Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction.
Neurochem Res
; 34(10): 1729-37, 2009 Oct.
Article
de En
| MEDLINE
| ID: mdl-19495967
ABSTRACT
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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Récepteurs du N-méthyl-D-aspartate
/
Protéines et peptides de signalisation intracellulaire
/
Transfert d'énergie
/
Motifs et domaines d'intéraction protéique
/
Mesures de luminescence
/
Protéines membranaires
Type d'étude:
Evaluation_studies
Limites:
Animals
/
Humans
Langue:
En
Journal:
Neurochem Res
Année:
2009
Type de document:
Article
Pays d'affiliation:
Danemark