Covalent labeling of cell-surface proteins for in-vivo FRET studies
Bruno H. Meyer1, Karen L. Martinez2, Jean-Manuel Segura, Pedro Pascoal, Ruud Hovius,
Nathalie George, Kai Johnsson, Horst Vogel*
Ecole Polytechnique Fe ´de ´rale de Lausanne (EPFL), Institut de Science et Inge ´nierie Chimiques, CH-1015 Lausanne, Switzerland
Received 23 November 2005; revised 11 January 2006; accepted 6 February 2006
Available online 17 February 2006
Edited by Irmgard Sinning
powerful technique to reveal interactions between membrane pro-
teins 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 con-
trol of donor–acceptor ratio and high background fluorescence
arising from intracellular FPs. Here we show that these short-
comings can be overcome by using the acyl carrier protein label-
ing technique. FRET revealed interactions between cell-surface
neurokinin-1 receptors simultaneously labeled with a controlled
ratio of donors and acceptors. Moreover, using FRET the spe-
cific binding of fluorescent agonists could be monitored.
? ? 2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Fluorescence resonance energy transfer (FRET) is a
Keywords: ACP labeling; FRET microscopy; GPCR; Donor–
G protein-coupled receptors (GPCRs) constitute the largest
family of transmembrane cell-surface proteins involved in sig-
nal transduction and are the most important targets in the
search for novel therapeutic compounds . With the develop-
ment of various labeling strategies, such as the site-selective
introduction of fluorescent amino acids into the sequence of
proteins , the modification of cysteines in suitable receptor
mutant proteins  or the production of fusion constructs with
fluorescent proteins (FP) , fluorescence techniques  have
rapidly gained importance in the study of the function of
GPCRs in vitro and in living cells. In particular, fluorescence
resonance energy transfer (FRET), owing to its strong distance
sensitivity in the range of 10–100 A˚, was shown to be ideally
suited to study both structures  and structural changes with-
in , and interactions between [7–9] GPCR-signaling proteins
tagged with suitable donor and acceptor fluorophores. In addi-
tion, FRET is of increasing importance as a readout signal for
genetically encoded sensors  and as a means to monitor
ligand binding to membrane proteins [11,12].
Restrictions often encountered in FRET studies using FPs
are the choice of suitable fluorophores, the incomplete process-
ing of fusion constructs leading to high background signals,
and difficulties in achieving defined donor–acceptor (DA)
ratios. In recent years several promising alternatives to FPs
have been developed [13–19]. They are based on the covalent
or non-covalent post-translational labeling of a fusion protein.
One of these methods, the acyl carrier protein (ACP) labeling
technique, makes use of the enzymatic transfer of a 4’-phos-
phopantetheine from coenzyme A (CoA) labeled with a fluoro-
phore to a serine residue of ACP fused to the protein of
interest . Because the enzyme and the CoA substrate re-
quired for the reaction are not membrane permeable, only pro-
teins correctly localized in the cell membrane are labeled,
avoiding background signals from intracellular proteins. In
addition, the ACP labeling allows the choice of optimal fluoro-
phores for each experimental situation and thus the panel of
applications is substantially broader.
Here we show the feasibility and versatility of ACP labeling
for in vivo FRET studies by investigating a prototypical
GPCR, the neurokinin-1 receptor (NK1R). FRET between
labeled receptors and fluorescently labeled agonists allowed
accurate recording of binding kinetics. Furthermore, FRET
between NK1Rs simultaneously labeled with donors and
acceptors at a controlled DA ratio revealed close proximity
of NK1Rs in the plasma membrane.
2. Materials and methods
Enhanced cyan (ECFP) and yellow (EYFP) fluorescent proteins
were from Clontech; Dulbecco’s modified Eagle’s medium (DMEM),
fetal calf serum (FCS), Dulbecco phosphate-buffered saline (D-PBS)
and pCEP4 expression vector from Invitrogen; hygromycin B from
Calbiochem; bovine serum albumin (BSA) from Fluka; substance P
(SP) and N-terminally acetylated SP from K. Servis (University of
Lausanne, Lausanne, CH); bacitracin from Serva.
2.2. Plasmid constructs and cell culture
Fusion of ACP to the extracellular N-terminus of NK1R (ACP-
NK1R) is described elsewhere . ECFP and EYFP were fused to
the C-terminus of NK1R through a TSGGGG linker yielding
NK1R-ECFP and NK1R-EYFP, respectively, and were subcloned
Adherent HEK293 cells were grown in DMEM containing 2.5 v%
FCS at 37 ?C in a humidified atmosphere with 5% CO2. For confocal
microscopy, HEK293 cells were seeded (105cells/ml) into 6-well plates
Abbreviations: DA, donor–acceptor; FRET, fluorescence resonance
energy transfer; ECFP, enhanced cyan fluorescent protein; EYFP,
enhanced yellow fluorescent protein; FP, fluorescent protein; NK1R,
neurokinin 1 receptor; GPCR, G protein-coupled receptor; ACP, acyl
carrier protein; SP, substance P; DMEM, Dulbecco’s modified Eagle’s
medium; FCS, fetal calf serum; D-PBS, Dulbecco phosphate-buffered
saline; BSA, bovine serum albumin
*Corresponding author. Fax: +41 2169 36190.
E-mail address: email@example.com (H. Vogel).
1Present address: Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland.
2Present address: Nano-Science Center, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen, Denmark.
0014-5793/$32.00 ? 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 580 (2006) 1654–1658
containing a 25 mm glass coverslip and 2 ml DMEM/FCS. About 16–
20 h after splitting, cells were transfected with 2.5 lg of plasmid DNA
per well using the calcium phosphate technique. Imaging was
performed 24–55 h after transfection in D-PBS (with 0.1% w/v BSA
for ligand binding). Stable HEK293 cell lines were produced from
transiently transfected cells by selection with 200 lg/ml hygromycin B.
2.3. Calcium signaling
HEK293 cells stably expressing NK1R constructs were seeded into a
clear-bottom 96-well plate (Greiner) in DMEM/FCS and incubated at
37 ?C and 5% CO2. After 24 h the cells were loaded with a calcium-sen-
sitive fluorophore (Calcium 3 assay kit, Molecular Devices) during
30 min at 37 ?C. The change in fluorescence intensity at 525 nm upon
addition of SP in buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl,
5 mM MnCl2, 1 mg/ml BSA, and 100 lg/ml bacitracin) was recorded
on a FLEX station (Molecular Devices) upon excitation at 485 nm.
The dependence of the amplitude of the calcium signal on the SP con-
centration was fitted with a Hill-equation.
2.4. Synthesis of SP-Cy5
N-terminally acetylated SP was reactedwith 0.9 equivalentof Cy5-N-
hydroxysuccinimide in 500 ll N,N-dimethylformamide and 50 ll
100 mM sodium carbonate, pH 8.2. The product was purified by thin
layer chromatography on silica G60 (Merck) using MeOH:NH4OH
(95:5) as eluent and extraction with MeOH:H2O:5 M HCl (85:14:1).
nist with pEC50= 9.2 ± 0.6 for wtNK1R and 8.9 ± 0.4 for ACP-NK1R.
2.5. ACP labeling
Synthesis of CoA-Cy3 and CoA-Cy5 substrates and purification of
AcpS were described previously . The cells were first washed with
D-PBS and then labeled in D-PBS supplemented with 10 mM MgCl2,
1 lM AcpS and 5 lM CoA-substrate at 19 ?C for 40 min. For double
labeling CoA-Cy3 and CoA-Cy5 were mixed beforehand in the desired
ratio to a total concentration of 5 lM. After labeling the cells were
washed three times with D-PBS.
2.6. Confocal microscopy and FRET imaging
Laser-scanning confocal micrographs were recorded using the 458/
488 nm Ar-ion or the 543/633 nm HeNe laser lines on an LSM 510
microscope (Zeiss) with a 63· (1.2 NA) water immersion objective.
Detection of fluorescence signals was achieved at following settings
(excitation wavelength, dichroic mirror, emission bandpass filter):
ECFP (458 nm, HFT 453, BP 465-495); EYFP (488 nm, HFT 488,
BP 510-560). FRET efficiencies using Cy3 as donor and Cy5 as accep-
tor were measured by sensitized acceptor emission. Settings for the
acceptor emission Af (acceptor channel) were (633 nm, HFT 633, LP
650). Donor and FRET images were recorded simultaneously with
excitation at 543 nm and a dichroic mirror HFT 543, the emission
beam was split with a NFT 635 dichroic mirror onto two detectors
with a BP 560-615 filter for donor emission Df (donor channel) and
a LP 650 filter for acceptor emission Ff (FRET channel), respectively.
Intensities were corrected for background.
In the following, we use the terminology of Gordon et al. . The
apparent FRET efficiency Eapp,sewas calculated according to a method
called 3-cube FRET . After applying a threshold on the donor im-
age to select the membrane comprising the labeled ACP-NK1, FRET
ratios (FR), the fractional increase in acceptor emission due to FRET,
were calculated on a pixel-by-pixel basis:
FR ¼Ff ? S1Df
where S1and S4are cross-talk factors correcting for the donor emis-
sion detected in the FRET channel and the emission due to direct exci-
tation of the acceptor, respectively. No additional correction factors
were necessary as Cy3 is not excited at 633 nm and Cy5 does not emit
in the donor channel. A histogram of FR values was then built and fit-
ted with a Gaussian to yield the mean FR. This procedure minimized
possible artefacts introduced by the threshold at low FR values. Eapp,se
were calculated using:
Eapp;se¼ ðFR ? 1Þ½eAðkÞ=eDðkÞ?;
where eA(k) and eD(k) are the molar extinction coefficients of donor
and acceptor, respectively, at the donor excitation wavelength
(543 nm). For Cy3-Cy5, the ratio at 543 nm is 0.11. The experimentally
determined Eapp,seis a function of both the true FRET efficiency E and
the fraction of acceptor-labeled molecules bound to donor-labeled
Image analysis was performed using IGOR Pro 5 (Wavemetrics).
The fusion of ACP to the N-terminal part of NK1R and its
subsequent labeling did not alter the functionality of the recep-
tor as shown in Fig. 1. First, ACP-NK1R stably expressed in
HEK293 cells was still able to activate downstream signaling
upon agonist binding eliciting a calcium response in a dose
dependent manner (Fig. 1A). pEC50values measured for SP
were 10.7 ± 0.3 and 10.6 ± 0.3 for ACP-NK1R and wild-type
NK1R, respectively, in close agreement with published data
. Second, exposure to the agonist induced internalization
of the Cy5-labeled ACP-NK1R within 15 min (Fig. 1B), which
is consistent with previous reports on NK1R internalization by
Grady et al. . No fluorescence from either CoA-Cy5 or
Fig. 1. SP activation of ACP-NK1R signaling. (A) Concentration–response curves of SP measured by calcium signaling on wild-type NK1R (black)
and ACP-NK1R (red) expressed in HEK293 cells, yielding pEC50of 10.6 ± 0.3 and 10.7 ± 0.3, respectively. (B) Fluorescence confocal micrograph
showing HEK293 cells stably expressing ACP-NK1R labeled with Cy5. After 15 min of incubation with 100 nM SP at 19 ?C, the receptor was
internalized as revealed by the presence of bright intracellular foci. Scale bar is 10 lm.
B.H. Meyer et al. / FEBS Letters 580 (2006) 1654–1658
CoA-Cy3 was observed on cells not expressing ACP-NK1R,
showing that the receptors were specifically labeled by the
ACP-labeling technique (see Supplementary data). Together
these data demonstrate that the ACP-NK1R behaves as the
wild-type receptor and that fluorescent labeling of NK1R
through the ACP fusion allows observation of the authentic
GPCR signal transduction processes.
GPCR-mediated signaling is initiated by ligand binding.
This can be easily monitored on a living cell in real-time by
FRET when receptor and ligand are labeled with donor and
acceptor. The versatility of the ACP labeling method for this
approach was demonstrated by labeling ACP-NK1R with
Cy3 (donor) and subsequently adding SP-Cy5 (acceptor)
(Fig. 2A–B). Complete co-localization of both fluorescence sig-
nals showed that all Cy3-ACP-NK1R are accessed by SP-Cy5,
confirming that the ACP method exclusively labels the recep-
tors on the cell surface. Non-specific binding of SP-Cy5 to
the plasma membrane was far less than 5% of the specific bind-
ing to the NK1R as assessed from competition experiments
with other non-fluorescent NK1R-specific ligands and the
colocalisation with ACP-NK1R (see Supplementary data). In
contrast to these results obtained using ACP labeling, similar
experiments using NK1R fused to FPs suffered from strong
background from intracellular receptors (see below) and from
the restricted choice of the spectral region (data not shown).
The high quality of the fluorescence signals allowed monitor-
ing of the time course of repetitive binding and dissociation of
SP-Cy5 to Cy3-ACP-NK1R (Fig. 2). The time course of the in-
crease of the fluorescence of SP-Cy5 at the cell membrane
shows two distinct processes (Fig. 2C, middle): the rapid phase
stems from ligand addition to the sample (Fig. 2C, top), the
slower phase corresponds to the specific binding of SP-Cy5
to the NK1R as revealed by the concomitant Cy3-Cy5 FRET
signal (Fig. 2C, bottom). It was not possible to determine the
specific binding of SP-Cy5 to NK1R only from the fluores-
cence trace of SP-Cy5 (Fig. 2C, middle) because in addition
to both specific and unspecific binding, it comprises a contribu-
tion from free SP-Cy5 in solution. Addition of increasing SP-
Cy5 concentrations resulted in both a further increase of the
fluorescence of membrane associated SP-Cy5 and a decrease
of the Cy3-ACP-NK1R signal (see Supplementary data). The
time course of specific binding measured by FRET revealed
that after removal of SP-Cy5, about one-third of the specifi-
cally bound agonists did not dissociate from their cognate
receptors (Fig. 2C, bottom). The origin of this effect might
be the internalization of NK1R-bound SP-Cy5 (see also
Fig. 1B) or the existence of a state of the NK1R with extremely
low dissociation rate , which might represent receptors pre-
coupled to G proteins.
Besides the monitoring of ligand binding, FRET is increas-
ingly used to detect protein–protein interactions in living cells.
For example, in GPCR research, FRET (and bioluminescence
resonance energy transfer) is a method of choice to investigate
receptor oligomerization in the plasma membrane [8,9]. In this
type of experiments, quantitative determination of the degree
of oligomerization critically relies on the possibility to vary
precisely the DA ratio and to investigate its influence on the
FRET signal . This is usually done by cotransfection of
plasmids encoding the protein of interest fused to ECFP and
We compared the standard FP method using ECFP- and
EYFP-NK1R fusions to our approach of double labeling by
the ACP technique using ACP-NK1R. Representative confocal
micrographs in Figs. 3A–C show the difficulties related to the
Fig. 2. SP-Cy5 binding to Cy3-labeled ACP-NK1R. Fluorescence confocal micrographs showing HEK293 cells expressing ACP-NK1R labeled with
Cy3 (A) and receptor-bound SP-Cy5 upon a short incubation with 100 nM ligand (B). Scale bar is 10 lm. (C) A 116 nM solution of SP-Cy5 was
applied at a few micrometers away from the cell with the pipette of a perfusion system at 19 ?C. Application duration denoted by the bar length was
84 s. The time traces represent fluorescence intensities for the same region of interest using the acceptor channel of SP-Cy5 in buffer (top) and bound
to the cell membrane (middle), and using the donor channel of Cy3-labeled ACP-NK1R (bottom).
B.H. Meyer et al. / FEBS Letters 580 (2006) 1654–1658
use of FPs for double labeling: (i) a considerable amount of the
FP-labeled NK1Rs is retained within the cell, thereby introduc-
ing a strong fluorescence background. (ii) Although a 1:1 mix-
ture of the corresponding plasmids was used for transfection,
expression levels and localizations of the receptor constructs
in the cells were finally very different for both fusion proteins,
making a precise determination of the DA ratio difficult. In
contrast, ACP labeling using a mixture of Cy3 and Cy5 sub-
strates resulted in the simultaneous double labeling of only sur-
fluorescence from receptors inside the cells (Fig. 3D–E). Fur-
thermore, cellular autofluorescence did not interfere with the
measurements when using dyes with longer wavelengths.
The DA ratio of Cy3- and Cy5-labeled ACP-NK1R could be
precisely controlled by simultaneous double labeling using dif-
ferent mole fractions of the CoA-substrates. This is demon-
strated in Fig. 3H, which shows that the fluorescence
intensity of Cy5-ACP-NK1R depended linearly on the accep-
tor mole fraction. The final DA ratio after labeling can thus
be varied in a controlled way by simply mixing the two dye-
conjugated substrates to the desired ratio beforehand.
The high quality of the measurements on ACP-NK1R in
Fig. 3 together with the absence of non-specific binding of
the fluorescent CoA analogues to the plasma membrane al-
lowed a quantitative evaluation of the FRET signal. Figs.
3D–F are typical images taken with the donor, acceptor and
FRET settings, respectively. The FRET ratio (FR) was calcu-
lated on a pixel-by-pixel basis after application of an intensity
threshold to select the membranes comprising the labeled
NK1Rs (Fig. 3G). The average FR value of 5.75 ± 0.02 corre-
sponds to an apparent FRET efficiency of 52.3%. This high
FRET signal indicates that the NK1Rs on the cell surface
are in close proximity, which could result from either GPCR
oligomerization  or high receptor surface densities.
Our results show that the ACP labeling technique is highly
suitable for FRET studies of plasma membrane proteins in liv-
ing cells. Compared to the very popular labeling with FPs, ACP
labeling offers access to a wide range of labels and to the exclu-
sive labeling of cell-surface proteins, thereby allowing an opti-
mized choice of fluorophores and measurements free of the
beled proteins. This is crucial for accurate FRET experiments.
measurements of specific ligand binding in a FRET-based ap-
proach as pioneered by Turcatti et al. in native membranes
using suppressor tRNA technology  and Vollmer et al. using
Fig. 3. Controlled double labeling of NK1R by the ACP technique for FRET. HEK293 cells were transiently transfected with a 1:1 DNA ratio of
ECFP and EYFP. Fluorescence of ECFP (A) and EYFP (B) was detected using appropriate settings. (C) Transmission image with superimposed
fluorescence signals from A and B. (D–F) HEK293 cells transiently expressing ACP-NK1R were labeled simultaneously with an 80%/20% mixture of
Cy3 and Cy5. Cells were imaged with the appropriate settings for donor (D), acceptor (E) and FRET (F). Scale bars are 10 lm. (G) FRET ratio (FR)
image calculated from the images D–F. The average FR value was 5.75 ± 0.02, corresponding to an apparent FRET efficiency of 52.3%. The color
scale represents the FR value. (H) Normalized acceptor intensity (a.u.) as a function of the acceptor mole fraction used for labeling. Data points are
means of 10 cells (±S.D.).
B.H. Meyer et al. / FEBS Letters 580 (2006) 1654–1658
receptor-FP fusions in live cells . For experiments in live Download full-text
cells, the ACP method presents two major advantages com-
pared to FP fusions: (i) Improved signal-to-noise ratios can be
obtained owing to the exclusive labeling of the cell-surface
receptors, and (ii) an extended panel of FRET pairs can be
tested with the same fusion protein, reducing tedious cloning ef-
forts. This is of utmost interest in this context because only a
limited number of FPs suitable as FRET pairs is available .
We further showed the benefits of ACP labeling for monitor-
ing protein–protein interactions. The simultaneous two-color
labeling of ACP-NK1R with well-defined ratios of donors and
acceptors revealed a high apparent FRET efficiency, showing
close proximity of the NK1Rs in the plasma membrane, whose
origin will need further investigations. Similar studies using FPs
suffer from poorer signal-to-noise ratios and require the co-
expression of two fusion proteins at the same time in the same
cell in a defined ratio, which is often difficult to achieve and is
usually controlled by the DNA ratio used for transfection.
Therefore, we are confident that the many advantages of-
fered by the ACP labeling technique, in particular the possibil-
ity of double labeling with controlled DA ratios, make it a
generally applicable method for investigating cell-surface pro-
teins by novel fluorescence techniques  including molecular
interactions, especially homo-oligomerisation, by FRET with
potential single-molecule sensitivity. We are presently extend-
ing this approach to monitor conformational changes of iono-
tropicreceptors, which are
Acknowledgements: This project was financially supported by the Swiss
National Science Foundation (NRP 47, Project No. 4047-057572), the
TopNano21 program (Project Nos. 5636.2 and 6266.1), the European
Commission (contract LSGH-CT-2004-504601/MEP), and internal
grants from EPFL.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.febslet.2006.02.007.
 Wise, A., Gearing, K. and Rees, S. (2002) Target validation of G
protein-coupled receptors. Drug Discov. Today 7, 235–246.
 Turcatti, G., Nemeth, K., Edgerton, M., Meseth, U., Talabot, F.,
Peitsch, M., Knowles, J., Vogel, H. and Chollet, A. (1996)
Probing the structure and function of the tachykinin NK2
receptor through biosynthetic incorporation of fluorescent amino
acids at specific sites. J. Biol. Chem. 271, 19991–19998.
 Gether, U., Lin, S. and Kobilka, B.K. (1995) Fluorescent labeling
of purified beta 2 adrenergic receptor. Evidence for ligand-specific
conformational changes. J. Biol. Chem. 270, 28268–28275.
 Tsien, R.Y. (1998) The green fluorescent protein. Annu. Rev.
Biochem. 67, 509–544.
 Hovius, R., Vallotton, P., Wohland, T. and Vogel, H. (2000)
Fluorescence techniques. Shedding light on ligand-receptor inter-
actions. Trends Pharmacol. Sci. 21, 266–273.
 Vilardaga, J.P., Bunemann, M., Krasel, C., Castro, M. and
Lohse, M.J. (2003) Measurement of the millisecond activation
switch of G protein-coupled receptors in living cells. Nat.
Biotechnol. 21, 807–812.
 Bunemann, M., Frank, M. and Lohse, M.J. (2003) Gi protein
activation in intact cells involves subunit rearrangement rather
than dissociation. Proc. Natl. Acad. Sci. USA 100, 16077–16082.
 Bouvier, M. (2001) Oligomerization of G-protein-coupled trans-
mitter receptors. Nat. Rev. Neurosci. 2, 274–286.
 Stanasila, L., Perez, J.B., Vogel, H. and Cotecchia, S. (2003)
Oligomerization of the alpha 1a- and alpha 1b-adrenergic
receptor subtypes. Potential implications in receptor internaliza-
tion. J. Biol. Chem. 278, 40239–40251.
 Miyawaki, A. and Tsien, R.Y. (2000) Monitoring protein
conformations and interactions by fluorescence resonance energy
transfer between mutants of green fluorescent protein. Methods
Enzymol. 327, 472–500.
 Vollmer, J.Y., Alix, P., Chollet, A., Takeda, K. and Galzi, J.L.
(1999) Subcellular compartmentalization of activation and desen-
sitization of responses mediated by NK2 neurokinin receptors. J.
Biol. Chem. 274, 37915–37922.
 Palanche, T., Ilien, B., Zoffmann, S., Reck, M.P., Bucher, B.,
Edelstein, S.J. and Galzi, J.L. (2001) The neurokinin A receptor
activates calcium and cAMP responses through distinct confor-
mational states. J. Biol. Chem. 276, 34853–34861.
 Griffin, B.A., Adams, S.R. and Tsien, R.Y. (1998) Specific
covalent labeling of recombinant protein molecules inside live
cells. Science 281, 269–272.
 Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H.
and Johnsson, K. (2003) A general method for the covalent
labeling of fusion proteins with small molecules in vivo. Nat.
Biotechnol. 21, 86–89.
 Marks, K.M., Braun, P.D. and Nolan, G.P. (2004) A general
approach for chemical labeling and rapid, spatially controlled
protein inactivation. Proc. Natl. Acad. Sci. USA 101, 9982–9987.
 Yin, J., Liu, F., Li, X. and Walsh, C.T. (2004) Labeling proteins
with small molecules by site-specific Posttranslational modifica-
tion. J. Am. Chem. Soc. 126, 7754–7755.
 Guignet, E.G., Hovius, R. and Vogel, H. (2004) Reversible site-
selective labeling of membrane proteins in live cells. Nat.
Biotechnol. 22, 440–444.
 George, N., Pick, H., Vogel, H., Johnsson, N. and Johnsson, K.
(2004) Specific labeling of cell surface proteins with chemically
diverse compounds. J. Am. Chem. Soc. 126, 8896–8897.
 Chen, I., Howarth, M., Lin, W.Y. and Ting, A.Y. (2005) Site-
specific labeling of cell surface proteins with biophysical probes
using biotin ligase. Nat. Meth. 2, 99–104.
 Gordon, G.W., Berry, G., Liang, X.H., Levine, B. and Herman,
B. (1998) Quantitative fluorescence resonance energy transfer
measurements using fluorescence microscopy. Biophys. J. 74,
 Erickson, M.G., Alseikhan, B.A., Peterson, B.Z. and Yue, D.T.
(2001) Preassociation of calmodulin with voltage-gated Ca(2+)
channels revealed by FRET in single living cells. Neuron 31, 973–
 Dery, O., Defea, K.A. and Bunnett, N.W. (2001) Protein kinase
C-mediated desensitization of the neurokinin 1 receptor. Am. J.
Physiol. Cell Physiol. 280, C1097–C1106.
 Grady, E.F., Garland, A.M., Gamp, P.D., Lovett, M., Payan,
D.G. and Bunnett, N.W. (1995) Delineation of the endocytic
pathway of substance P and its seven-transmembrane domain
NK1 receptor. Mol. Biol. Cell 6, 509–524.
 Ayoub, M.A., Couturier, C., Lucas-Meunier, E., Angers, S.,
Fossier, P., Bouvier, M. and Jockers, R. (2002) Monitoring of
ligand-independent dimerization and ligand-induced conforma-
tional changes of melatonin receptors in living cells by biolumi-
nescence resonance energy transfer. J. Biol. Chem. 277, 21522–
 Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans,
B.N.G., Palmer, A.E. and Tsien, R.Y. (2004) Improved mono-
meric red, orange and yellow fluorescent proteins derived from
Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–
 Dahan, D.S., Dibas, M.I., Petersson, E.J., Auyeung, V.C.,
Chanda, B., Bezanilla, F., Dougherty, D.A and Lester, H.A
(2004) A fluorophore attached to nicotinic acetylcholine receptor
beta M2 detects productive binding of agonist to the alpha delta
site. Proc. Natl. Acad. Sci. USA 101, 10195–10200.
 Ilegems, E., Pick, H., Deluz, C., Kellenberger, S. and Vogel, H.
(2005) Ligand binding transmits conformational changes across
the membrane-spanning region to the intracellular side of the 5-
HT(3) serotonin receptor. Chembiochem 6, 2180–2185.
B.H. Meyer et al. / FEBS Letters 580 (2006) 1654–1658