After the processing of a 28 residue signal peptide, the mature human PRL molecule is secreted as a polypeptide with 199 amino acid residues, whereas mouse PRL is two residues shorter (Cooke 1989). In both species, six cysteines form three intramolecular disulfide bridges. The molecular weight of human PRL is approximately 23 kDa, but a 26-kDa glycosylated form is also produced. PRL circulates in blood as monomers of 23 to 26 kDa (Lewis, Sigh, Sinha, and Vanderlaan 1985). Larger "macroprolactins" (big-prolactin and big-big prolactin) represent both homo-oligomeric aggregates and immunoglobulin-complexed PRL. Furthermore, the physiological proteolysis of PRL to a C-terminally truncated 16 kDa variant results in a molecule with distinct biological activities that may activate unique receptors (Mittra 1980; Clapp and Weiner 1992). PRL is a tetrahelical cytokine most closely related to growth hormone and placental lactogenes. It binds to specific prolactin receptors that belong to the WS-motif cytokine receptor family.
The pituitary PRL secretory rate is approximately 18.6 nmol/day (400 ^/day). The hormone is cleared by the liver (75%) and the kidney (25%), and its half-life in plasma is approximately 50 min (Thorner et al. 1998).
Human PRL gene is present as a single copy on chromosome 6 (Owerbach, Rutter, Cooke, Martial, and Shows 1981). It is located close proximity to the HLA complex. This colocalization is of interest because of a possible association between prolactin-secreting adenomas and specific HLA alleles (Farid, Noel, Sampson, and Russell 1980). Meanwhile, the mouse PRL gene maps to chromosome 13, clustered with genes encoding mouse placental lactogenes and other prolactin-like genes (Jackson-Grusby, Pravtcheva, Ruddle, and Linzer 1988).
The human PRL gene is more than 15 Kb long and contains six exons (Truong, Duez, Belayew, Renard, Pictet, Bell, and Martial 1984). It is known that transcriptional control of the distal nonpituitary start site in endometrial stromal cells is linked to decidual differentiation during the secretory phase of the ovulatory cycle (DiMattia, Gekkersen, Duckworth, and Freisen 1990). Two consensus-binding sites for CCAAT/enhancer-binding proteins (C/EBP) mediate the cAMP/PKA-induced activation of this nonpituitary PRL gene promoter in human decidual cells (Pohnke, Kempf, and Gellersen 1999). Cyclic AMP, alone or in synergy with PHA, also stimulates the activation of this upstream PRL gene promoter in Jurkat T cells, possibly through activation of C/EBP proteins (Reem, Ray, and Davis 1999).
PRL activates the transcription factor STAT5 in most target cells and tends to interact positively and in a redundant manner with other cytokines that also activate STAT5 as IL-2, IL-3, IL-5, IL-7, IL-9, IL-15, GM-CSF, erythropoietin, thrombopoietin, and growth hormone (Kirken, Rui, Malabarba, and Farrar 1994).
PRL protein undergoes posttranslational modifications: oxidation, proteolysis, glycosylation, and phosphorylation. Mature prolactin, formed by proteolytic removal of a 28-kDa signal peptide, can be further modified by proteases. Cathepsin-D proteolysis at position 133 generates two fragments of 16 and 8 kDa respectively, which may exist as both disulfide-linked heterodimers and monomers (Mittra et al. 1980; Cole, Nichols, Lauziere, Edmunds, and McPeherson 1991). The six cystein residues in PRL undergo oxidation and form stable, successive intramolecular disulfide bonds. Besides, a proportion of pituitary and circulating PRL is glycosylated in most species. Approximately 20% of circulating human PRL is glycosylated through N-linkage at position 31 (Lewis et al. 1985; Champier, Claustrat, Sassolas, and Berger 1987). The physiological function of glyscosylation of PRL may be to reduce biological potency, while extending the half-life of the molecule (Hoffmann, Penel, and Ronin 1993). Finally, a significant proportion of PRL molecules are phosphorylated on their serine and threonine residues. Phosphorylation of PRL is associated with reduced bioactivity, but does not affect the biological half-life (Wang and Walker 1993). Because PRL is phosphorylated in secretory granules during its release from pituitary lactotrophs, it is possible that phosphorylation serves to reduce local bioactivity during secretion.
PRL could be considered to be a monogamous cytokine in that it binds exclusively to PRL receptors. The PRL receptor (PRLR) exists in all vertebrates in many isoforms, soluble or membrane-bound. It is expressed in a wide variety of tissues where PRL activates PRLR by inducing its homodimerization, the first step required for triggering signaling cascades. No other membrane chain is required for signaling. PRLR is a single-pass transmembrane chain, with the N-terminus outside the cell. In mammals, the overall length of the PRLR varies from 200 amino acids for the soluble-binding protein up to 600 residues for the long membrane isoforms (Goffin and Kelly 2001). The gene encoding the human PRLR is located on chromosome 5(p13-14) and contains at least 10 exons for an overall length >100 kb (Arden, Boutin, Djiane, Kelly, and Cavenee 1990).
It is usually observed that the soluble binding protein (PRLbp) has a higher affinity (10 times) than the membrane-bound PRLR for a given ligand (Postel-Vinay et al. 1991). The affinity of the PRLR will vary depending on the type and species of origin of the ligand considered. It is usually in the range of Kd = 10-9 to 10-10 M. The PRLR is activated by dimerization (Goffin, Shiverick, Kelly, and Martial 1996), which involves two regions (called binding sites 1 and 2) of the ligands, each interacting with one molecule of PRLR. It is known that both binding sites interact with virtually overlapping epitopes within the receptor (Goffin et al. 2001). The level of expression of the PRLR varies from 10 to 2000 fmol/mg of membrane protein. The expressions of short and long forms of receptor have been shown to vary as a function of the stage of the estrous cycle, pregnancy, and lactation. PRL is able to both up- and down-regulate its receptor, the latter process probably being due to an acceleration of internalization of hormone/receptor complexes. The effect of PRL on its receptor is also a function of the hormone concentration and time of exposure of the tissue. Growth hormone is also able to up-regulate the PRLR (Kelly, Djiane Postel-Vinay, and Edery 1991). The JAK/STAT signaling pathway is the most widely described cascade for the PRLR. Activation of the JAK2 occurs very rapidly after hormonal stimulation (within 1 min), suggesting that this Janus kinase occupies a central and very upstream role in the activation of several signaling pathways of the PRLR (Chang, Ye, and Clevenger 1998). However, many other signaling proteins were found to be activated by the PRLR. The well-known MAP kinase pathway involves the She/SOS/Grb2/Ras/Raf/MAP kinase cascade and this pathway has been demonstrated to be activated by the PRLR in various cell systems (Piccoletti, Marioni, Bendinelli, Bernelli-Zazzera 1994; Erwin, Kirken, Malabarba, Farrar, and Rui 1996). Although the JAK/STAT and the MAP kinase cascades were initially regarded as independent pathways, recent data rather suggest that these pathways are interconnected (Chida, Wakao, Yoshimura, and Miyajima 1998) (Fig. 5.2).
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