Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.
The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.
Biener-Ramanujan
Eva
E
The Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel.
Ramanujan
V Krishnan
VK
Herman
Brian
B
Gertler
Arieh
A
D002478:Cells, Cultured; D019281:Dimerization; D004705:Endocytosis / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D013006:Growth Hormone / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D021381:Protein Transport; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D014018:Tissue Distribution
D005456:Fluorescent Dyes; D011986:Receptors, Somatotropin; D050799:STAT5 Transcription Factor; D013006:Growth Hormone; D008156:Luciferases