Covalent labeling of cell-surface proteins for in-vivo FRET studies.
Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.
Ecole Polytechnique Fédérale de Lausanne (EPFL), Institut de Science et Ingénierie Chimiques, CH-1015 Lausanne, Switzerland.
Bruno H
BH
Meyer
Karen L
KL
Martinez
Jean-Manuel
JM
Segura
Pedro
P
Pascoal
Ruud
R
Hovius
Nathalie
N
George
Kai
K
Johnsson
Horst
H
Vogel
D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000235:genetics; D002232:Carbocyanines / Q000737:chemistry; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013373:Substance P / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000213:Acyl Carrier Protein; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1; D011993:Recombinant Fusion Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5; D013373:Substance P