Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3.
HEK-293T cells transiently transfected with ovine (o) GH receptor (GHR) and prolactin receptor (PRLR) constructs respectively tagged downstream with cyan or yellow fluorescent proteins were used to study ovine placental lactogen (oPL)-stimulated heterodimerization by fluorescence resonance energy transfer (FRET) microscopy. The oPL-stimulated transient heterodimerization of GHR and PRLR had a peak occurring 2.5-3 min after oPL application, whereas oGH or oPRL had no effect at all. The results indicate none or only little dimerization occurring before the hormonal stimulation. The effect of heterodimerization was studied by comparing activation of Janus kinase 2, signal transducer and activator of transcription (STAT)1, STAT3, STAT5, and MAPK in Chinese hamster ovary cells stably transfected with chimeric genes encoding receptors consisting of cytosolic and transmembrane parts of oGHR and oPRLR, extracellular domains of human granulocyte and macrophage colony-stimulating factor (hGM-CSF) receptor alpha or beta, and cells transfected with the two forms (alpha or beta) of PRLR and GHR. Functionality of those proteins was verified by hGM-CSF-induced phosphorylation of both intracellular PRLR and GHR domains and hGM-CSF-induced heterodimerization was documented by chimeric receptor coimmunoprecipitation. Homodimerization or heterodimerization of PRLRs and GHRs had no differential effect on activation of STAT5 and MAPK. However, heterodimerization resulted in a prolonged phosphorylation of STAT1 and in particular STAT3, suggesting that the heterodimerization of alpha-oGHR and beta-oPRLR is able to transduce a signal, which is distinct from that occurring on homodimeric associations.
Biener
Eva
E
The Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Martin
Cyril
C
Daniel
Nathalie
N
Frank
Stuart J
SJ
Centonze
Victoria E
VE
Herman
Brian
B
Djiane
Jean
J
Gertler
Arieh
A
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004622:Embryo, Mammalian; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008894:Milk Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010928:Placental Lactogen / Q000494:pharmacology*; D011981:Receptors, Prolactin / Q000737:chemistry* / Q000235:genetics; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D012756:Sheep; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D008894:Milk Proteins; D011981:Receptors, Prolactin; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D010928:Placental Lactogen; D020928:Mitogen-Activated Protein Kinases