Functional activity of the human prolactin receptor and its ligands.
Prolactin receptors (PRLR) have been identified in a number of human tissues and cell lines, although little is known about the human receptor protein. The cloning of the human PRLR cDNA has enabled further characterization of the receptor protein in transfected cells. Since the human cDNA is expressed at lower levels than the rat cDNA, we have constructed a hybrid cDNA (pECE r5'hPRLR) containing nucleotides of the 5' untranslated region and signal peptide of the rat PRLR and the protein coding and 3' untranslated portion of the human receptor. Expression of the hybrid receptor was increased more than two-fold compared to the human receptor as detected by specific binding of 125I-human growth hormone (GH) to transfected COS-7 cells. The relative molecular mass of the receptor was 93,000 Da, as determined by chemical cross-linking studies. Transcriptional assays were used to show the human PRLR was able to activate two milk protein genes; ovine beta-lactoglobulin and rat beta-casein. Transfected cells expressing the human PRLR receptor, treated with human GH or prolactin (PRL), induced a dose-dependent increase in transcriptional activation of the beta-casein/luciferase fusion gene. Glycosylated, and non-glycosylated human PRL, and ovine PRL were equally effective in activating the beta-casein promoter. Human placental lactogen and bovine PRL could also induce a greater than 10-fold induction, whereas insulin did not significantly stimulate the beta-casein promoter. The results show that the human PRLR can activate both beta-lactoglobulin and beta-casein milk gene promoters and that these reporter genes can be used to evaluate the functional activity of agonists and antagonists of the human PRLR.
Lochnan
H A
HA
INSERM U344-Endocrinologie Moléculaire, Faculté de Médecine Necker, Paris, France.
Buteau
H
H
Richards
S
S
Edery
M
M
Kelly
P A
PA
D000818:Animals; D002364:Caseins / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007782:Lactoglobulins / Q000235:genetics; D008024:Ligands; D008156:Luciferases / Q000235:genetics; D011388:Prolactin / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011981:Receptors, Prolactin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D014162:Transfection
D002364:Caseins; D018076:DNA, Complementary; D007782:Lactoglobulins; D008024:Ligands; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D011388:Prolactin; D008156:Luciferases