Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action.
Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-beta-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-beta-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2.5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.
Gertler
Arieh
A
The Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel. gertler@agri.huji.ac.il
Biener
Eva
E
Ramanujan
Krishnan V
KV
Djiane
Jean
J
Herman
Brian
B
D000818:Animals; D002477:Cells / Q000187:drug effects / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D013006:Growth Hormone / Q000494:pharmacology; D006801:Humans; D020738:Leptin / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D010928:Placental Lactogen / Q000494:pharmacology; D011388:Prolactin / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011981:Receptors, Prolactin / Q000737:chemistry; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000502:physiology; D012756:Sheep; D015533:Transcriptional Activation
D016207:Cytokines; D013006:Growth Hormone; D020738:Leptin; D010928:Placental Lactogen; D011388:Prolactin; D018719:Erb-b2 Receptor Tyrosine Kinases; D011981:Receptors, Prolactin; D011986:Receptors, Somatotropin