INTERNALIZATION OF PROLACTIN RECEPTOR AND PROLACTIN IN TRANSFECTED CELLS DOES NOT INVOLVE NUCLEAR TRANSLOCATION

Prolactin (PRL) interacts with a specific, well characterized plasma m embrane receptor (PRLR) that is coupled to signal transduction pathway s involving Jak2, Fyn, and MAP kinases, and signal transducers and act ivators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocyte s, PRL-dependent Nb2 lymphoma cell lines and 235-1 lactotrophs, the me chanisms of nuclear targeting remain unknown and conflicting results h ave been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PR LR and PRL in various cell lines transfected with an expression plasmi d encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing a n oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature recep tor (named N-terminal or C-terminal Flag-tagged PRLR). The correspondi ng receptors function as the PRLR in transfected cells : they are expr essed at the plasma membrane and in compartments of the secretory path way, they bind PRL with normal affinity (K-d = 4x10(-10) M) and have t he same capacity to stimulate the transcriptional activity of a milk p rotein (beta-casein) gene as wild-type PRLR. In addition, the tagged r eceptors are much more efficiently immunodetected using anti-Flag anti bodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluoresce nce combined with detailed confocal laser microscopy showed that addit ion of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibro blasts induces rapid internalization of the receptor (long form), with out any translocation to the nucleus. Using PRL-R tagged both in the N -terminal or C-terminal regions of the mature receptor excludes the po ssibility of a cleaved fragment which could have been subsequently imp orted into the nucleus. An absence of nuclear translocation of PRLR wa s also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressi ng the Nb2 form of the receptor or in BGME mammary gland epithelial ce lls upon overexpression of a Flag-tagged PRLR. Similarly, the short fo rm of the PRLR was not detected in nuclei of transfected COS cells upo n PRL treatment. Clearly, our results provide evidence that internaliz ation of the plasma membrane PRLR does not lead to nuclear translocati on 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. Acti vation of STATs transcription factors and MAP kinases, as well as tran slocation of these proteins to the nucleus following their phosphoryla tion, probably remains the intracellular mechanism coupling stimulatio n to nuclear events.