Detecting protein-protein interactions in living cells: development of a bioluminescence resonance energy transfer assay to evaluate the PSD-95/NMDA receptor interaction.
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.
Gottschalk
Marie
M
Department of Medicinal Chemistry, The Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
Bach
Anders
A
Hansen
Jakob Lerche
JL
Krogsgaard-Larsen
Povl
P
Kristensen
Anders S
AS
Strømgaard
Kristian
K
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D004735:Energy Transfer / Q000502:physiology*; D015696:Gene Products, tat / Q000737:chemistry / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism*
C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D015696:Gene Products, tat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins; D049410:Luciferases, Renilla