Molecular characterization of prolactin receptor (cPRLR) gene in chickens: Gene structure, tissue expression, promoter analysis, and its interaction with chicken prolactin (cPRL) and prolactin-like protein (cPRL-L)

Highlights

• The unique gene structure of chicken PRLR (cPRLR) was revealed.

• Two types of cPRLR transcripts with distinct tissue expression patterns were found.

• cPRLR gene transcription is controlled by multiple promoters.

• cPRL-L is a novel functional ligand of cPRLR.

• Membrane-proximal ligand-binding domain of cPRLR can interact with cPRL and cPRL-L.

Abstract

In this study, gene structure, tissue expression, and promoter usage of prolactin receptor (PRLR) and its interaction with prolactin (PRL) and the newly identified prolactin-like protein (PRL-L) were investigated in chickens. The results showed that (1) PRLR gene was found to consist of at least 25 exons by 5′-RACE and RT-PCR assays; (2) multiple PRLR 5′-UTR sequences different in exon composition were isolated from chicken liver or intestine by 5′-RACE and could be subdivided into type I and type II transcripts according to the first exon used (exon 1G or exon 1A); (3) PRLR Type I transcripts with exon 1G were detected to be predominantly expressed in adult kidney and small intestine by RT-PCR, implying their expression is likely controlled by a tissue-specific promoter (P1). By contrast, PRLR type II transcripts containing exon 1A are widely expressed in adult and embryonic tissues examined and their expression is controlled by a generic promoter (P2) near exon 1A, which was demonstrated to display promoter activities in cultured DF-1, HEK293 and LoVo cells by the dual-luciferase reporter assay; (4) Using a 5 × STAT5-luciferase reporter system, cPRLR expressed in HepG2 cells was shown to be activated by recombinant cPRL and cPRL-L via interaction with PRLR membrane-proximal ligand-binding domain, suggesting that like cPRL, cPRL-L is also a functional ligand of cPRLR. Collectively, characterization of cPRLR gene helps to elucidate the roles of PRLR and its ligands in birds and provides insights into the regulatory mechanisms of PRLR expression conserved in birds and mammals.

Introduction

Prolactin (PRL) is a single-chain peptide hormone synthesized primarily by the anterior pituitary and belongs to the prolactin/growth hormone (GH) family (Freeman et al., 2000, Ben-Jonathan et al., 2008). It is well documented that PRL participates in a variety of physiological processes in vertebrates, including reproduction, osmo-regulation, growth and development, metabolism, immuno-regulation, energy balance, and behavior (Freeman et al., 2000, Ben-Jonathan et al., 2008). The biological actions of PRL are reported to be mediated by a prolactin receptor (PRLR), which shares relatively high degree of structural similarity with growth hormone receptor (GHR) and belongs to the class I cytokine receptor superfamily (Kitamura et al., 1994, Bole-Feysot et al., 1998). Activation of PRLR by PRL binding can initiate the intracellular signaling cascades, including activation of the intracellular JAK2-STAT5 signaling pathway (Bole-Feysot et al., 1998, Brooks, 2012). In consistence with the diverse actions of PRL in vertebrates, PRLR is reported to be widely expressed in various tissues of vertebrates (Bole-Feysot et al., 1998, Ben-Jonathan et al., 2008). Moreover, there is evidence showing that in mammals, PRLR gene expression is controlled by multiple promoters, which direct either ubiquitous or tissue-specific expression (Moldrup et al., 1996, Hu et al., 1998a, Hu et al., 2002, Ben-Jonathan et al., 2008).

In birds, PRL has been reported to be involved in the regulation of many important physiological processes including crop milk production, egg laying, induction and maintenance of incubation behavior, osmo-regulation, immune-modulation, gonadal development and functions (Sharp et al., 1979, Doneen and Smith, 1982, Zadworny et al., 1989, Skwarlo-Sonta, 1990, el Halawani et al., 1991, Youngren et al., 1991, March et al., 1994, Sockman et al., 2006). As in other vertebrates, PRLR has been shown to be capable of mediating the actions of PRL and widely expressed in various tissues of avian species, including chickens (Tanaka et al., 1992, Ohkubo et al., 1998a, Ohkubo et al., 1998b), turkeys (Zhou et al., 1996), and pigeons (Chen and Horseman, 1994). Interestingly, unlike the structure of PRLR in other vertebrates, which has only one single ligand-binding domain in their large extracellular region, avian PRLR contains two putative ligand-binding domains – membrane-distal and membrane-proximal domains – arranged tandemly in its large extracellular region (Tanaka et al., 1992). In vitro study showed that the membrane-proximal ligand-binding domain is capable of binding PRL in pigeons (Chen and Horseman, 1994), however, whether the putative membrane-distal ligand-binding domain is functional needs further clarification.

Although PRLR has been cloned in several avian species, including chicken (Tanaka et al., 1992), pigeons (Chen and Horseman, 1994), turkeys (Zhou et al., 1996), geese (Xing et al., 2011), and ducks (Wang et al., 2009), the information regarding their detailed gene structure, promoter usage, and spatio-temporal expression patterns during the embryonic stages remains limited in birds. Moreover, a novel PRL-like protein (PRL-L) gene has been reported to exist in chickens and other non-mammalian species in our recent study (Wang et al., 2010), also raising an interesting question whether PRL-L can function as an additional ligand of PRLR in non-mammalian vertebrates including birds. Therefore, using chicken as an experimental model, our present study was undertaken to: (1) reveal the gene structure of PRLR; (2) examine tissue expression of PRLR at both embryonic and adult stages; (3) identify the promoter(s) responsible for the control of PRLR expression; (4) examine whether PRLR could be activated by PRL-L specifically; (5) investigate by which ligand-binding domain(s) the avian PRLR employed in its interaction with PRL-L and PRL. The results from our present study would not only contribute to the better understanding of spatio-temporal expression pattern of PRLR and its underlying regulatory mechanisms in birds, but also provide new insights into the roles of PRLR and its ligand(s) in vertebrates, such as its roles in embryogenesis.