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 trophic factor since the amount of both prolactin mRNA and immunoreactive prolactin declines over 24–48 h in mammary gland explants (992). The mammary gland may also act as a posttranslational processing site for prolactin. In both human (499) and rat (888, 889) milk, the number of prolactin variants far exceeds that found in serum. Indeed, the mammary gland is the site of formation of the important 16-kDa variant of prolactin mentioned previously (332). Although prolactin, produced locally by mammary epithelial cells promotes proliferation, the 16-kDa cleaved prolactin variant inhibits local angiogenesis, which makes this proteolytic step a possible target of breast cancer research (636). The physiological role for milk-borne prolactin has only been described in the rat, which is born immature relative to many other mammals. Indeed, during a brief window of neonatal life, the gastrointestinal tract lacks the ability to digest protein and likewise possesses the ability to absorb intact protein. This is particularly important since the rat pituitary gland is relatively quiescent during this period. Approximately 20% of the prolactin ingested in milk passes to the neonatal circulation (686). It has been shown that milk prolactin participates in the maturation of the neuroendocrine (1596, 1629) and immune (687, 702) systems. E. The Immune System A great deal of evidence suggests that lymphocytes can be a source of prolactin as well (599, 882, 1214, 1516). Indeed, immune-competent cells from thymus and spleen as well as peripheral lymphocytes contain prolactin mRNA and release a bioactive prolactin that is similar to pituitary prolactin (445, 612, 613, 1214–1216, 1523). Not only is an immunoreactive 22-kDa prolactin found in murine (1214) and human (1886) immune-competent cells, but size variants of prolactin have been described as well (1215, 1398, 1523, 1592). Although the control of pituitary prolactin secretion differs from that of lymphocytic origin, there is abundant evidence that lymphocytes contain dopamine receptors that may be involved in the regulation of lymphocytic prolactin production/release (432). Pharmacological characterization of lymphocytic dopamine receptors suggests that rather than the classical D2 type receptors found on lactotrophs, both the D4 and 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. F. Prolactin-Secreting Cell Lines To study the synthesis, processing and secretion of prolactin at the cellular and molecular level, cell lines derived from pituitary tumors have been developed. The first cell line was a mammosomatotroph (MtT/W5) isolated from a radiation-induced pituitary tumor produced in a Wistar-Furth rat (1724, 1907). Because these cell lines secreted mostly growth hormone, they were designated as GH cells. It was subsequently found that some of the subclones were pluripotent and heterogeneous (1721, 1722). For example, GH3 cells may release growth hormone only (somatotrophs), prolactin only (mammotrophs), both hormones (mammosomatotrophs), or neither hormone (189, 192). Similarly, the GH1 and GH4C1 cell lines produce both prolactin and growth hormone but in varying ratios (1721). The most obvious advantage of using cell lines rather than primary pituitary lactotrophs is that clonal cells are usually immortal, can be easily stored, and thus provide a perpetual supply of cells without sacrificing animals and purifying primary pituitary cultures. To critically use these cells, one should recognize their dissimilarity to primary cultures of pituitary cells. For example, unlike pituitary cells, the vast majority of the prolactin synthesized by GH cell lines is rapidly released and not stored (1722); thus there is no intracellular degradation of prolactin (381). Moreover, GH cells lack functional dopamine receptors, and thus they are resistant to the prolactininhibiting 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). 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 prolactin exclusively, expresses functional dopamine D2 receptors (881), and behaves in a manner similar to (but not the same as) that of normal lactotrophs (567, 699). IV. PROLACTIN RECEPTORS A. Prolactin Receptor: Gene, Splicing Variants, and Isoforms The prolactin-R is a single membrane-bound protein that belongs to class 1 of the cytokine receptor superfamily (131, 132, 926, 997). Just like their respective ligands, prolactin and growth hormone receptors share several structural and functional features despite their low (30%) sequence homology (632, 633). Each contains an extracellular, transmembrane, and intracellular domain (1899). The gene encoding the human prolactin-R is located on chromosome 5 and contains at least 10 exons (131, 132). Transcriptional regulation of the prolactin-R gene is accomplished by three different, tissue-specific promoter regions. Promoter I is specific for the gonads, promoter II for the liver, and promoter III is “generic,” present in both gonadal and nongonadal tissues (812). Numerous prolactin-R isoforms have been described in different tissues (24, 386, 1031). These isoforms are results of transcription starting at alternative initiation sites of the different prolactin-R promoters as well as alternative splicing of noncoding and coding exon transcripts (809, 812). Although the isoforms vary in the length and composition of their cytoplasmic domains, their extracellular domains are identical (184, 926, 1031). The three major prolactin-R isoforms described in rats are the short (291 amino acids), intermediate (393 amino acids), and long (591 amino acids) forms (184). In mice, one long and three short forms have been described (338, 386). In addition to the membrane-bound receptors, soluble prolactin-binding proteins were also described in mammary epithelial cells (158) and milk (1430). These soluble forms contain 206 NH2-terminal amino acids of the extracellular domain of the prolactin-R (159). The soluble prolactin binding proteins are also products of the same prolactin-R gene, but it is still uncertain whether they are results of alternative splicing of the primary transcript or products of proteolytic cleavage of the mature receptor (or both) (184). B. Activation of Prolactin-R and the Associated Signal Transduction Pathways 1. Prolactin-R domains and receptor activation A) EXTRACELLULAR DOMAIN: LIGAND-INDUCED DIMERIZATION. The extracellular domain of all rat and human prolactin-R isoforms consists of 210 amino acids (196, 197) and shows sequence similarities with other cytokine receptors (cytokine receptor homology domain, CRH) (1872). The extracellular domain can be further divided into NH2-terminal D1 and membrane-proximal D2 subdomains (926, 1872). Both D1 and D2 show analogies with the fibronectin type III molecule, which drives the receptor-ligand interactions in the majority of cytokine receptors (1872). The most conserved features of the extracellular domain are two pairs of disulfide bonds (between Cys12-Cys22 and Cys51-Cys62) in the D1 domain and a “WS motif” (Tpr-Serx-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 prolactinR (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). 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) 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 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 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 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). 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 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 COOHterminal transactivating domain (552). According to the consensus model of STAT activation (184, 552), a phosFIG. 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 hormonereceptor 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). 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 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 (g-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). 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. 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 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 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)-a 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) 39-kinase (103, 152, 1453) have been described. Both IRS-1 and PI 39- kinase seem to be associated with the prolactin-R complex. It has been proposed that prolactin-induced activation 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 inositol hexakisphosphate (InsP6)} that open voltage-independent Ca21 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 prolactinR 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. 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