Erythroid progenitors carry erythropoietin receptor (EpoR) homodimers which change their conformational state upon activation by its ligand Epo. This initiates intracellular signalling required for proliferation and differentiation of progenitors. It has been hypothesized that non-erythroid cells - particularly certain tumor cells - may express the EpoR and respond to Epo administration by increased tumor growth. The aim of this work is therefore to find evidence for EpoR homodimers in non-erythroid cells and study their functional relevance.

Methods:

Cells were transfected with plasmids encoding for EpoR fused to either ECFP or EYFP and subjected to fluorescence resonance energy transfer (FRET) microscopy on our custom built confocal FRET analysis system.

Results:

Despite excellent co-localization of EpoR chimeras we failed to detect significant levels of energy transfer between both fluorophores. Significant FRET between ECFP and EYFP was only observed for truncated EpoR chimeras that lacked most of the intracellular domain. This truncated EpoR variant has been earlier discussed to exist on early-stage erythroid progenitors as a form of EpoR unresponsive to Epo. Interestingly, we were also not able to detect any conformational changes upon Epo stimulation.

Conclusion:

The absence of FRET is not sufficient to rule out physical interaction between two molecules. Despite physical interaction of the transmembrane domains the distance between the C-termini may be too large for FRET. Experiments with constructs where the fluorophores were inserted near the transmembrane domain are currently in progress and will allow for more sensitive detection of possible homodimers.

Supported by European Commision under the 7th Framework Programme (FP7-HEALTH-2011-single-stage Grant Agreement Number 282551EpoCan) to Joachim Fandrey. This report reflects only the authors' views, and the European Community is in no way liable for any use that may be made of the information contained herein.