolactin actions in mammals (1268). What may be truly surprising is that the receptor and its mRNA are also found in numerous parts of the CNS. The distribution of mRNA for the long form of the prolactin-R has been characterized in the rat brain (112). Abundant message is found in the choroid plexus, bed nucleus of the stria terminalis, amygdala, the central gray of the midbrain, thalamus, hypothalamus, cerebral cortex, and olfactory bulb (586, 587). Recently, prolactin binding sites have been described in the area postrema, which is one of the main chemosensitive areas of the brain lacking the blood-brain barrier (1112). Prolactin receptors are also present in a wide range of peripheral organs like the pituitary gland, heart, lung, thymus, spleen, liver, pancreas, kidney, adrenal gland, uterus, skeletal muscle, and skin (184, 1268).

	V. BIOLOGICAL ACTIONS OF PROLACTIN

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Our goal in this section is to summarize the most relevant actions of prolactin in the mammalian body. An extensive summary of prolactin's effects in different vertebrate species and organ systems can be found in the recent review written by Kelly et al. (184).

Although it has long been accepted that prolactin is involved in the development of the mammary gland (154), recent elegant techniques have confirmed such findings. Indeed, targeted disruption of the prolactin gene (prolactin knockout) (790) or knockout of the prolactin receptor (1358) results in abnormal mammogenesis characterized by complete absence of lobuloalveolar units in adult homozygous females. Prolactin knockout heterozygotes appear to have nearly normal mammogenesis that is indistinguishable from wild type (790). Transplantation of mammary epithelium from prolactin receptor knockouts into mammary fat pads of wild-type mice revealed that prolactin affects mammary morphogenesis in two different ways: it controls ductal side branching and terminal end bud regression in virgin animals via indirect mechanisms, but acts directly on the mammary epithelium to produce lobuloalveolar development during pregnancy (223). Lactogenesis clearly requires pituitary prolactin, since hypophysectomy during pregnancy prevents subsequent lactation (1306). Due to impairment of mammogenesis, both prolactin knockout (790) and prolactin receptor knockout (1358) homozygous mice fail to produce milk. Interestingly, although the heterozygous prolactin knockouts have normal mammogenesis (790), the F₁ generation heterozygous prolactin receptor knockouts are unable to lactate for their first litter (1358). However, milk is delivered perfectly normally to the F₁'s second litter (1358). The phenotype of the F₂ generation was similar (1358). With further reproductive cycles, mammogenesis in both the F₁ and F₂ generations proceeds sufficiently to support lactation, indicating that the defect in heterozygotes is one affecting the rate of mammary gland development. These experiments further indicate that two functional alleles of the prolactin receptor are required for full lactation.

Although there are dramatic differences between mammals in the hormonal requirements for galactopoiesis, the common absolute requirement is prolactin. Aqueous extracts of anterior pituitary gland containing prolactin (1093) initiate lactation in pseudopregnant rabbits (1694). Replacement of prolactin to hypophysectomized rabbits will fully restore lactation (364). On the other hand, while hypophysectomy of rats and mice stops lactation (1587), the replacement cocktail should minimally consist of prolactin and glucocorticoid or adrenocorticotrophin to maintain sufficient nurturing of the pups (173). Addition of growth hormone permits the maintenance of maximal lactation (1094).

It should be noted that none of these actions is solely due to prolactin, but the hormone is merely a player in an orchestra of hormones and growth factors that affect the mammary gland. A great deal of evidence from hypophysectomized-ovariectomized-adrenalectomized rodents suggests that the mammary gland's lobuloalveolar growth and development in vivo requires prolactin, estrogen, progesterone, and glucocorticoids (837). During pregnancy, the extensive branching of the ducts and development of the alveoli is a function of progesterone and prolactin or placental lactogen (837). There is evidence that insulin, growth hormone, thyroid hormone, parathyroid hormone, calcitonin, several growth factors, and even oxytocin may also play a role in galactopoiesis in various mammals (1779).

In the process of lactogenesis, prolactin stimulates uptake of some amino acids, the synthesis of the milk proteins casein and -lactalbumin, uptake of glucose, and synthesis of the milk sugar lactose as well as milk fats (118, 1779).

Controlled exclusively by promoter III of the rat prolactin-R gene (812), the mammary gland expresses mainly the long form of prolactin-R (1268). The activation of promoter III involves binding of C/ERB (CCAAT-enhancer binding protein) and Sp1 (recognizing GC boxes of DNA) transcription factors to their respective binding elements and activation of a downstream sequence element resembling the consensus AP2 binding site (812). As in vitro studies indicate (750) and in vivo studies confirm (854), prolactin-R in the mammary gland is phosphorylated upon prolactin binding (1868) and activates the Jak2/STAT5 pathway responsible for both mammo- and lactogenesis. STAT5 (especially STAT5a) activated by the long form of the prolactin-R induces transcription of milk protein genes (151). Null mutation of the STAT5a or STAT5b gene is detrimental to tubuloalveolar development of the mammary gland and results in inability to lactate in homozygous (/) females (184). The signal transduction pathways over which prolactin induces mammary gland growth and development have been extensively studied in vitro and reviewed recently (750).

In most rodents, prolactin acts as a luteotrophic hormone by maintaining the structural and functional integrity of the corpus luteum for 6 days after mating (1232). This "luteotrophic" action of prolactin, which has been best described in the rat, is characterized by enhanced progesterone secretion (580). Progesterone is essential for the implantation of the fertilized ovum (along with estrogen), maintenance of pregnancy, and inhibition of ovulation (580). In the absence of prolactin, the dominant steroid produced by the corpus luteum of the rat is 20-hydroxyprogesterone, whose synthesis from progesterone is catalyzed by 20-hydroxysteroid dehydrogenase (1508). This metabolite of progesterone is "inactive" in most progesterone bioassays. Prolactin enhances progesterone secretion two ways: prolactin potentiates the steroidogenic effects of luteinizing hormone (LH) in granulosa-luteal cells (1471) and inhibits the 20-hydroxysteroid dehydrogenase enzyme, which inactivates progesterone (580). In other rodents such as hamsters, prolactin is part of a "luteotrophic complex" consisting of LH, follicle stimulating hormone (FSH), and prolactin (672). There is some evidence that prolactin may also be part of a luteotrophic complex in dogs (1322) and primates (1472). In humans, high levels of prolactin inhibit granulosa cell luteinization (10, 1170) and steroidogenesis (1017). Further evidence of luteal dependence on prolactin is found in prolactin receptor knockouts who lack normal luteal function and thus are sterile due to decreased ovulation rate, aberrant oogenesis, and implantation failure (184). Prolactin is essential for progesterone biosynthesis and luteal cell hypertrophy during pregnancy. In addition to luteal function, the prolactin-R mediates numerous functions in granulosa cells and oocytes as well (184).

Aside from its luteotrophic role, there is evidence in the rat that prolactin may be luteolytic as well (1111, 1887) by inducing programmed cell death in the corpora lutea (901, 1156). Prolactin's luteolytic effect seems to be mediated by CD3-positive lymphocytes, which increase the expression of the membrane form of the Fas ligand, known to mediate luteal cell death through the Fas receptor (989). In the rat, as many as three generations of corpora lutea may appear on the ovary. There is evidence that prolactin may perform a "housekeeping function" by inducing the structural regression of the oldest of these. It should be emphasized that the corpora lutea are nonfunctional at the time that prolactin exerts this effect. The mechanism by which prolactin can be both luteotrophic and luteolytic is still uncertain. One suggestion is that at some critical time between periods of exposure to prolactin during the estrous cycle, the corpora lutea of the rat acquire the capacity to express monocyte chemoattractant protein-1, which subsequently interacts with prolactin on proestrus to induce luteal cell death (200, 1773).

Both short and long isoforms of the prolactin-R are present in the ovaries (337, 1621). Transcription of the prolactin-R in the ovaries is controlled by intricate developmental and hormonal regulation (340, 1730). Regulation of transcription of the prolactin-R gene in the ovaries is accomplished by the gonad-specific promoter I and the "generic" promoter III. Essential transcriptional activator of prolactin-R's promoter I is steroidogenic factor-1 (SF-1)-binding consensus element, which is activated by SF-1 (810, 811). SF-1 is a specific zinc finger DNA binding protein, also known as Ad4BP (1089).

Recently, expression of a prolactin-R associated phosphoprotein (PRAP) has been described in luteal cells (468). PRAP binds to the intracellular domain of the long form of the prolactin-R, but not to the short form. Expression of PRAP is upregulated by estrogen and prolactin (469). Structurally, PRAP shows 89% homology with a newly characterized form (type 7) of 17-hydroxysteroid dehydrogenases/17-ketosteroid reductases (17-HSD), suggesting that PRAP may be an enzyme catalyzing the conversion of estrone to estradiol (1324).

A) FEMALE RECEPTIVITY. There are data suggesting that prolactin influences reproductive behavior (476). In humans, high prolactin levels are associated with psychosomatic reactions including pseudopregnancy (1653). There are prolactin-R in the ventromedial nucleus of the hypothalamus (375), an area which controls female sexual behavior. Coincidentally, iontophoresis of prolactin to this area increases local neuronal electrical activity (743). However, in rats, prolactin's precise action has been confounded by the multiplicity of experimental designs. For example, when given in the third ventricle of estrogen and progesterone-primed ovariectomized rats, prolactin diminishes lordosis frequency, an index of sexual receptivity (470). Though, when given in the midbrain of estradiol-treated ovariectomized rats, prolactin enhances sexual receptivity (738). Enhancement of endogenous prolactin secretion in response to dopamine antagonism has been reported to have no effect on mating behavior in females (1654), whereas elevation of prolactin secretion in response to the nursing stimulus diminishes sexual behavior (1655). In contrast, when the rat is sexually receptive in the afternoon of proestrus, suppression of the spontaneous release of prolactin with a dopamine agonist dramatically attenuates sexual receptivity (1884). Finally, although a null mutation of the prolactin-R gene in the mouse produces most of the defects associated with a deficiency of prolactin, such receptor-deficient females appear to mate normally with heterozygote or wild-type males (1358, 1680). Thus these data, taken together, do not provide a firm basis for assigning a well-defined role for prolactin in female sexual behavior. In contrast, it is clear that prolactin suppresses stereotypical male sexual behavior in rats (451, 896) and sheep (629).

B) PARENTAL BEHAVIOR. Probably the best-characterized prolactin-driven behaviors are the parental behaviors. In mammals, maternal behavior is the most extensively studied (218, 221, 1086, 1530). These include nest building as well as gathering, grouping, cleaning, crouching over, and nursing of the young by the mother. Although most widely described in rats, there is also an extensive literature on the effects of prolactin on the induction and maintenance of these maternal behaviors in mice, rabbit, hamsters, and sheep (219, 1329). It should be emphasized that prolactin, by itself, does not initiate maternal behavior, but merely decreases the latency to the onset of maternal behavior. Intracerebroventricular infusion of prolactin decreases the latency to initiation of maternal behavior in steroid-primed rats (220). The basic observation was made that nulliparous female rats treated with a pregnancy-like regimen of estrogen and progesterone for 10 days showed maternal behaviors with a mean latency of 5-6 days. Superimposition of prolactin treatment on the ovarian steroid regimen reduces the latency of maternal behavior to 1-2 days (217). In addition, hypophysectomized rats failed to display a facilitation of maternal behavior in response to the sequential steroid treatment. On the other hand, prolactin-hypersecreting pituitary transplants placed beneath the kidney capsule of hypophysectomized female rats kept on a maternal ovarian steroid replacement regimen, dramatically advanced the onset of maternal behaviors (218). Suppression of endogenous prolactin release with bromocryptine prevents the onset of maternal behavior, whereas superimposition of prolactin promotes it (222). Prolactin may be exerting this effect by acting within the medial preoptic area of the hypothalamus (220, 1701).

Pup contact has been shown to induce transcription of the long-form prolactin-R mRNA in the brain of female rats. The effect of pup contact on prolactin-R expression is prevented by ovariectomy and hypophysectomy (1701). It seems that the effect of pup contact is not sex specific; the same induction of brain prolactin-R long-form mRNA expression and maternal behavior can be observed in pup-contacted male rats. Administration of prolactin promotes, while female contact suppresses, the effect of pup contact in males (1530). In mice carrying a germ line null mutation of the prolactin receptor gene, homozygous mutant and heterozygous mutant nulliparous females show a deficiency in pup-induced maternal behavior (1086). Moreover, primiparous heterozygous females exhibit a profound deficit in maternal care when challenged with foster pups. Such data suggest that pup contact is required for transcription of the prolactin receptor whose stimulation by prolactin eventuates in maternal behavior (1086).

Although not as widely studied, prolactin may have a role in paternal care as well. The data for this role are most convincing in fish and birds but somewhat less convincing in mammals (1574). Indeed, because prolactin is an "old" hormone, it could be that this role in our most recent ancestors has become somewhat redundant. This is emphasized by the significant stereotypical paternal role of nonmammalian vertebrates (e.g., the male sea horse is the incubator) and the almost nonexistent role in most mammals.

C) PROLACTIN-R IN THE HYPOTHALAMUS. Although the brain contains mainly the long isoform of the prolactin-R, the hypothalamus contains both the long and short forms (323). Within the hypothalamus, prolactin-R mRNA-containing neurons have been found in the anterior as well as the mediobasal hypothalamus (323-325). Also, the mRNA of the long form of prolactin-R is found within the periventricular, paraventricular, supraoptic, arcuate, and ventromedial nuclei of the hypothalamus as well as the medial preoptic area (112). Immunocytochemical data support these observations by showing that these hypothalamic areas contain prolactin-R protein as well (1028, 1415, 1495). Expression of prolactin-R in the brain increases with age (325, 993, 1241), exposure to estrogens (1264, 1598), elevation in serum prolactin levels, and by pup contact (1700).

Few studies have examined the signal transduction pathways specifically activated upon binding of prolactin to its receptor in the CNS. Preliminary data from our laboratory indicate that systemic administration of prolactin results in nuclear translocation of STAT5 in neurons of the mediobasal hypothalamus. This suggests that the signal transduction pathways coupled to prolactin-R in CNS neurons are similar to those described in peripheral tissues. Prolactin also increases the expression of NGFI-A, NGFI-B, c-fos, and c-jun in numerous populations of CNS neurons, among them the tuberoinfundibular (TIDA) neurons of the arcuate nucleus (1524).

Prolactin is a common mediator of the immunoneuroendocrine network, where nervous, endocrine, and immune systems communicate with each other (631). Prolactin plays a significant role in regulation of the humoral and cellular immune responses in physiological as well as pathological states, such as autoimmune diseases (253, 1295, 1850).

The earliest evidence that prolactin plays a role in the immune response was the demonstration in 1972 that exogenous prolactin enhanced thymic function in prolactin-deficient dwarf mice (314). Shortly thereafter, Nagy and Berczi (1269) found that hypophysectomy or suppression of prolactin secretion with bromocryptine (1273) led to attenuation of humoral or cell-mediated immunity that could be reversed by treatment with exogenous prolactin. A large number of immune perturbations were found to be associated with prolactin deficiency (148, 1269-1273).

As noted in two recent reviews (1145, 1871), prolactin stimulates mitogenesis in both normal T lymphocytes (1828) and the Nb2 lymphoma cell line (1622). It should not be surprising that prolactin affects lymphocytes since prolactin-R has been detected on human peripheral lymphocytes (1517-1519) and their mRNA expression is regulated by prolactin itself (442). Moreover, effects of prolactin on lymphocytes may involve interleukin (IL)-2 since T-lymphocyte activation by IL-2 requires prolactin (344, 712). Interestingly, prolactin's site of action for modifying the effects of IL-2 on lymphocytes appears to be the nucleus (343). Prolactin is also required for mitogen-stimulated proliferation of lymphocytes (741, 757, 758). Nb2 cells, derived from immature T lymphocytes, are dependent on the mitogenic activity of prolactin (1622, 1713). Indeed, this property has served as the basis for a highly sensitive, specific bioassay for prolactin. However, there is not uniform agreement on the role of prolactin in hematopoiesis. Although targeted disruption of the prolactin gene leads to numerous defects in prolactin-dependent events such as lactation, there is no difference between homozygotes and heterozygotes in the frequency of B- and T-cell antigen expression (790). Such results argue that prolactin does not play an indispensable role in primary lymphocyte differentiation or its absence during development can be compensated by other factors.

The role of prolactin in the immune response of the organism is a matter of continuing concern. It appears that immune responses in vivo are enhanced by prolactin. For example, prolactin-secreting pituitary grafts placed beneath the kidney capsule (1270) or administration of prolactin (1272) restores dinitrochlorobenzene-induced contact dermatitis impaired by hypophysectomy. On the other hand, skin allograft transplants elevate serum prolactin (725). During graft rejection, lymphocytic prolactin gene expression is also upregulated (1601). Moreover, elevated serum prolactin levels induced by skin allografts can be suppressed by either bromocryptine or an antilymphocytic serum (1505). However, only antilymphocytic serum prolongs the survival time of the graft (1505). These data suggest that lymphocytic prolactin plays a specific role in skin graft rejection and may play a role in other transplantation responses as well (764).

Immunocytochemical demonstration of prolactin-R on T and B lymphocytes (1769) was followed by detection of mRNA encoding the short and long prolactin-R isoforms in the thymus, spleen, lymph nodes, and bone marrow of both rats and mice (1020). Expression of prolactin-R isoforms was more extensively mapped in rat splenocytes and thymocytes from birth to adulthood (703), as well as during the estrous cycle, pregnancy, and lactation (704). Prolactin's functions are the most extensively described and reviewed in the Nb2 cell line (1920). This cell line also expresses an intermediate (393 amino acid) isoform of prolactin-R (184). In Nb2 lymphocytes, activation of the prolactin-R is associated with (945) 1) rapid tyrosine phosphorylation of STAT5a, STAT5b, STAT1, and STAT3; 2) rapid and selective formation of STAT5a/b heterodimers; 3) marked Ser, but not Thr phosphorylation of STAT5a and STAT5b; and 4) the appearance of two qualitatively distinct STAT5 protein complexes that discriminate between oligonucleotides corresponding to the prolactin response elements of the -casein and interferon regulatory factor-1 gene promoters (945).

One of the least understood actions of prolactin is regulation of solute and water transport across mammalian cell membranes (1602). Studies in this area were motivated by the finding in lower vertebrates that prolactin stimulates solute transport across cell membranes and thus could be an osmoregulatory hormone (153). Some of the actions in mammals are easier to envision in a physiological perspective than others. For example, prolactin exerts a host of activities on transport of solute across mammary epithelial cel