Genetic Analysis: Biomolecular Engineering
From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models
Introduction
Prolactin (PRL) along with growth hormone (GH) and placental lactogen (PL) form a family of hormones which probably evolved from the duplication of an ancestral gene [1]. PRL and GH are mainly synthesized by the pituitary in all vertebrates, whereas placental lactogen is produced by placenta in mammals. PRL has more actions than all other pituitary hormones combined. The initial step in the action of PRL is the binding to a specific membrane receptor, the PRL receptor (PRLR). Similar to the ligand, the PRLR has also been shown to be a member of the same family as the GH receptor, and as well part of the larger class of receptors, known as the class-1 cytokine receptor superfamily.
In this review, we will discuss many of the molecular steps by which PRL exerts its various functions. This includes the sites of PRL synthesis, PRL structure, the interaction of PRL with its receptor and the main signaling cascades activated in target cells. In an recent review [2] we have expanded the original list of 85 different actions of PRL in vertebrates [3] to more than 300 separate biological functions. In addition, we have recently generated a mouse model deficient in the PRLR which allowed us to detect unexpected phenotypes, indicating that PRL is involved in other functions than those reported to date. The main phenotypes of PRLR knockout (KO) mice will be discussed, along with those described for other related knockout.
The amino acid sequence of sheep PRL was determined in 1969 and was shown to be a protein of 199 amino acids [4]. Genetic, structural, binding and functional studies of PRL, GH and PL as well as of the more recently identified somatolactin and PRL-related proteins have clearly confirmed that they all belong to a unique family of proteins [5]. The gene encoding human PRL (hPRL) is located on chromosome 6 [6]. It is composed of five exons and four introns with an overall length of ∼10 kilobases [7]. The hPRL cDNA is composed of 914 nucleotides and contains a 681 nucleotide open reading frame encoding a pre-hormone of 227 amino acids (aa), including a signal peptide of 28 aa [8]. With the exception of fish, in which PRL is smaller, the length of mature PRL proteins is around 200 amino acids for a molecular weight of 23 000. Post-translational modifications of mature PRL, including glycosylation, phosphorylation or proteolytic cleavage, have been reported and recently reviewed [9]. To date, attempts to determine the three-dimensional (3D) structure of PRL via experimental techniques (X-ray, NMR) have been unsuccessful, and the only available structural data are theoretical 3D models of human [10] and rabbit [11] PRL. It is assumed that PRL adopts the four helix bundle fold described for the closely related GH [12].
PRL is mainly synthesized by lactotroph cells of the anterior pituitary and its expression is essentially controlled by the negative regulation of dopamine, although numerous other factors are able to stimulate or inhibit PRL synthesis/secretion. In addition to being synthesized by pituitary gland, PRL is also produced by numerous other cells and tissues [13]. For example, PRL gene expression has been confirmed in various regions of the brain, decidua, myometrium, lacrimal gland, thymus, spleen, circulating lymphocytes and lymphoid cells of bone marrow, mammary epithelial cells and tumors, skin fibroblasts, and sweat glands. Interestingly, hypophysectomized rats retain ∼20% of biologically active PRL in the circulation, which increases to ∼50% of normal levels with time. Neutralization of circulating PRL with anti-PRL antibodies results in immune dysfunction and death [14], suggesting that PRL from extrapituitary origin is important, and under some circumstances, can compensate for pituitary PRL.
More than two decades ago, the PRLR was identified as a specific, high affinity, membrane-anchored protein. In 1988, the cDNA encoding the rat PRLR was isolated in our laboratory [15] and, as is true for the closely related receptor for GH (GHR), is a single-pass transmembrane chain. In the early 1990’s, sequence comparisons with newly-identified membrane receptors led to the identification of a new family of receptors including both PRLR and GHR [16]. Termed Class-1 cytokine receptors, this superfamily includes receptors for several interleukins, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), leukemia inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), gp130 and the obesity factor, leptin [17]. Although all these membrane chains are apparently genetically unrelated, they contain stretches of highly conserved amino acids, mostly in the extracellular domain.
The gene encoding human PRLR is located on chromosome 5 (p13→14) and contains at least ten exons for an overall length>100 kb [18], [19]. Multiple isoforms of membrane-bound PRLR resulting from alternative splicing of the primary transcript have been identified in several species [2]. These different PRLR isoforms differ in the length and composition of their cytoplasmic tail and are referred to as short, intermediate or long PRLR with respect to their size (Fig. 1). In mice, one long and three short isoforms have been identified, the short forms only differing by a few amino acids in the C-terminal part of their cytoplasmic tail [20]. In addition to the membrane-anchored PRLR, soluble isoforms have also been identified (PRL binding protein, or PRLBP), but whether they result from alternative splicing of the primary mRNA or proteolytic cleavage of membrane-bound PRLR (or both) is uncertain. In all the cases, however, the extracellular, ligand-binding domain is identical, whatever the isoform.
Typically, a cytokine receptor extracellular domain (ECD) is composed of a module of ∼200 aa, referred to as the cytokine receptor homology region [17]. This region can be divided into two subdomains of ∼100 aa, each showing analogies with the fibronectin type III module [21]. Two highly conserved features are found in the cytokine receptor ECDs: the first is two pairs of disulfide-linked cysteines in the N-terminal subdomain, and the second is a pentapeptide termed ‘WS motif’ (Trp–Ser–any amino acid–Trp–Ser) found in the membrane proximal region of the C-terminal subdomain (Fig. 1). The cytoplasmic domain of cytokine receptors displays more restricted sequence similarity than the extracellular domain. Two regions, called Box 1 and Box 2 [2] are relatively conserved. Box 1 is a membrane-proximal region composed of amino acids highly enriched in prolines and hydrophobic residues (aa 243–250 in PRLR; Fig. 1). Other structural features of the PRLR have been discussed elsewhere [2], [22].
The 3D structure of genetically engineered hPRLR ECD (i.e. hPRLBP) has been determined by crystallographic analysis [23]. It folds in two domains of 7 β-strands forming a sandwich of two anti-parallel β-sheets. As anticipated from sequence comparison [24] this folding pattern is likely to be shared by several, if not all, cytokine receptors (reviewed in Ref. [2]). Determining the structure of PRLR cytoplasmic domains is one of the projects currently in progress in our laboratory.
Prolactin receptors have been identified in a number of cells and tissues of embryonic and adult mammals. The expression of short and long isoforms of receptor have been shown to vary as a function of the stage of the estrous cycle, pregnancy and lactation [2]. PRL binding sites or receptors are widely distributed throughout vertebrate tissues. We have recently determined the cellular distribution and developmental expression of the PRLR in the late gestational fetal rat by in situ hybridization, immunocytochemistry and radioligand binding [25]. Sense and antisense strand probes were prepared encoding the long and short isoforms of the rat PRLR and hybridized to various fetal tissues obtained at the end of pregnancy (days 17.5–20.5). These studies showed that the mRNA encoding the short and long isoforms was widely expressed in tissues from all three germ layers and that, in addition to the classical target organs of PRL, tissues not known previously to contain PRLR, such as olfactory neuronal epithelium and bulb, fetal adrenal cortex, gastrointestinal and bronchial mucosae, renal tubular epithelia, choroid plexus, thymus, liver pancreas and epidermis, also express the PRLR mRNA. The wide distribution of the PRLR is obviously correlated with the extremely large spectrum of activities of its ligand (for more details, see Ref. [2]).
Section snippets
Interaction between PRL and its receptor
Although stoichiometric analysis of the interaction between different PRLR ECDs and lactogenic hormones achieved 1:1 [26] or 1:2 [27] complexes depending on the species involved, dimerization of the PRLR upon ligand binding has now been clearly established. We have shown that at least two regions of hPRL are involved in the binding of the hormone to the PRLR. The first, referred to as binding site 1, encompasses several residues belonging to helices 1 and 4, while the second, termed binding
The JAK–Stat pathway
All of the actions of PRL result from the interaction of PRL with its receptor on the numerous target cells, which leads to the activation of a cascade of intracellular events. The cytoplasmic tail of the PRLR, whatever the isoform, is devoid of any consensus sequence for enzymatic activity, including kinase activity [33]. However, hormonal stimulation of the PRLR leads to tyrosine phosphorylation of several cellular proteins, including the receptor itself [34]. In 1994, JAK2, one of the four
Biological functions of PRL: lessons from PRLR knockout mice
PRL was originally isolated by its ability to stimulate mammary development and lactation in rabbits and soon thereafter to stimulate the production of crop milk in pigeons. PRL was also shown to be luteotrophic, that is to promote the formation and action of the corpus luteum. Subsequently, a number of additional activities have been associated with this hormone in various vertebrate species. In the now classical review by Nicoll and Bern [3], 85 different biological functions were attributed
Identification of new target genes of PRL
As mentioned above, PRL is implicated in a number of physiological processes, with perhaps a large number still yet to be determined. Although the effects of PRL and the genes specifically induced by PRL are best known during lactation and pregnancy, it is also well established that other extrapituitary sites synthesize and secrete PRL, and that the PRLR is expressed at various levels in many organs. In most cases, the functions of PRL in these different target-tissues, such as the liver, the
Pathologies associated with PRL
To date, no pathology linked to mutations in genes encoding PRL or the PRLR have been reported. Either these genes are not important, or they are essential to the survival of the species. As described above, knockout of the PRLR gene in mice is not lethal, but does produce major reproductive defects in females, which would of course affect reproductive function and survival. Despite the lack of known genetic disease related to PRL or its receptor, PRL is linked with some pathological states,
Conclusion
PRL receptors or binding sites are widely distributed throughout the body. In fact, it is difficult to find a tissue that does not express any PRL receptor mRNA or protein. In agreement with this wide distribution of receptors is the fact that now over 300 separate actions of PRL have been reported in various vertebrates, including effects on water and salt balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, and immunology and protection. Clearly, a
References (98)
- et al.
J. Biol. Chem.
(1981) - et al.
Cell
(1988) - et al.
Molecular aspects of prolactin and growth hormone receptors
- et al.
FEBS Lett.
(1993) - et al.
J. Biol. Chem.
(1993) - et al.
J. Biol. Chem.
(1996) - et al.
J. Biol. Chem.
(1995) - et al.
J. Biol. Chem.
(1998) - et al.
Biochem. Biophys. Res. Commun.
(1992) - et al.
J. Biol. Chem.
(1992)
Trends Genet.
J. Biol. Chem.
Mol. Cell Endocrinol.
J. Biol. Chem.
FEBS Lett.
J. Biol. Chem.
Cell
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
Cell
Mol. Cell Endocrinol.
Trends Genet.
Baillieres Clin. Endocrinol. Metab.
Baillieres Clin. Endocrinol. Metab.
Biochem. Biophys. Res. Commun.
Horm. Behav.
Gene
Trends Genet.
Endocrinol. Metab. Clin. North Am.
Pharmacol. Ther.
J. Biol. Chem.
Endocr. Rev.
Endocr. Rev.
On the actions of PRL among the vertebrates: is there a common denominator?
Nature
Endocr. Rev.
Science
EMBO J.
Endocr. Rev.
Prot. Eng.
Proteins
Science
Endocr. Rev.
Endocrinology
Biochem. Biophys. Res. Commun.
Annu. Rev. Biochem.
Cytogenet. Cell Genet.
Cited by (74)
New insights into progesterone actions on prolactin secretion and prolactinoma development
2019, SteroidsCitation Excerpt :Although PRL was originally named for its ability to promote lactation, it is now accepted that its biological actions are not limited solely to reproduction. With more than 300 functions, PRL has been shown to have a role in the immune response, animal behavior, osmoregulation, growth and metabolism [3–6]. In mammals, PRL is mainly produced by lactotrophs, a distinct group of cells in the anterior pituitary gland.
The role of sex differences in inflammation and autoimmune diseases
2019, Sex Differences in Cardiovascular Physiology and PathophysiologyContribution of sex steroids and prolactin to the modulation of T and B cells during autoimmunity
2018, Autoimmunity ReviewsCitation Excerpt :The JAK/Stat pathway is the main signaling pathway used by all members of this receptor family. To activate this pathway, PRL binds to the PRL-R causing the receptors to dimerize [104,105] that leads to the activation of PRL-R-associated JAK2 protein tyrosine kinases (PTK). Activated JAK2 then phosphorylates downstream targets on tyrosine residues, including the PRL-R ICD, Stat proteins, and other SH2-containing signaling molecules.
Prolactin receptor targeting in breast and prostate cancers: New insights into an old challenge
2017, Pharmacology and TherapeuticsCitation Excerpt :This explains why PRL has been implicated in a wide array of pathophysiological processes (reviewed by Bole-Feysot, Goffin, Edery, Binart, & Kelly, 1998; Goffin & Touraine, 2015). In most cases, however, PRL appears to participate in the modulation of biological processes more than playing an essential role, as reflected by the relatively mild phenotypes of PRLR-deficient mice (reviewed by Goffin et al., 1999; Kelly, Binart, Lucas, Bouchard, & Goffin, 2001). In fact, mice deficient for PRL or its receptor have shown that PRLR signaling is essential for adaptive functions related to reproduction, including mammary gland differentiation and mammopoiesis (Horseman et al., 1997; Ormandy, Camus, et al., 1997; reviewed by Chen et al., 2012; Hennighausen & Robinson, 2005), maternal behavior (Lucas, Ormandy, Binart, Bridges, & Kelly, 1998; Shingo et al., 2003) or beta-cell proliferation during pregnancy (Karnik et al., 2007).
Neuroendocrine control of photoperiodic changes in immune function
2015, Frontiers in NeuroendocrinologyRole of prolactin in B cell regulation in multiple sclerosis
2014, Journal of NeuroimmunologyCitation Excerpt :Prl activity is mediated by Prl-Rs, found in monocytes and in activated lymphocytes. Different isoform receptors exist, including the long (85–90 kDa) and short (42 kDa) Prl-R, which result from differential splicing of 3′ end cytoplasmic domain exons from a single gene (Goffin et al., 1999). Moreover, estrogens have been implicated in the regulation of Prl-R gene expression (Leondires et al., 2002; Adamson et al., 2008).