NR AREM
AU Knetsch,H.; Gauczynski,S.; Weiss,S.
TI Life cell Imaging of Prion Proteins, Prion-Receptor Molecules and their subcellular localization
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Poster GL-21
PT Konferenz-Poster
AB
We identified the PrP interacting 37 kDa/67 kDa laminin receptor (LRP/LR) (1) as the cell surface receptor for the cellular prion protein (PrPc) (2) and HSPGs as co-factors / co-receptors for PrPc (3). Recently, we demonstrated that LRP/LR is required for PrPsc propagation in neuronal cells (4).
In order to investigate the subcellular localization of PrP and LRP, we cloned murine LRP as a fusion with EGFP, ECFP, EYFP and DsRed and murine PrP as a fusion with EYFP and DsRed. In addition we used pEGFP-moPrP, a kind gift of Dr. Kil Sun Lee, Brasil, for cell transfections.
As markers specific for several cell compartments of the secretory and endocytic pathway we used the following constructs: pECFP-Golgi, pECFP-Mem, pECFP-Endo, pECFP-Cav1 and pClathrin-LC-ECFP.
BHK cells and HeLa cells were transfected with these constructs and the protein expression was analyzed by western blotting. To examine the subcellular distribution of these fusion proteins, transfected cells were analyzed by confocal microscopy. Co-localization studies might clarify if LRP and PrP share same regions of the cell surface or within specific compartments of the cell.
Fluorescence resonance energy transfer (FRET) represents a promising methodology for the detection of protein-protein interactions because it surpasses the optical limitations of the light microscope (5).
We are going to use this technique to investigate PrP/LRP interactions in living cells in more detail.
1. Rieger, R., Edenhofer, F., Lazémas, C. I., and Weiss S. (1997) Nat Med 3, 1383-1388
2. Gauczynski,S., Peyrin, J. M., Haik, S., Leucht, C., Hundt , C., Rieger, R., Krasemann, S., Deslys, J. P., Dormont, D., Lazémas, C. I., and Weiss S. (2001) EMBO J 20, 5863-5875
3. Hundt , C., Peyrin, J. M., Haik, S., Gauczynski, S., Leucht, C., Rieger, R., Riley, M.L., Deslys, J. P., Dormont, D., Lazémas, C. I., and Weiss S. (2001) EMBO J 20, 5876-5886
4. Leucht, C., Simoneau, S., Rey, C., Vana, K., Rieger, R., Lazémas, C. I., and Weiss S. (2003) EMBO Rep 4, 290-295
5. Sekar, R.B., Periasamy, A. (2003) J Cell Biol. 160, 629-633
AD Heike Knetsch, Sabine Gauczynski and Stefan Weiss, Laboratorium für Molekulare Biologie - Genzentrum - Institut für Biochemie der LMU München, Feodor-Lynen-Str. 25, 81377 München, Germany
SP englisch
PO Deutschland
OR Tagungsband