No abstract

The biological activities of long and short forms of the prolactin receptor have been compared. These two receptors expressed in mammalian cells were shown to bind prolactin with equal high affinity. The ability of these different forms to transduce the hormonal message was estimated by their capacity to stimulate transcription by using the promoter of a milk protein gene fused to the chlormphenicol acetyltransferase (CAT) coding sequence. Experiments were performed in serum-free conditions to avoid the effect oflactogenic factors present in serum. An 417-fold induction of CAT activity was obtained in the presence of prolactin when the long form of the prolactin receptor was expressed, whereas no induction was observed when the short form was expressed. The present results clearly establish that only the long form of the prolactin receptor is involved in milk protein gene transcription.

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...

The prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained. These clones had 100% sequence identity in the overlapping region (893 bp) and were used to deduce the complete amino acid sequence of sbPRLR. The receptor spans 2640 bp and encodes a protein of 537 amino acids. Features characteristic of PRLR, two pairs of cysteines, WS box, hydrophobic transmembrane domain, box 1, and box 2, were identified and showed a high degree of sequence identity to PRLRs from other vertebrate species. SbPRLR is 29 and 32% identical to tilapia (Oreochromis niloticus) and goldfish (Carassius auratus) PRLRs, respectively. In the sea bream two PRLR transcripts of 2.8 and 3.2 kb were detected in the intestine, kidney, and gills and a single transcript of 2.8 kb was detected in skin and pituitary by Northern blot. Spermiating gonads (more than 95% male tissue; gonado-somatic index of 0.6) contained, in addition to the 2.8-kb transcript, three more transcripts of 1.9, 1.3, and 1.1 kb. RT-PCR, which is a far more sensitive method than Northern blot, detected PRLR mRNA in gills, intestine, brain, pituitary, kidney, liver, gonads, spleen, head-kidney, heart, muscle, and bone. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected PRLR in several epithelial tissues of juvenile sea bream, including the anterior gut, renal tubule, choroid membrane of the third ventricle, saccus vasculosus, branchial chloride cells, and branchial cartilage.