《动物生理学》课程教学资源（文献资料）催乳素综述 Prolactin - Structure, Function, and Regulation of Secretion

Prolactin:Structure,Function and Regulation of Secretion MARC E FREEMAN.BELA KANYICSKA.ANNA LERANT.AND GYORGY NAGY Se Brain creting cell lines rv. m of prol n s Prolac CNS an.Marc E.Bela Kanvicska.Anna Lerant,and Gyorgy Nagy.Prolactin:Structure Function.and ion of Secretion.Physio Rev 80: -1631,200 Prolactin is a protein hormone of the anterior pituitary within the central n rous system.the immune sv the mammary gland itsef Moreover,its biological actions are not limited solely to reproduction because t has been *The sequence of authorship does not imply importance of but s presented alphabetically http://physrev.physiology.org 31-33/00$the American Physiological Society

Prolactin: Structure, Function, and Regulation of Secretion MARC E. FREEMAN, BE´ LA KANYICSKA, ANNA LERANT, AND GYO¨ RGY NAGY* Department of Biological Science, Florida State University, Tallahassee, Florida; Department of Anatomy, The University of Mississippi Medical Center, Jackson, Mississippi; and Neuroendocrine Research Laboratory, Department of Human Morphology, Semmelweis University School of Medicine, Budapest, Hungary I. Introduction 1524 II. Prolactin Chemistry and Molecular Biology 1524 A. Prolactin: gene, primary structure, and species specificity 1524 B. Secondary and tertiary structure of prolactin 1524 C. Prolactin variants 1524 III. Sites of Synthesis and Secretion of Prolactin 1525 A. Anterior pituitary gland 1525 B. Brain 1526 C. Placenta, amnion, decidua, and uterus 1527 D. Mammary gland and milk 1527 E. The immune system 1528 F. Prolactin-secreting cell lines 1528 IV. Prolactin Receptors 1529 A. Prolactin receptor: gene, splicing variants, and isoforms 1529 B. Activation of prolactin-R and the associated signal transduction pathways 1529 C. Distribution of prolactin-R 1532 V. Biological Actions of Prolactin 1533 A. Reproduction 1533 B. Homeostasis 1536 VI. Patterns of Pituitary Prolactin Release 1537 A. Circadian rhythm of prolactin secretion 1537 B. Patterns of prolactin secretion in different reproductive states 1538 C. Prolactin release in response to exteroceptive stimuli 1540 VII. Regulation of Pituitary Prolactin Secretion 1542 A. CNS 1542 B. Intrapituitary regulation 1573 C. Peripheral organs 1579 VIII. Epilogue 1585 Freeman, Marc E., Be´la Kanyicska, Anna Lerant, and Gyo¨rgy Nagy. Prolactin: Structure, Function, and Regulation of Secretion. Physiol Rev 80: 1523–1631, 2000.—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 * The sequence of authorship does not imply importance of contribution but is presented alphabetically. PHYSIOLOGICAL REVIEWS Vol. 80, No. 4, October 2000 Printed in U.S.A. http://physrev.physiology.org 0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society 1523

1524 FREEMAN,KANYICSKA,LERANT,AND NAGY Volume 80 rigin ides inhibitor control ov peripheral organs the purbts I.INTRODUCTION a the tin prohormone of 227 amino acids.The signal peptide pituitary gland,the lactotrophs.The hormone was given contains 28 amin acids;thus the mature human prolactin its name based on the fact that an extract of bovine is co e growth crop sac an pr with thre a single chair of amin la mote lactation in rabbits (1477).However we now ap ciate that prolactin has over 300 separate biological ac- and Cys -Cys1 in humans)(357).The sequence homol- tivities(18)not represented by its name.Indeed,not only ogy can vary from the striking9 among pr 【0a does prolact erve multiple roles in static roles in the organism.Futher amino acids whereas in sheen (1036),pig(1035),cattle aware that synthesis and secretion of prolactin is not (1851),and humans(1624)it consists of 199 amino acids restricted to the anterior pituitary gland,but other organs with a molecular mass of ~23,000 Da and sues in t body have th Indeed,the B.Secondary and Tertiary Structure of Prolactin tilin." In this review we integrate the burgeoning informa- n the secondary str of prolactin hav shown that 50%6 of the amino acid chain is arr ed in onon prolactin structure (sect.)sy and re a-helices,while the rest of it forms loops(169).Although ism of th was predicted earlier (1311), there are still no direc functions(sect.v),and the patterns (sect.vi)and regula- tion of its secretion (sect.vu). mology modeling approach (635).based on the structura similarities between prolactin and other helix bundle pro IL. teins,especially growth hormone )According to the long a-helicmension pr s arranged lel A.Prolactin:Gene,Primary Structure,and 438). Species Specificity C.Prolactin Variants ased on its gen rowth hormone placental lact family ig roup I of the Although the major form of prolactin found in the is 23 kD helix bundle protein hormones (195 791)1 Genes encod. nts of prolactin have been ing prolactin,growth hormone,and placental lactogen lactin variants plicing of the 131 mmon ance al ge ot by gen primary transcript,proteolytic cleavage and other post- ages ooge translational modifications of the amino acid chain. 358).In the human genome,a single found on e 6,encodes prolactin (1363) The prolactin 1.Alternative splicing chromosom and is compe d or exons and Altemative splicing of prolactin mRN regulated by two inder The evidence si proximal 5,000-bp region directs pituitary-specific expres- spliced prolactin variant of 137 amino acids has been sion (160),while a more upstream promoter region is described in the anterior pituitary(501,1882).In addition

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. I. INTRODUCTION Prolactin is a polypeptide hormone that is synthe￾sized 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 pro￾mote lactation in rabbits (1477). However, we now appre￾ciate that prolactin has over 300 separate biological ac￾tivities (184) not represented by its name. Indeed, not only does prolactin subserve multiple roles in reproduction other than lactation, but it also plays multiple homeo￾static 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 “versa￾tilin.” In this review we integrate the burgeoning informa￾tion on prolactin’s structure (sect. II), synthesis and re￾lease 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 regula￾tion 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 func￾tional properties, prolactin belongs to the prolactin/ growth hormone/placental lactogen family [group I of the helix bundle protein hormones (195, 791)]. Genes encod￾ing prolactin, growth hormone, and placental lactogen evolved from a common ancestral gene by gene duplica￾tion (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 expres￾sion (160), while a more upstream promoter region is responsible for extrapituitary expression (159). The hu￾man prolactin cDNA is 914 nucleotides long and contains a 681-nucleotide open reading frame encoding the prolac￾tin 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 homol￾ogy 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 a-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 ho￾mology modeling approach (635), based on the structural similarities between prolactin and other helix bundle pro￾teins, especially growth hormone (2, 438). According to the current three-dimensional model, prolactin contains four long a-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. Pro￾lactin variants can be results of alternative splicing of the primary transcript, proteolytic cleavage and other post￾translational modifications of the amino acid chain. 1. Alternative splicing Alternative splicing of prolactin mRNA has been pro￾posed 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, 1524 FREEMAN, KANYICSKA, LERANT, AND NAGY Volume 80

PROLACTIN 1525 alterative splicing involving retention of introns is also fore exocytosis and involves esterification of hydroxvl possible.However,alternative splicing is not considered a groups of serine and threonine residues(670).Phosphor- major source of prolactin variants. ylated prolactin isoforms have beer e(1337)pituitary gla 2.Proteolytic cleavage sphorylae Of the cleaved forms that have been characterized. total pituitary prolactin in catle( 14- 16 and 2 2-kDa prolactin variants have been most prolactin has been shown to be secreted in vitro,it is not The sig widely s known if it is secreted into the plasma in vivo. posttra pro in th tactins has (736.hC ated pre ctin has much lower hiological activity than non tivity with the 16-kDa fragment (335 1765)Both s m to phosphorylated prolactin (1859).However. DOS lated prolactin may subserve a unique role as an autocrine ae虹 prolacun-(I oI p. tin se (a642207 an supp (1643)pituitary glands as well as in human plasma (643) (768).Phosphor rvlation of pr The 16-kDa prolactin is a product of kallikrein enzymatic ratio of phosphorvlated to nonphosphorylated isoforms activity. ced seems to be regulated throughout the estrous cycle (769) an estrogen-indu trypsin-lik ugn the ce of th f 1433 cleave rat prolactin in a thiol-dependent manner ist to the simal transduction pathways (362)and proliferative activities alters the conformation of prolactin such that kallikrein init iated by unmodified prolactin on Nb2 lymphoma cells 81 ves xypep of nl /11780 spl cells agment can be detected in pituitary and tis blot using an antiserum produced specifically against the c)GLYCOSYLATON.Glycosylated prolactin has been -kDa prolactin fragment(45).Its ems that the produc found in the pituitary glands of a wide variety of mamma ti lian,amphibian,and avia ,spee (1640 The degree o a. inhibition by dopa mine (45)Althe an fragments have been found in pituitary gland and serum (1640).The linkage of the earbobydrate moiety may be more work is required to de ermine their physiologica ility rem they may be harie2atioDmeCa either through nitrogen (N-glycosylation)or oxygen (O ohydrate residue s of th varyt diffe b ecies physiological and pathologieal states 3.Other posttranslational modifieations 1640).Lk othe prolactin variants,glycosylation also Besides prote tin olhtedeavagethenaioiotpmolhg ogical activity (1127 of the itary gland or the plasma.These include dimerization and poly merization,phosphorylation,glycosylation,sulfation, and deamidation (1702) activity or clearance of the molecule A) tion with binding proteins.such as immund SITES OF SYNTHESIS AND SECRETION covalent and noncovalent bonds may result in high-mo OF PROLACTIN lecular-weigh forms cular A.Anterior Pituitary Gland detection and difrerential diagno sis of different 1.Morpholog of lactotrophs lactinemias is targeted primarily in clinical studies(299). B).Phosphorylation of prolactin oc The cells of the anterior pituitary gland which syn- curs within the secretory vesicle of lactotrophs just be- thesize and secrete prolactin were initially described by

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 ac￾tivity with the 16-kDa fragment (335, 1765). Both seem to possess a unique biological activity, which will be de￾scribed later. The 16-kDa fragment [prolactin-(1O148)] 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-(1O173)]. 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 produc￾tion 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 prolac￾tin variants can be the result of other posttranslational processing of the mature molecule in the anterior pitu￾itary 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 aggrega￾tion with binding proteins, such as immunoglobulins, by covalent and noncovalent bonds may result in high-mo￾lecular-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 pro￾lactinemias is targeted primarily in clinical studies (299). B) PHOSPHORYLATION. Phosphorylation of prolactin oc￾curs within the secretory vesicle of lactotrophs just be￾fore exocytosis and involves esterification of hydroxyl groups of serine and threonine residues (670). Phosphor￾ylated 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 phospho￾prolactin has been shown to be secreted in vitro, it is not known if it is secreted into the plasma in vivo. The sig￾nificance of phosphorylated and nonphosphorylated pro￾lactins has been reviewed in detail (736). Phosphorylated prolactin has much lower biological activity than non￾phosphorylated prolactin (1859). However, phosphory￾lated 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 phos￾phorylated 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 mamma￾lian, 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 oligosac￾charide 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 prolac￾tin 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 syn￾thesize and secrete prolactin were initially described by October 2000 PROLACTIN 1525

1526 FREEMAN,KANYICSKA,LERANT,AND NAGY Volume 80 light mic lation phs are not hor orphology motrophs,comprise 20-50%of the cellular population of hormonal phenotype,distribution,or function. physiol wer B.Brain e(109 (110.127.and human111.725.1387 using so The first observation that prolactin is produced in the brain was by Fuxe et al.(594)who found prolactin im- specific prolactin antibodies.Ontogenetically,lactotrophs Pitl-depend y was in the te n th 643.1382.1599) tum (433),caudate putamen (502,737),brain stem The morphology and distribution of lactotrophs have (433.737.cerebellum (1589).spinal cord (737.1630) been best described in the rat (1768) where prolactin- containing ce distributed in the choroid plexi,and the circumventricular organs (1741). are 127 heterogeneous a aring as either nolyhe dral or a Prolactin immunoreactivity is found within numer but at times rounde ed or oval (429).With the use of either shypothalamic areas in a variety of mammals(29,677 velo se me 67 132 .1630.1741) un th rat hypo their dial vor ia【676. anule size and content (1650)as well as on the (735)nuclei Several an ches have been taken to amount of prolactin and prolactin mRNA present (1813) 2.functional heterogeneity of lactotrophs nt ofi ie hy Aside from morphological heteroge heity.lactotrophs display functional heterogeneity as well.Development of the reverse hemolytic plaque assay (572,576,1300)led t hypoth nd( ith the and tide mapping eted from a distinct cell type in the pituitar othalamic eDNA from inta and hy gland,the lactotroph,both prolactin and growth hormone ysectomized rats (1882)it has been established that the can also be secreted from the interm primary structure of prolactin of hypothalamic and pitu opns (272 Ines rigin i the neonatal rats (770)differentiate into lactotr nhs in the olactin gene of the anter r pituitary (501 1882) presence of estrogen (191).Mammosomatotrophs also Although the role of prolactin of hypothalamic origin differentiate into lactotrophs in pups in the pre nce of a in the central nervous system (CNS)is not apparent,it appears 1429 nyp ap There also an ars to he functional het into 16 and 14-kDa fragments (435).We do not know if among lactotrophs with regard to their regio nal distrib ctin of neural origin exerts its central effect as a tion within the anterior lobe (1246)as well as to the neuromodula of tue ond to thy releasing horme in art bec e it is difficult to differ ntiate h reen the (TRH than those of the inner zone adia ent to the inter. effects of p olactin of pituitary versus hypothalamic ori mediate lobe of the pituitary gland (188) On the other gin in the CNS One cause of thes difficulties is tha and, dopami ary p bypa oity is alsc roid reflected in the discordance between prolactin gene tran- plexi have a very high density of prolactin receptors(pro scription and prolactin release in some lactotroph popu- lactin-Rs)as demonstrated by autoradiography (1113

light microscopy using conventional staining techniques (753). These cells, designated lactotrophs or mam￾motrophs, comprise 20–50% of the cellular population of the anterior pituitary gland depending on the sex and physiological status of the animal. Lactotrophs were sub￾sequently identified unequivocally by immunocytochem￾istry 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 lateroven￾tral 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 discontin￾uous 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 distribu￾tion 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 inter￾mediate 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 tran￾scription and prolactin release in some lactotroph popu￾lations (296, 1562). Taken together, it is clear that lac￾totrophs are not homogeneous in their morphology, hormonal phenotype, distribution, or function. B. Brain The first observation that prolactin is produced in the brain was by Fuxe et al. (594) who found prolactin im￾munoreactivity in hypothalamic axon terminals. Prolactin immunoreactivity was subsequently found in the telen￾cephalon 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). 1. Hypothalamus Prolactin immunoreactivity is found within numer￾ous hypothalamic areas in a variety of mammals (29, 677, 678, 737, 1321, 1630, 1741). Within the rat hypothalamus, prolactin immunoreactivity is detectable in the dorsome￾dial, ventromedial (676), supraoptic, and paraventricular (735) nuclei. Several approaches have been taken to prove that prolactin found in the hypothalamus is synthe￾sized locally, independent of prolactin synthesis in the pituitary gland. Indeed, hypophysectomy has no effect on the amount of immunoreactive prolactin in the male hy￾pothalamus 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 hypoph￾ysectomized rats (1882), it has been established that the primary structure of prolactin of hypothalamic and pitu￾itary 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 ap￾propriate 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 as￾cribe 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 ori￾gin 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 cho￾roid plexi of the brain ventricles. Coincidentally, choroid plexi have a very high density of prolactin receptors (pro￾lactin-Rs) as demonstrated by autoradiography (1113, 1526 FREEMAN, KANYICSKA, LERANT, AND NAGY Volume 80

PROLACTIN 1527 1853.1854).immunocytochemistry (1495).standard re. prolactin-like protein J(1752).which is produced by the ceptor binding assays(1242),reverse transcriptase PCR, decidua during early pregnancy.Each of these prolactin- and ribonuclease protection assay (590).Interestingly. the 9731.1745 1746ae hlood to the cerebrosninal fluid by way of the choroid tin-rele asing factors (PRE).P esterone has also been plexus,pituitary prolactin may also reach the brain by identified as a potent stimulator of decidual prolactin retrograde blood flow from the anteror pituitary to the production (1143) In addition to stimulatory factors ypo in th5 the ac 731 decidual prolactin release and compe tes with the decid to its effects in the CNS without attributing a source. ual PRF (731).Recently,the N5 endometrial stromal cell line.which expresses the prolact gen driven by the 2.Regulation of hypothalamic prolactin synthesis d as a possibl Some well-established stimulat (210)Ample evidence indicates that decidual prolactin lactin se duction.For example,ovarian steroids modulate hypotha- diffuses into the amniotic fluid (1473,1474,1476,1501) release prolactin (43 437 Although the function of amniotic prolactin is uncertain n suggest al（S may serve ar e with Pestradiol (436).suggesting that these bryo/fetus 8o8uha have estrogen receptors.Ovariectomy lowers hypotha- Finally,the nonpregnant uterus has been shown to be lamic prola a source of prolactin as well.Indeed,a decidual-like pro conten n In rom pituitary prolactin (611 fact 15511mt o stim lates the production of decidual prolactin.it appears to be lar iniection of vasoactive intestinal peptide will i otent inhibitor of myometrial prolactin production (61 ogical role for myometrial prolactin has ever,other ously.much more work is needed to establish the conto of hypothalamic prolactin synthesis and release. D.Mar mary Gland and Milk C.Placenta,Amnion,Decidua,and Ute ru n can be det The placenta,in addition to its bidirectional fetoma ortion of the ternal metabolic transport functions,has a wide array of prolactin found in the milk originate in the pituitary endocrine functions as well.Among its ma gland and reaches the mammary gland through thec 253537 74,14sz 88,149 n h some or a mm 151 1605.1896.hamster874,1662-1664).cowC46.1612. Indeed a sic mincant amount of radiolabeled prolactin pig (568),and human (728).The rat placenta produces a introduced into the circulation appears in milk (685, bewildering array of prolactin-like molecule that bear 1253).Apparently,prolactin 058, reaches the milk by first PL mary eplt PLP)have been variously identined as PLL PLI PLIm within the mammary epithelial cell.and is ultimately (mosaic),PL-Iv (variant)(349,1487,1490),or PLP-A,-B. transported by exocytosis through the apical membrane -D-E.-F,and-G(356.392,851).n ddition into the alv lar lumen (1352,1583) a in (PLF in additi from the blood,th 1057 The decidua on the other hand prolactin.The s an of ce of nrolactir like molecule,that is indistinguishable from pituitary pro mRNA(992,1682)as well as synthe sis of immunoreactive lactin in human (35,342,147 1707),but is somewhat prolactin by mammary epithe al cells of lactating rats has dissimilar in rat (688).A novel member of this family is been described (1063 1064).It is possible that de novo

1853, 1854), immunocytochemistry (1495), standard re￾ceptor 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 ac￾tions 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 pro￾lactin secretion also affect hypothalamic prolactin pro￾duction. For example, ovarian steroids modulate hypotha￾lamic synthesis and release of prolactin (436, 437). Approximately 33% of the prolactin immunoreactive neu￾rons in the medial basal hypothalamus can be labeled with [3 H]estradiol (436), suggesting that these neurons have estrogen receptors. Ovariectomy lowers hypotha￾lamic 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 intracerebroventricu￾lar injection of vasoactive intestinal peptide will increase the amount of hypothalamic prolactin mRNA (212). How￾ever, other established stimulators of pituitary prolactin secretion such as TRH are without effect (437). Obvi￾ously, 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 fetoma￾ternal 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 pro￾lactin 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) prolac￾tin-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 decid￾ual 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 pro￾lactin, indistinguishable from pituitary prolactin (611), has been identified in the myometrium of nonpregnant rats (1855). Interestingly, although progesterone stimu￾lates 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 circu￾lation. 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 mem￾brane, 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 October 2000 PROLACTIN 1527

1528 FREEMAN,KANYICSKA,LERANT,AND NAGY Volume 80 immunoreactive prolactin declines over 24-48 h in mam- jection in mice.Administration of bromocryptine,a D mary gland explants(992).The mammary gland may also diminishes act a sa po ilk.prol anima d in has ahat ituta important 16-kDa variant of prolactin mentioned previ modulate the elaboration of lymphocytic prolactin and ously (33 prolactin,prod tha rther in which makes this proteolytic step a possible target of gation. breast cancer r rch(636) he phy 1010g for milk-born e prolactin has F.Prolaetin-Secreting Cell lines d in window of ne onatal life.the gastrointestinal tract lacks the ability to digest protein and likewise po s the derived from pituitary tumors have beer developed.The ability to a nta (MtT/W5)）is d ell line was a mamm ing this ately 20%6 of the ina wistar-Furth rat (724.1907).B ted in milk n to the neonatal circulation (66 owth hormone,they were designated It has been shown that milk prolactin participates in the as GH cells.It was subsequently found that some of the the neuroendocrine (1596,1629)and im- and heterogeneous (172 systems motronhs)hoth hormones mammos matotronhs E.The Immune System neither hormone(189,192).Similarly,the GH and GHC cell lines produce prolactin and growth hormone bu o172 e of using cell lines rathe Indeed immune- nt cells from thymus and snle than primary pituitary lactotrophs is that clonal cells are as well as peripheral lymphocytes contain prolactin usually immortal,can be easily stored,and thus provide a mRNA and release a bioa e prola n th perpetual supply of cells witho 1(44 612, 121 10 rine (1214)and human (1886)immune petent cells primary cultures of pituitary cells.For example,unlike but size variants of prolactin have been described as well pituitary cells,the vast majority of the prolactin synthe (1215,1398,1523,1592) sized by GH cell lines is rapidly releas ed and not stored ere Is n 0 evidence that Iymnbe cvtes contain dopamine receptors tors and thus they are resistant to the prolactin that may be involved in the regulation of lymphocytic inhibiting actions of dopamine (538).This can be viewed prolactin prodt ction/release(432).Pharmac cological char as eithe advantage or a disadvantage ause cel recepto onhs both the D and D dom ate on ly ons that a ly to normal lactor phs on the basis of data cytes(186.187.13611470.1545.1824.oreover mrna collected from cell lines on the other hand with knowl for the D,.D.and Ds receptors have been identified in rat edge of the defect bore by cell lines,one can study the an abs phenotype in ar fun of the role for ocvtic prolactin in the immune nse It is inter. nine rec tor a e thus isolating and examining the esting to note that pituitary prolactin gene expression role of that particular dopamine receptor subtype in lac (1601),bioassayable serum prolactin (1601),immunoas- totroph function(22.244.249.491.666.1784.1807.1924

synthesis of mammary prolactin requires a systemic tro￾phic factor since the amount of both prolactin mRNA and immunoreactive prolactin declines over 24–48 h in mam￾mary 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. In￾deed, the mammary gland is the site of formation of the important 16-kDa variant of prolactin mentioned previ￾ously (332). Although prolactin, produced locally by mam￾mary 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 impor￾tant 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 im￾mune (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 mu￾rine (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 char￾acterization of lymphocytic dopamine receptors suggests that rather than the classical D2 type receptors found on lactotrophs, both the D4 and D5 predominate on lympho￾cytes (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 inter￾esting to note that pituitary prolactin gene expression (1601), bioassayable serum prolactin (1601), immunoas￾sayable serum prolactin (1505), and lymphocyte number (1505, 1601) are elevated during acute skin allograft re￾jection in mice. Administration of bromocryptine, a D2 receptor agonist, or antilymphocytic serum diminishes circulating levels of prolactin in grafted animals and pro￾longs 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 require￾ment for graft survival (1131). Indeed, such a role for prolactin in transplant rejection warrants further investi￾gation. 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) iso￾lated 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 hor￾mone only (somatotrophs), prolactin only (mam￾motrophs), 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 synthe￾sized by GH cell lines is rapidly released and not stored (1722); thus there is no intracellular degradation of pro￾lactin (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 conclu￾sions that apply to normal lactotrophs on the basis of data collected from cell lines. On the other hand, with knowl￾edge of the defect borne by cell lines, one can study the role of an absent phenotype in control of cellular func￾tion. 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 lac￾totroph function (22, 244, 249, 491, 666, 1784, 1807, 1924). 1528 FREEMAN, KANYICSKA, LERANT, AND NAGY Volume 80

PROLACTIN 1529 Not all clonal lactotroph lines deviate as markedly from isoforms consists of 210 amino acids (196.197 and shows primary cells.For example,the MMQ cell line derived from sequence similarities with other cytokine receptors(cy- the estrogen-induced rat pituitary tumor 7315a secretes pro- tokine receptor homology domain,CRH)(1872).The ex tos（881. and betexpr nain can be fur D2 92 same as)that of nommal lactotrophs (567.6) 1872).Both Dl and D2 show analogies with the fibrone tin type III molecule,which drives the receptor-ligand IV.PROLACTIN RECEPTORS interactions in the majority of cytokine receptors(1872). eptor:Gene,Splicing Variants, Cys-cvs in the andaws motir Cor-ser x-Tr-Ser)in the D2 domain (1872)The disulfide bonds oundprotein and the WS motif are essential for the proper folding and a ily (131,132,926,997).Just like their respe le fo ing o ceptor,althou are not etive ligands prolactin and growth hormone receptors share several olactin-R involves ligand-induced sequential receptor dimerization (184)(ig 1)Each prolactin molecule con structural and fu ctio tains two binding sites (site I involves helices I and 4 cellular. 1s99 I and 3).F st, The gne eneoding the human prolactin-R ocated on hi encompa (634)The formation of this initial chromosome 5 and contains at least 10 exons (131 132) plex is the prerequisite for the interaction of binding site Transcriptional regulation of the prolactin-R gene is ac R(184).D prolac for the live r and nr omoter II is“gen "DF nt in both gonadal and nongonadal tissues (812).Numerous prolac- mplex (2 eceptors.1 hor mone)is formed (184.631). as are res B)INTRACELLULAR DOMAIN:ACTIVATION OF JAK2 AND RECEPTOR PHC intracellular do lactin-R ati ng of no coding and coding exon transerints (809.812).Although tin-R i (8A)The intracellular domain however is a key plaver the isoforms vary in the length and composition of their doma in the initiation of the signal transduction mechanisms ated with the prolactin 34).The t291 ans are c h intermediate (393 amino acids)and long (591 amino ac. nd shom ids)forms (184).In mice,one long and three short forms imilarities with other cytokine receptors(18).Howeve the are two relatively conserved regions termed box i have been described(③38， 6).In addition to the mem p0n s,s milk (1430).These soluble forms contain 206 NH-termi by th nal amino acids of the extracellular domain of the prolac. (18).Box 2 is less conserved and is missing in the short tin-R (159).The soluble prolactin binding proteins are isoform of the prolactin receptor(632,926) Alth ough the intracellular any of the primary transerint or products of p oteolvtic cleav. age of the mature receptor (or both)(184) proteins (1479),including the receptor itself(926,1267) The B.Activation of Prolactin-R and the Associated membrane proximal region of the intracellular do Signal Transduction Pathways very (l.e by ith 1.Prolactin-R domains and receptor activation within I min after prolactin binding,suggesting a majo A)EXTRACELLULAR DOMAIN: LIGAND-INDUCED DIMERIZATIO upstream role for Jak2 (1014)(Fig.1).Experimental data The extracellular domain of all rat and human prolactin-R suggest two major prerequisites for Jak2 activation:1)

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 pro￾lactin exclusively, expresses functional dopamine D2 recep￾tors (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 superfam￾ily (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 extra￾cellular, 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 ac￾complished 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 prolac￾tin-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 pro￾lactin-R promoters as well as alternative splicing of non￾coding 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 ac￾ids) forms (184). In mice, one long and three short forms have been described (338, 386). In addition to the mem￾brane-bound receptors, soluble prolactin-binding proteins were also described in mammary epithelial cells (158) and milk (1430). These soluble forms contain 206 NH2-termi￾nal amino acids of the extracellular domain of the prolac￾tin-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 cleav￾age 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 (cy￾tokine receptor homology domain, CRH) (1872). The ex￾tracellular domain can be further divided into NH2-termi￾nal D1 and membrane-proximal D2 subdomains (926, 1872). Both D1 and D2 show analogies with the fibronec￾tin 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 respon￾sible for binding the ligand itself (632). Activation of the prolactin-R involves ligand-induced sequential receptor dimerization (184) (Fig. 1). Each prolactin molecule con￾tains two binding sites (site 1 involves helices 1 and 4, while site 2 encompasses helices 1 and 3). First, prolac￾tin’s binding site 1 interacts with a prolactin-R molecule (634). The formation of this initial hormone-receptor com￾plex 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 initi￾ated only when a trimeric complex (2 receptors, 1 hor￾mone) is formed (184, 634). B) INTRACELLULAR DOMAIN: ACTIVATION OF JAK2 AND RECEPTOR PHOSPHORYLATION. I) Transmembrane and intracellular do￾mains. The role of the 24-amino acid-long transmem￾brane 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 enzy￾matic 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 do￾main 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) October 2000 PROLACTIN 1529

1530 FREEMAN,KANYICSKA,LERANT,AND NAGY Vobme 80 Ligand-induced dimerization Phosphorylation of Jak and PRL-R Step2 Step 3 P on of p or-ligand i e 2 on the 1872.Th f the initi rolactin-R box I x21200 both the of the pro rich box (1014)and stric ted with th a2 es (of the 1514 -rich box 1 stoichiometr of the ligand-induced prolactin-R dimers unon activation of the short form of the prolactin R de (307,549,550).Although the association of Jak2 with spite the presence of four Tyr residues in its intracellular been undoubtedly proven (266,1013 domain (660).Cer ain cellular functi ociation is of the prolac 1906 the tynical SH3 (sre kinase homoloay domain a)folding The long form of the prolactin-R also contains numerous (1464),no matching SH3 region is found in the sequence Tyr residues,many of which are phosphorylated upon of either the presence of an adaptero prolactin-R activation (1412). Acnt from ulation uon recentor dimerization which 2.Signal transduction pathways associated ith the prolactin-F brings two Jak2 molecules close to each other (550). Experiments with chimerc receptors suggest that mer A)STAT PROTEINS.The signal transducer and activator aposit 306 gu of tran cellular domain is also roquired sting (8).The STAT family currently consist cance of the COOH-terminal residues (550). bers.Four of them,STATI,STAT3,and especially STAT5a ID Phosphorylation of the prolactin-R.Jak2 kinases and STAT5b,have been identifie as tr nsducer mole 852 STA Ty (1514)(Fig.1).Pho domain an SH2-like domain.and an NH.-and a COOH they are potential binding/docking sites for transducer terminal transactivating domain (552).According to the molecules containing SH2 domains.Although phosphory- consensus model of STAT activation (184.552).a phos

presence of the proline-rich box 1 motif in the intracellu￾lar 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 pro￾tein 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 intra￾cellular domain is also required, suggesting the signifi- cance 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 phosphory￾lation 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, de￾spite the presence of four Tyr residues in its intracellular domain (660). Certain cellular functions, like prolifera￾tion, 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). 2. Signal transduction pathways associated with the prolactin-R 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 mem￾bers. Four of them, STAT1, STAT3, and especially STAT5a and STAT5b, have been identified as transducer mole￾cules 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 phos￾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 transphos￾phorylate 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). 1530 FREEMAN, KANYICSKA, LERANT, AND NAGY Volume 80

PROLACTIN 1531 PRL-R ong form of PRLR nomic BIOLOGICAL EFFECTS (834).STA ak/STA ST an SHa-like domain ain of a s e docked at n the t gene (18 the STA 8a47 ng s o the (392 N 23 )Th 1658) are in te with STAT for docking sites on pmolactin re or (144 DNA motif ree gnized by STATI STAT3 and STAT5 ho mo or heterodime is termed GAS (interferon acti STAT,while docked at the receptor,is phosphorylated by vated sequence)(791)(Fig.2).GAS consists of a palin the receptor-associated Jak kinase.The phosphorylated dromic sequence: 791) Numerous s from th o or n ontain the GAs con ate th the sH2 domain of another sTAT mole vitro (548.658).It has been pr nosed that sTAT interact cule(184)(Fig.2).Finally,the STAT dimer translocates to with other signal transducers(e.g.,glucocorticoid recep the nucleus and activates a STAT DNA-binding motif in tor)to initiate a cell-and cytokine-specific response the promoter of a target gene (184,291).The consensus 1687,1688)

phorylated 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 ho￾modimerizes through its phosphotyrosine residues with the SH2 domain of another phosphorylated STAT mole￾cule (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 ho￾mo- or heterodimers is termed GAS (g-interferon acti￾vated sequence) (791) (Fig. 2). GAS consists of a palin￾dromic 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 recep￾tor) to initiate a cell- and cytokine-specific response (1687, 1688). 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 (g-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. Phospho￾tyrosine 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 K1 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 Ca21 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). October 2000 PROLACTIN 1531

1532 FREEMAN,KANYICSKA,LERANT,AND NAGY Volume 80 Of the STATI,STAT3,and STAT5 STAT5 than in GH or other cytokines (23,147, (earlier k 490.141i14451770 nized as the most important transducer of the long and A newly revealed facet of cytokine receptor signaling intermediate isoforms of the prolactin-R(106).STAT5 is iden -containing protein families inhib two STAT5b ng th es or ly in the nal do in Both isof (cIS)1042,1144.1914)and of ev ess a Tvr-694 which is phosphorvlated by Jak2 (659) naling(S0CS)603,762,1289,1312,1672.Their main In addition to Tyr phosphorylation,activation of STAT mechanism of action in prola receptor signaling has we 133 sOCs-1and Socs a 4u socs1 and switch tein kinase C (PKC-o and casein kinase II have off the prolactin receptor-mediated signaling by inhibiting pro osed as s activating STATa the ca ic activity of Jak2 and activation of STAT pro data inhibi- teins (1411) The and -2 gen of gen may fulfil SOCS-2 s 、prola .Although Jak/STAT are the most re eptor stimulation probably by suppre ng SOC-I's in pathways initiated by activation of the prolactin-R,a num- hibitory effect (1411). of reports 518 307132,41D.Ph0sp Distribution of Prolactin-R the nrolactinR can ser sites for adante teins(Shc/Grb2/SOS)connecting the otor to the R APK casc a ath were rega of prolactin-R ing that thes se pathways connected (698) For proper surface targeting,glycosylation of the of the s(A the Oh kinases c-sre and Fym. Several recent lactin reports indicate prola in-induce mbers og though pr olactin-R is mainly a cell-surface receptor,de- phorylation of insulin re substrate-1 (RS1)and glycosylated forms of prolactin-R can accumulate in the cetylglu (103 Golgi apparatus(256) whic h is 152 have been described.Both IRS-1 and PI 3 e It ho tion of these newly glycosylated receptors to the cell tion of PI3'-kinase is mediated by Fyn (3la)(Fig 2). surface(183). endocytosis of prolac ID Iatracellular ion concentration at least two she wn in prolactin-R ha events and two regions of the prolactin-R are involved in ed i in-R R to the sine kinas endent kt channels by Jak2 (435) types (344,1028,1450).Nuclear translocation of prolac whereas the COOH terminal of the intracellular domain is tin-R can b involved in the production of the intra engers AT MAT h 1,3,45Pla inde kinase)(1404)do not require nuclear tr ranslocation of pendent Ca2 chan nels3511451650a prolactin-R,the mechanism and in vivo importance of internalization and nuclear actions still re- C)DOWNREGULATION OF PROLACTIN-R SIGNAL TYR PH IA- TASES AND INHIBITOR PROTEINS.Because activation of prolac man to Tyr 2.Distribution of prolactin-R in the mammalian body pathways involves T Tyr phosphatases(184).Expe It is not surprising that prolactin-R and its messag data indicate that SH2 containing Tyr phosphatases SHP-1 are found in the mammary gland and the ovary,two of the and sHP-2 play less of a role in downregulation of pro- best-characterized sites of prolactin actions in mammals

Of the STAT1, STAT3, and STAT5 proteins, STAT5 (earlier known as mammary gland factor, MGF) is recog￾nized 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 differ￾ences 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). Pro￾tein kinase C (PKC)-a and casein kinase II have been proposed as serine/threonine kinases activating STAT5 (133). Novel data indicate that STAT5 may fulfill inhibi￾tory 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 num￾ber of reports implicate activation of the mitogen-acti￾vated 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 ini￾tially the Jak/Stat and MAPK pathways were regarded as independent or parallel pathways, there are data suggest￾ing 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 phos￾phorylation of insulin receptor substrate-1 (IRS-1) and a subunit of the phosphatidylinositol (PI) 39-kinase (103, 152, 1453) have been described. Both IRS-1 and PI 39- kinase seem to be associated with the prolactin-R com￾plex. It has been proposed that prolactin-induced activa￾tion of PI 39-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 K1 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 ino￾sitol hexakisphosphate (InsP6)} that open voltage-inde￾pendent Ca21 channels (351, 1452, 1659) (Fig. 2). C) DOWNREGULATION OF PROLACTIN-R SIGNAL: TYR PHOSPHA￾TASES AND INHIBITOR PROTEINS. Because activation of prolac￾tin-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 pro￾lactin 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 inhib￾iting the Jak/STAT pathways. These protein families are referred to as cytokine-inducible SH2-containing protein (CIS) (1042, 1144, 1914) and suppressors of cytokine sig￾naling (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 pro￾teins (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 in￾hibitory 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 extracel￾lular domain of the prolactin-R is crucial, although not an absolute requirement for prolactin-R activation (256). Al￾though prolactin-R is mainly a cell-surface receptor, de￾glycosylated forms of prolactin-R can accumulate in the Golgi apparatus (256). Nitric oxide activates N-acetylglu￾cosamine transferase, which is responsible for glycosyla￾tion of these intracellular receptors and promotes migra￾tion 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). Surpris￾ingly, even translocation of prolactin (1451) and prolactin￾R to the nucleus has been demonstrated in different cell types (344, 1028, 1450). Nuclear translocation of prolac￾tin-R can be accompanied by nuclear actions like stimu￾lation 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 re￾main to be determined. 2. Distribution of prolactin-R in the mammalian body 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 1532 FREEMAN, KANYICSKA, LERANT, AND NAGY Volume 80