Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation.
Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235-1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells: they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4x10(-10) M) and have the same capacity to stimulate the transcriptional activity of a milk protein (beta-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-localization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.
Perrot-Applanat
M
M
INSERM U 344, Faculté de Médecine Necker-Enfants Malades, Paris, France.
Gualillo
O
O
Buteau
H
H
Edery
M
M
Kelly
P A
PA
D016475:3T3 Cells / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D008024:Ligands; D008321:Mammary Glands, Animal / Q000166:cytology; D051379:Mice; D018613:Microscopy, Confocal; D016296:Mutagenesis / Q000502:physiology; D011388:Prolactin / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011981:Receptors, Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D008024:Ligands; D011981:Receptors, Prolactin; D011388:Prolactin