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.

Human PRL receptor cDNA clones from hepatoma (Hep G2) and breast cancer (T-47D) libraries were isolated by using a rat PRL receptor cDNA probe. The nucleotide sequence predicts a mature protein of 598 amino acids with a much longer cytoplasmic domain than the rat liver PRL receptor. Although this extended region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction. This cDNA will be a valuable tool to better understand the role of PRL in the development and growth of human breast cancer.

Two Agtll clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.The anterior pituitary hormone prolactin (PRL) is involved in an impressive list of biological actions in all vertebrates (1) and specific receptors for this hormone have been identified in numerous organs (2). PRL is structurally related to growth hormone (GH) and chorionic somatomammotropic or placental lactogen, which are encoded by the same gene family (3). In mammals, PRL is primarily responsible for the development of the mammary gland and lactation. It stimulates the expression of milk protein genes by increasing both gene transcription and mRNA half-life (4). These actions are initiated by an interaction of PRL with specific, high-affinity receptors located in cell membranes. This receptor has been partially purified from rabbit mammary gland (5) and monoclonal antibodies against it have been developed (6). After binding of PRL to its receptors, the mechanism(s) by which the hormonal signal is transferred inside the cell remains unknown. We have shown (7) that one monoclonal antibody against the PRL receptor is a potent agonist of PRL actions in mammary cells, suggesting that PRL itself is probably not necessary beyond its binding and that the receptor is able, under certain experimental conditions, to operate alone probably by coupling to a second messenger system. However, as is true for GH, none of the classical second messengers (cAMP, cGMP, inositol phospholipids, or calcium ions) appears to be involved in PRL signal transduction (8). The knowledge of the primary structure of the receptor and the identification of functional domains may facilitate a better understanding of these phenomena. To that end, we have previously cloned cDNAs of PRL receptor from rat liver cDNA libraries (9), deduced primary structure of the protein, and established that this receptor has regions of strong sequence homology with the GH receptor, also cloned (10), but contains a much shorter cytoplasmic domain. The mammary gland is a major target organ for PRL, where in contrast to the liver, biological effects are well established (4). We have reported (9) that the major mRNA species for the PRL receptor is much larger in the mammary gland [4 kilobases (kb)] than in the liver (2.2 kb), suggesting heterogeneity in the coding sequence or in the untranslated region ...

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We have recently cloned a cDNA encoding a mutant form of PRL receptor (PRL-R) from Nb2 cells, a PRL-dependent T lymphocyte-derived cell line. This cDNA is identical to the long form of the rat PRL-R, except for a deletion of 594 base pairs in the cytoplasmic domain, resulting in a mature receptor protein of 393 amino acids. Although a segment containing three cytoplasmic regions of moderate to high amino acid sequence identity with members of the PRL/GH receptor family is missing in this receptor form, the region of highest (70%) identity is retained. In the following studies, a homologous functional assay was developed to test the activity of three forms of receptor with respect to their ability to transmit a lactogenic signal. In this system, CHO cells were transiently transfected with a construct containing 2300 base pairs of the 5'-flanking sequence of the rat beta-casein gene fused to the chloramphenicol acetyltransferase (CAT) gene and an expression vector containing the various forms of rat PRL-R cDNA. The transfected cells were grown in serum-free medium in the absence or presence of PRL. In cells transfected with the long form of the PRL-R and beta-casein/CAT construct, a 7.2- +/- 0.9-fold induction (n = 3) of CAT activity was seen when cells were cultured in the presence of 400 ng/ml PRL and 1 micrograms/ml hydrocortisone. This level of stimulation was similar to that observed for the ovine beta-lactoglobulin/CAT construct in which a 5.7- +/- 1.2-fold (n = 3) effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)