Prolactin signal transduction to milk protein genes: carboxy-terminal part of the prolactin receptor and its tyrosine phosphorylation are not obligatory for JAK2 and STAT5 activation.
In this study, we have developed several Chinese Hamster ovary (CHO) cell clones stably expressing various deletion mutant forms of the rabbit prolactin receptor (rbPRL-R) to better define the domains of the receptor involved in JAK2 kinase interaction, STAT5 activation, and to assess the role of tyrosine phosphorylation of the PRL-R in signal transduction. We observed that the box 1 region of the receptor was critical for productive interaction with JAK2 and its tyrosine phosphorylation after PRL stimulation. However, this region appeared to require the presence of additional cytoplasmic domain region(s), such as box 2, to exert its complete effect. In addition, we found that a mutant form lacking the 141 C-terminal residues lost the capacity to be tyrosine phosphorylated in response to PRL but remained able to activate JAK2 kinase and STAT5 transcription factor, indicating that it contained the minimal sequence required for STAT5 activation. The absence of tyrosine phosphorylation of this C-terminal rbPRL-R mutant upon PRL stimulation indicated that the phosphorylation of the PRL-R normally occured in the last 141 animo acids (aa) containing three tyrosines and was not absolutely necessary for induction of these early events in PRL signal transduction. Transfectant cell lines expressing wild type (WT) PRL-R and this C-terminal mutant form were able to induce CAT activity upon PRL stimulation when transiently transfected with the ovine-beta-lactoglobulin promoter, containing STAT5 recognition sites, fused to the CAT reporter gene. The comparison between transcriptional activity of these two receptor forms leads to the conclusion that the C-terminal region of the rbPRL-R, containing the physiological sites for tyrosine phosphorylation, is probably responsible for an amplification of the PRL signal to milk protein genes.
Goupille
O
O
Unité d'Endocrinologie Moléculaire, Institut National de la RechercheAgronomique, Jouy en Josas, France.
Daniel
N
N
Bignon
C
C
Jolivet
G
G
Djiane
J
J
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002364:Caseins / Q000235:genetics; D002457:Cell Extracts; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053614:Janus Kinase 2; D007782:Lactoglobulins / Q000235:genetics; D008894:Milk Proteins; D008970:Molecular Weight; D010766:Phosphorylation; D011388:Prolactin / Q000378:metabolism* / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011817:Rabbits; D051381:Rats; D011981:Receptors, Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D017384:Sequence Deletion; D012756:Sheep; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D002364:Caseins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D007782:Lactoglobulins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011981:Receptors, Prolactin; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; D011505:Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2