FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.
The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligand-induced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute approximately 1% of the cell membrane and to be sensitive to cholesterol depletion.
Meyer
Bruno H
BH
Ecole Polytechnique Fédérale de Lausanne (EPFL), Institut des Sciences et Ingénierie Chimiques, CH-1015 Lausanne, Switzerland.
Segura
Jean-Manuel
JM
Martinez
Karen L
KL
Hovius
Ruud
R
George
Nathalie
N
Johnsson
Kai
K
Vogel
Horst
H
D000213:Acyl Carrier Protein / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D002784:Cholesterol / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D018040:Receptors, Neurokinin-1 / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics
D000213:Acyl Carrier Protein; D018040:Receptors, Neurokinin-1; D011993:Recombinant Fusion Proteins; D002784:Cholesterol