Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy

Eva Biener-Ramanujan, V. Krishnan Ramanujan, Brian Herman, Arieh Gertler

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)247-257
Number of pages11
JournalGrowth Hormone and IGF Research
Volume16
Issue number4
DOIs
StatePublished - Aug 1 2006

Keywords

  • Dimerization
  • FLIM
  • FRET
  • Growth hormone receptor
  • Live-cell imaging
  • STAT5

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