Expression cloning of a cDNA encoding a fish prolactin receptor.

Abstract

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (-37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.Prolactin (PRL) is a pituitary polypeptide hormone that is implicated in many physiological actions in vertebrates including fish (1). Since the pioneering work by Pickford and Phillips in 1959 (2) demonstrating that hypophysectomized Fundulus heteroclitus require PRL for survival in freshwater, numerous studies have confirmed that PRL is one of the major hormones regulating the maintenance of water and electrolyte homeostasis on osmoregulatory surfaces (3-5). In tilapia, two distinct PRL forms have been well characterized (tiPRL188 or tiPRL, and tiPRL177 or tiPRLII), which share only 69% identity (6-8).These two tilapia PRLs (tiPRLs) were shown to be differentially regulated during adaptation to a hyperosmotic environment (9-11) and to exhibit both common and distinct biological effects and potencies (6, 10, 12). These effects are mediated by a specific cell membrane receptor. In tilapia, initial studies using ovine PRL (oPRL) as a ligand revealed the presence of PRL receptors in various tissues (13-15). Using bioactive recombinant tiPRL forms (16), high specific binding of PRL (up to 45% with tiPRL188) has recently been shown in gill and kidney, involving only one class of receptors, which binds tiPRL188 with higher affinity than tiPRL177 (17).PRL receptor cDNAs have been cloned from several mammalian species and sources (18)(19)(20) and two avian species (21, 22). These receptors belong to a superfamily including the receptors for growth hormone (GH), cytokines, and erythropoietin (18). This superfamily has several common structural features (23) including two pairs of conserved extracellular cysteine residues, a single transmembrane domain, and an intracellular proline-rich region (24). In lower vertebrates, however, no PRL receptor cDNA has been identified.We report in this paper the isolation of a tiPRL receptor cDNAI by an expression cloning approach (25). The characterized tiPRL receptor is a mature protein of 606 amino acids with a single extracellular unit and a long cytoplasmic domain. Moreover, tissue distribution studies indica...

Cloning, Characterization, and Tissue Distribution of Prolactin Receptor in the Sea Bream (Sparus aurata)

“…The expression of PRLR was analyzed in tilapia by Northern blot and the results obtained demonstrated that the osmoregulatory organs, gills, kidney, and intestine expressed PRLR at higher levels. The ovary, testis, and headkidney (which has hematopoietic function in fish) also expressed PRLR but at much lower levels (Sandra et al, 1995(Sandra et al, , 2000. A similar distribution of PRLR was also observed in the goldfish (Tse et al, 2000).…”

“…Despite the presence of all the characteristic features of PRLRs, the amino acid sequence of sbPRLR shares a relatively low level of identity with tiPRLR (Sandra et al, 1995) and gfPRLR (Tse et al, 2000). However, sequence identity is higher (46%) in tilapia and goldfish PRLR.…”

“…In tilapia there is a single transcript of 3.2 kb which shares greatest similarity with the long forms of mammalian PRLRs, although the overall homology is low (37%) (Sandra et al, 1995(Sandra et al, , 2000. In the goldfish two transcripts, of 3.5 and 4.6 kb, were identified.…”

“…PRLRs have also been cloned in an amphibian (Yamamoto et al, 1998), in a euryhaline teleost, Nile tilapia (Oreochromis niloticus) (tiPRLR) (Sandra et al, 1995), and in a fresh water teleost, the goldfish (Carassius auratus) (gf PRLR) (Tse et al, 2000). In tilapia there is a single transcript of 3.2 kb which shares greatest similarity with the long forms of mammalian PRLRs, although the overall homology is low (37%) (Sandra et al, 1995(Sandra et al, , 2000.…”

Prolactin177, prolactin188 and prolactin receptor 2 in the pituitary of the euryhaline tilapia, Oreochromis mossambicus, are differentially osmosensitive

“…We also explored the effects of acclimation salinity and extracellular osmolality on the mRNA expression of two Prl receptors (prlr) in the RPD to explore possible feedback mechanisms on Prl expression/ release. Originally cloned by Sandra et al (1995) in Nile tilapia (Oreochromis niloticus), the tilapia Prl receptor (PrlR1) is expressed in an array of tissues, including gill, intestine and kidney (Pierce et al 2007). prlr1 mRNA expression is sensitive to salinity, with enhanced expression occurring in the gill when Mozambique tilapia are transferred from SW to FW (Breves et al 2011) and down-regulated expression after transfer from FW to SW (Breves et al 2010a).…”