Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction
Research output: Contribution to journal › Journal article › Research › peer-review
Standard
Detecting protein-protein interactions in living cells : development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction. / Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche; Krogsgaard-Larsen, Povl; Kristensen, Anders Skov; Strømgaard, Kristian.
In: Neurochemical Research, Vol. 34, No. 10, 2009, p. 1729-1737.Research output: Contribution to journal › Journal article › Research › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Detecting protein-protein interactions in living cells
T2 - development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction
AU - Gottschalk, Marie
AU - Bach, Anders
AU - Hansen, Jakob Lerche
AU - Krogsgaard-Larsen, Povl
AU - Kristensen, Anders Skov
AU - Strømgaard, Kristian
N1 - Keywords: BRET assay; BRET; NMDA receptor; Protein-protein interactions; PSD-95; PDZ domain
PY - 2009
Y1 - 2009
N2 - The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.
AB - The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.
KW - Faculty of Health and Medical Sciences
U2 - 10.1007/s11064-009-9998-4
DO - 10.1007/s11064-009-9998-4
M3 - Journal article
C2 - 19495967
VL - 34
SP - 1729
EP - 1737
JO - Neurochemical Research
JF - Neurochemical Research
SN - 0364-3190
IS - 10
ER -
ID: 17366071