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Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

Ali Işbilir1, J. Moeller1, Andreas Bock1, Ulrike Zabel1, Paolo Annibale1, Martin J. Lohse1 •
University of Würzburg1
29 Dec 2017-bioRxiv (Cold Spring Harbor Laboratory (U.S.A.))-pp 240903
TL;DR: The data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86, and the combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels.
Abstract: G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated. Here we assess the oligomerization behavior of three prototypical Class A GPCRs, the β1-ARs, β2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the β2-AR shows a slight degree of oligomerization. From a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86. Taken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels.

Summary (3 min read)

Jump to: [Introduction] – [C-terminally EYFP-labeled controls] – [N-terminally SNAP-tagged controls] – [Discussion] – [Molecular Cloning] – [Cell Culture] – [Transient transfection] – [SNAP-labeling] – [Temporal Brightness] – [Acknowledgments] and [Contributions]

Introduction

  • G protein-coupled receptors constitute the largest class of membrane-bound receptors with >800 members expressed in humans.
  • The impact of dimerization on GPCR function and signaling, is not completely understood.
  • One of the most promising and accurate approach developed over the last few years appears to be single molecule tracking (SMT) [10].
  • The major limitation of the method is the assessment of oligomerization at plasma membrane concentrations exceeding a few receptor molecules/µm2 [11, 12].

C-terminally EYFP-labeled controls

  • In order to assess the oligomeric nature of β1-ARs, β2-ARs and M2Rs the authors first devised a set of reference proteins to characterize the brightness of constitutively monomeric and dimeric EYFP-tagged membrane proteins [17].
  • This would be in line with previous findings of a preferred rather than exclusive monomeric organization of CD86 [30].
  • The second approach to generate a dimeric control was to C-terminally tag GPCRs with two EYFP molecules separated by a single α-helical spacer to limit selfassociation of EYFP molecules .
  • In their experimental conditions the brightness measured by SpIDA is 9 times larger than what the authors measure by TB, which accounts for the higher laser power (8x) and lower scan rate (0.25x) used in spatial brightness measurements .

N-terminally SNAP-tagged controls

  • In order to minimize potential contributions from cytosolic aggregates containing EYFP, the authors decided to further validate their findings with an alternative labeling strategy.
  • Towards this goal, the authors worked with N-terminally SNAP-tagged constructs labeled with cell membrane impermeable SNAP dyes.
  • Given the fact that brightness values are a weighted average of the brightness of each species present in a pixel, immobile background fluorescence skews any ε value towards one.
  • The authors used β1-ARs as a reference to compare the constitutive brightness vs. expression curves of the β2-AR and the M2R.

Discussion

  • The authors report here an experimental protocol based on two different methods and two labeling strategies to address the longstanding controversy of GPCR oligomerization in intact cells when high expression levels of the receptors are observed.
  • The first experimental approach, temporal brightness analysis, is an image based version of the Photon Counting Histogram method [23], which was recently used to characterize the oligomerization state of many Class A GPCRs, including the β2-AR and M2R used in their study [15].
  • Interestingly, the authors obtained similar or even lower brightness values when using Atto488-labeled SNAP-tagged constructs compared to EYFP.
  • The authors measurements are in favor of a predominantly monomeric organization for these three receptors.
  • The only reports supporting a higher oligomerization state for β1-ARs come from whole-cell BRET measurements [33] and the results may have been influenced by receptor interactions not on the plasma membrane.

Molecular Cloning

  • Two EYFP cDNAs, the second one with a stop codon, were sequentially subcloned to the C-terminus of the β1-AR after PCR amplification using primers for EYFP1: forward 5’- AATAATGGATCCGTGAGCAAGGGCGAGGAG-3’ and reverse 5’- AATAATGAATTCCTTGTACAGCTCGTCCATGCC-3’ and for EYFP2: forward 5’-AATAATTCTAGAGTGAGCAAGGGCGAGGAGCTG-3’ and reverse.
  • The mYFP construct was a kind gift of Roger Y. Tsien (University of California,San Diego, USA).
  • Plasmids coding for N-terminally SNAP-tagged CD86, CD28, β1AR, β2AR and 2xSNAP-CD86 were previously described [12].

Cell Culture

  • All experiments were performed with transiently transfected HEK293-AD cells (Cell Biolabs, San Diego, USA).
  • Cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) (PAN Biotech, Aidenbach, Germany), supplemented with 4,5 g/L Glucose, 2 mM L-Glutamine, 10% FCS , 100 units/mL penicillin and 0,1 mg/mL streptomycin and maintained at 37 °C and 5% CO2.
  • Cells cultured in 15-cm dishes were split at a 1:36 ratio into 6-well plates containing poly-D-lysine (PDL)coated 24 mm glass coverslips.

Transient transfection

  • Cells were transfected using Effectene Transfection Reagent (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
  • Cells seeded on PDL-coated coverslips in 6-well plates were transfected 16 hours after seeding with 0.6 µg plasmid/well.

SNAP-labeling

  • Cells transfected with SNAP-tagged receptors were labeled using the SNAP-Surface 488 Dye (New England Biolabs, Frankfurt am Main, Germany) according to the manufacturer’s instructions.
  • Cells were imaged with a 40x / 1.25 numerical aperture oil immersion objective.
  • For photobleaching experiments, a series of 3 images was acquired with the same acquisition settings.
  • The ROIs for image analysis using SpIDA were drawn carefully in free area selection mode, implemented to the original SpIDA function, in order to avoid vesicles and inhomogenously distributed membrane areas more effectively [19].
  • The molecular concentration of EYFP-tagged receptors was calculated by dividing the mol (calculated by multiplying the number of molecules with the Avogadro number) by the focal volume.

Temporal Brightness

  • For TP imaging the same setup was used as for SpIDA measurements.
  • EYFP-tagged constructs were imaged using a 514 nm laser power of 2.5 % and photobleached with 10 % laser power over 10 frames.
  • Data were analyzed using a custom-written Igor Pro routine as described previously [28].
  • The brightness values were calculated based on the average of the brightness values from each pixel within the region of interest.

Acknowledgments

  • The authors are grateful to Antoine Godin (CERVO, Brain Research Centre - Laval University, Canada) for discussion concerning SpIDA data analysis.
  • The authors would like to acknowledge the contribution of the students of the Master in Biohysics programme of the University of Würzburg, Germany, as well as that Jana Wächter and Sofia Krohne for the work performed during their internships.

Contributions

  • Dorsch, S., et al., Analysis of receptor oligomerization by FRAP microscopy.
  • Solid circles highlight two large aggregates/vescicles, giving rise to large brightness values due to their high concentration of fluorophores and small local motion.

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Figures (4)

Content maybe subject to copyright    Report

Visualization of class A GPCR oligomerization by
image-based fluorescence fluctuation spectroscopy
Ali Isbilir
1,2 †
, Jan Möller
1,2 †
, Andreas Bock
1,2
, Ulrike Zabel
1
, Paolo Annibale
1,2 *
,
Martin J. Lohse
1,2 *
1. Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078
Würzburg, Germany
2. Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Robert-Rössle-Strasse
10, 13125 Berlin, Germany
Abstract
G protein-coupled receptors (GPCRs) represent the largest class of cell surface
receptors conveying extracellular information into intracellular signals. Many GPCRs
have been shown to be able to oligomerize and it is firmly established that Class C
GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However,
the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-
adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated.
Here we assess the oligomerization behavior of three prototypical Class A GPCRs,
the β
1
-ARs, β
2
-ARs, and muscarinic M
2
Rs in single, intact cells. We combine two
image correlation spectroscopy methods based on molecular brightness, i.e. the
analysis of fluorescence fluctuations over space and over time, and thereby provide an
assay able to robustly and precisely quantify the degree of oligomerization of GPCRs.
In addition, we provide a comparison between two labelling strategies, namely C-
terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in
order to rule out effects arising from potential fluorescent protein-driven
oligomerization. The degree of GPCR oligomerization is expressed with respect to a
set of previously reported as well as newly established monomeric or dimeric control
constructs. Our data reveal that all three prototypical GPRCs studied display, under
unstimulated conditions, a prevalently monomeric fingerprint. Only the β
2
-AR shows
a slight degree of oligomerization.
From a methodological point of view, our study suggests three key aspects. First, the
combination of two image correlation spectroscopy methods allows addressing cells
transiently expressing high concentrations of membrane receptors, far from the single
molecule regime, at a density where the kinetic equilibrium should favor dimers and
higher-order oligomers. Second, our methodological approach, allows to selectively
target cell membrane regions devoid of artificial oligomerization hot-spots (such as
vesicles). Third, our data suggest that the β
1
-AR appears to be a superior monomeric
control than the widely used membrane protein CD86.
Taken together, we suggest that our combined image correlation spectroscopy method
is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells
at high expression levels.
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
*
!Correspondence to paolo.annibale@mdc-berlin.de, martin.lohse@mdc-berlin.de
†
These authors contributed equally to this work
!
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted December 29, 2017. ; https://doi.org/10.1101/240903doi: bioRxiv preprint
Introduction
G protein-coupled receptors (GPCRs) constitute the largest class of membrane-bound
receptors with >800 members expressed in humans. GPCRs relay extracellular stimuli
into intracellular signals and modulate almost every physiological process. The
fidelity of GPCR signaling is fine-tuned on three different levels. First, GPCRs can
possess distinct binding sites for extracellular ligands and some receptors get
activated by multiple endogenous ligands. Second, intracellular adaptor proteins such
as G proteins, GRKs and β-arrestins may channel the extracellular signal into distinct
cellular outcomes. Third, GPCRs signaling can be modulated within the plasma
membrane by forming dimers and/or higher-order oligomers [1-5].
Whereas Class C GPCRs function as obligate dimers [6], the situation for the larger
family of Class A GPCRs is less clear. Although Class A GPCRs are fully functional
as monomers [7], there are multiple lines of evidence that Class A GPCRs can also
form dimers[4]. However, the impact of dimerization on GPCR function and
signaling, is not completely understood. As receptor monomers and dimers may have
distinct functions on cell signaling, it is important to rigorously assess the
dimerization behavior in intact cells with appropriate methods.
Since the first bioluminescence resonance energy transfer (BRET) investigation on
the oligomerization behavior of β
2
-adrenergic receptors (β
2
-ARs) [8], a large number
of studies have addressed the oligomerization state of GPCRs with fluorescence
approaches [4].
However, there are conflicting data on the existence and abundance of GPCR dimers
in intact cells. In the extreme case, by using the same method and the same receptor,
such as in the case of BRET read-out of β
2
-AR oligomerization, certain reports
support the presence of oligomers [8], while others suggest that they are absent [9].
One of the most promising and accurate approach developed over the last few years
appears to be single molecule tracking (SMT) [10]. SMT provides precise information
on single molecule dynamics while at the same time offering insight into the specific
oligomerization state of a protein by using the intensity of the localized spots. The
major limitation of the method is the assessment of oligomerization at plasma
membrane concentrations exceeding a few receptor molecules/µm
2
[11, 12]. At
concentrations above this value, individual molecular point spread functions (PSF)
begin to significantly overlap and accurate localization and tracking becomes
impossible. This limits the exploration of higher expression levels, which, although
not always physiological, provide an interesting range where to test the law of mass
action and compare experimental data to predictions arising from coarse-grained
molecular simulations, which tend to be performed at much higher “in-silico”
concentrations [13]. Moreover, the majority of reports on GPCR dimerization have
been performed in overexpressed systems.
Fluorescence fluctuation spectroscopy techniques offer an effective tool to investigate
with good precision receptor dimerization at higher expression levels than SMT.
Single point fluorescence correlation spectroscopy was used in the past to characterize
GPCR diffusion, formation of hetero-complexes [14] and receptor homo-
dimerization, by analyzing the histogram of the collected photons [15, 16]. Time-
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted December 29, 2017. ; https://doi.org/10.1101/240903doi: bioRxiv preprint
based image fluorescence fluctuation spectroscopy methods, which rely on the
statistical analysis of many pixels of an image, have allowed to characterize the
oligomerization state of the GPI-anchored membrane receptor uPar [17] and the ErbB
[18] observing the agonist-dependent formation of dimers and oligomers. More
recently, GPCRs such as the 5-HT
2C
and the muscarinic M
1
receptor [19, 20] were
investigated by spatial intensity distribution analysis (SpIDA): the authors observed
an antagonist-promoted oligomerization in the case of M
1
receptors and, in contrast,
an antagonist-dependent disruption of 5-HT
2C
receptor oligomers.
The fundamental advantage of spatial-temporal brightness analysis over SMT is that a
spatially resolved view of the plasma membrane allows discarding from the analysis
those regions where receptor aggregation phenomena different than oligomerization,
such as recruitment by ‘endocytic machinery’, have occurred [21, 22].
In this report, we combine two image-based fluctuation spectroscopy methods,
namely temporal brightness (TB) [17, 23, 24] and SpIDA [25] to characterize the
oligomerization state of three prototypical GPCRs, the homologous β
1
-AR and β
2
-
ARs, and the muscarinic M
2
receptor (M
2
R) at expression levels of the order of tens
to hundreds of receptors/µm
2
, a concentration level where oligomerization driven
exclusively by physical kinetics should have already occurred. By comparing the
receptors with a set of monomeric and dimeric reference proteins, and by employing
two labeling strategies based on C-terminal fluorescent protein fusions and N-
terminal SNAP-tag labels, we find here clear evidence of a predominantly monomeric
state for all these three receptors in intact cells. The absence of a dominant dimeric
fraction at these concentrations suggests that kinetic oligomerization is inefficient,
characterized by slow on-rates and/or fast off-rates, resulting in a short lifetime of any
oligomeric complex.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted December 29, 2017. ; https://doi.org/10.1101/240903doi: bioRxiv preprint
Results
C-terminally EYFP-labeled controls
In order to assess the oligomeric nature of β
1
-ARs, β
2
-ARs and M
2
Rs we first devised
a set of reference proteins to characterize the brightness of constitutively monomeric
and dimeric EYFP-tagged membrane proteins [17]. Such controls carry the same
fluorophore as the target constructs, and should share a similar diffusion coefficient
and mode of motion (Supplementary Figure 1).
Based on previous reports we identified the single transmembrane peptide CD86 (also
known as B7-2) as a potential monomeric control. This construct is routinely used as
a monomeric reference in photo activated localization microscopy (PALM)
experiments [26] and was previously used by our group to calibrate SMT experiments
[12]. We chose to tag all our constructs C-terminally with EYFP (ex. 513 nm, em. 527
nm, EC 83400, QY 0.61) as we found no evidence for intrinsic dimerization of EYFP
in comparison to monomeric YFP (mYFP) [27] (Supplementary Figure 2).
Another important characteristic that the control construct should exhibit is a
homogeneous expression on the plasma membrane. If this is not the case, and a large
number of aggregates such as forming vesicles and mature endosomes are present,
then both spatial and temporal brightness measurements may be affected
(Supplementary Figure 3). In our hands, using HEK293-AD cells, CD86 displayed a
robust cell membrane expression but was also, albeit to a lesser extent, found in
cytosolic and near-membrane aggregates dotting the basal membrane (Figure 1). The
presence of such aggregates may affect the correct brightness readout (e.g. an
endosome will appear –in brightness- as a large oligomer (Figure 1a), and moving
vesicles can generate extra variance over space and time). This is one of the reasons
why we decided to employ two complementary approaches to measure molecular
brightness: measurement of brightness over time (TB) and over space (SpIDA).
Large, immobile or slow (nm/s) and bright features can be easily treated in TB
analysis by a boxcar filter detrend [28], while they have to be avoided from the region
of interest (ROI) used to extract SpIDA values (Figure 1b). On the other hand, since
SpIDA brightness values can be extracted from one image, this latter approach is less
sensitive to photobleaching, drift or defocus of the sample. Considering the different
acquisition parameters, in particular, the pixel dwell time, spatial and temporal
brightness values are expected to be distinct. The average apparent brightness of
CD86-EYFP measured on a stack of 50-100 images (from the variance of the
intensity of each pixel over time, i.e. temporal brightness) and on a pool of 21 cells is
ε
t
CD86
=1.118 ± 0.0075 (Figure 2a). The SpIDA analysis of CD86 yielded a brightness
value of ε
s
CD86
=2.14 ± 0.03 (Figure 2b).
To obtain a reference brightness dimer, we pursued two strategies. In the first
approach, we relied on CD28, a transmembrane protein displaying a disulfide-linked
dimeric structure. The temporal and spatial brightness obtained for CD28 are
ε
t
CD28
=1.18 ± 0.02 and ε
s
CD28
=3.15 ± 0.05, respectively (Figure 2 a, b). Both temporal
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted December 29, 2017. ; https://doi.org/10.1101/240903doi: bioRxiv preprint
and spatial brightness values are smaller than twice the values for ε
t/s
CD86
, viz. 1.24
and 3.28.
We then checked a reference GPCR, which in previous investigations from our own
group, [12, 29] displayed a monomeric fingerprint, the β
1
-AR. Interestingly, β
1
-AR-
EYFP displayed an apparent brightness lower than that of CD86-EYFP, ε
t
β1=1.095 ±
0.008 and ε
s
β1=2.02 ± 0.03 (Figure 2a, b). These values are compatible with half the
brightness for CD28-EYFP, suggesting that CD86 may dimerize to a certain extent in
intact cells. This would be in line with previous findings of a preferred rather than
exclusive monomeric organization of CD86 [30].
The second approach to generate a dimeric control was to C-terminally tag GPCRs
with two EYFP molecules separated by a single α-helical spacer to limit self-
association of EYFP molecules (see Materials and Methods and Supplementary
Figure 4e). The brightness measured for this β
1
-AR-2xEYFP construct is ε
t
β1-2x=1.21
± 0.01 and ε
s
β1-2x=3.01 ± 0.04, in full agreement with the values measured for CD28
and twice the values measured for β
1
-AR-EYFP.
To further test the quality of our measurements, we subjected cells expressing the
dimeric constructs CD28 and β
1
-AR-2xEYFP to whole cell photobleaching. Due to
the stochastic nature of photobleaching, many of the fluorophores of a dimer will be
photobleached (although the actual physical dimer is not affected), resulting in an
apparent increase of the monomer/dimer ratio. After sustained photobleaching, we
observe that the apparent brightness of CD28 and β
1
-AR-2xEYFP revert to the
approximately monomeric value: ε
t
β1-2x bleach =1.11 ± 0.01 and ε
t
CD28 bleach =1.105 ±
0.006, while for SpIDA we obtain ε
s
β1-2x bleach 2.11 ± 0.05 and ε
s
CD28 bleach =2.18 ± 0.06.
(Figure 2 a, b). In contrast, when bleached, β
1
-AR-EYFP displays a negligible
reduction in brightness (Supplementary Figure 6a).
The agreement between temporal and spatial brightness measurements is overall
excellent. In our experimental conditions the brightness measured by SpIDA is 9
times larger than what we measure by TB, which accounts for the higher laser power
(8x) and lower scan rate (0.25x) used in spatial brightness measurements (Figure 2c).
Finally, all our controls displayed correct membrane localization, as illustrated in the
panels of Figure 2d, as well as a diffusion coefficient in agreement with previous
observations, in the range of 0.1 µm
2
/s (Supplementary Table 1).
N-terminally SNAP-tagged controls
In order to minimize potential contributions from cytosolic aggregates containing
EYFP, we decided to further validate our findings with an alternative labeling
strategy. Towards this goal, we worked with N-terminally SNAP-tagged constructs
labeled with cell membrane impermeable SNAP dyes. Using this approach, the
extracellular SNAP-tag is labeled by incubating the cells with an organic dye, in our
case Atto488 (See Materials and Methods). We first tested SNAP-CD86 and a double
SNAP-tagged construct, 2xSNAP-CD86 as monomer and dimer controls,
respectively. We recorded
SNAP
ε
t
CD86=1.11 ± 0.01 and
SNAP
ε
s
CD86=1.66 ± 0.01 (Figure
3a, b). The dimer reference 2xSNAP-CD86 yielded
SNAP
ε
t
s2xCD86=1.18 ± 0.01 and
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted December 29, 2017. ; https://doi.org/10.1101/240903doi: bioRxiv preprint
Citations
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  • ...Image analysis was performed using the one-population mode of the SpIDA function using a custom-written MATLAB routine, as described previously (34)....

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  • ...We used a C-terminally EYFP-tagged β1AR as a monomeric control, as we had confirmed its monomeric behavior even at overexpression levels (9, 34)....

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Life Sciences Institute1, Max Delbrück Center for Molecular Medicine2
10 May 2019-ACS Chemical Biology
TL;DR: It is demonstrated here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR.
Abstract: High-end microscopy studies of G protein-coupled receptors (GPCRs) require installing onto the receptors bright and photostable dyes. Labeling must occur in quantitative yields, to allow stoichiometric data analysis, and in a minimally invasive fashion, to avoid perturbing GPCR function. We demonstrate here that the genetic incorporation of trans-cyclooct-2-ene lysine (TCO*) allows achieving quantitative single-residue labeling of the extracellular loops of the β2-adrenergic and the muscarinic M2 class A GPCRs, as well as of the corticotropin releasing factor class B GPCR. Labeling occurs within a few minutes by reaction with dye-tetrazine conjugates on the surface of live cells and preserves the functionality of the receptors. To precisely quantify the labeling yields, we devise a method based on fluorescence fluctuation microscopy that extracts the number of labeling sites at the single-cell level. Further, we show that single-residue labeling is better suited for studies of GPCR diffusion than fluorescent-protein tags, since the latter can affect the mobility of the receptor. Finally, by performing dual-color competitive labeling on a single TCO* site, we devise a method to estimate the oligomerization state of a GPCR without the need for a biological monomeric reference, which facilitates the application of fluorescence methods to oligomerization studies. As TCO* and the dye-tetrazines used in this study are commercially available and the described microscopy techniques can be performed on a commercial microscope, we expect our approach to be widely applicable to fluorescence microscopy studies of membrane proteins in general.

29 citations

Journal Article•DOI•
Chemokine Receptor Crystal Structures: What Can Be Learned from Them?

[...]

Marta Arimont1, Carsten Hoffmann, Chris de Graaf, Rob Leurs1•
VU University Amsterdam1
01 Dec 2019-Molecular Pharmacology
TL;DR: This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokin receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications.
Abstract: Chemokine receptors belong to the class A of G protein-coupled receptors (GPCRs) and are implicated in a wide variety of physiologic functions, mostly related to the homeostasis of the immune system. Chemokine receptors are also involved in multiple pathologic processes, including immune and autoimmune diseases, as well as cancer. Hence, several members of this GPCR subfamily are considered to be very relevant therapeutic targets. Since drug discovery efforts can be significantly reinforced by the availability of crystal structures, substantial efforts in the area of chemokine receptor structural biology could dramatically increase the outcome of drug discovery campaigns. This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications. SIGNIFICANCE STATEMENT: This short review summarizes the available data on chemokine receptor crystal structures, discusses the numerous applications from chemokine receptor structures that can enhance the daily work of molecular pharmacologists, and describes the challenges and pitfalls to consider when relying on crystal structures for further research applications.

19 citations

Cites background from "Visualization of class A GPCR oligo..."

  • ...…resonance energy transfer approaches (Percherancier et al., 2005; Goddard and Watts, 2012; Fumagalli et al., 2019; Heuninck et al., 2019), fluorescence fluctuation spectroscopy (Isbilir et al., 2017;Briddon et al., 2018), and spatial intensity distribution analysis (Pediani et al., 2018)....

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  • ..., 2019), fluorescence fluctuation spectroscopy (Isbilir et al., 2017;Briddon et al., 2018), and spatial intensity distribution analysis (Pediani...

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References
More filters
Journal Article•DOI•
Seven-transmembrane receptors.

[...]

Kristen L. Pierce1, Richard T. Premont1, Robert J. Lefkowitz1•
Howard Hughes Medical Institute1
01 Sep 2002-Nature Reviews Molecular Cell Biology
TL;DR: This paper showed that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain seven-transmembrane receptors' remarkably diverse biological actions.
Abstract: Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

2,300 citations

Journal Article•DOI•
Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

[...]

David A. Zacharias1, Jonathan D. Violin, Alexandra C. Newton, Roger Y. Tsien1•
University of California, San Diego1
03 May 2002-Science
TL;DR: Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts, and the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.
Abstract: Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.

2,217 citations

"Visualization of class A GPCR oligo..." refers background in this paper

  • ...61) as we found no evidence for intrinsic dimerization of EYFP in comparison to monomeric YFP (mYFP) [27] (Supplementary Figure 2)....

    [...]

Journal Article•DOI•
The photon counting histogram in fluorescence fluctuation spectroscopy.

[...]

Yan Chen1, Joachim D. Müller1, Peter T. C. So2, Enrico Gratton1•
University of Illinois at Urbana–Champaign1, Massachusetts Institute of Technology2
01 Jul 1999-Biophysical Journal
TL;DR: The photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume.

708 citations

"Visualization of class A GPCR oligo..." refers background or methods in this paper

  • ...The first experimental approach, temporal brightness analysis, is an image based version of the Photon Counting Histogram method [23], which was recently used to characterize the oligomerization state of many Class A GPCRs, including the...

    [...]

  • ...While the photon counting histogram is a powerful method which allows discriminating the number and brightness of up to two existing species mixed within a homogeneous sample [23], it is insensitive to such heterogeneities: if not properly corrected, the histogram of the photon counts may be affected by fluctuations that do not originate from the receptor diffusion within the membrane (Figure 1a,b)....

    [...]

  • ...In this report, we combine two image-based fluctuation spectroscopy methods, namely temporal brightness (TB) [17, 23, 24] and SpIDA [25] to characterize the...

    [...]

Journal Article•DOI•
Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).

[...]

Stephane Angers1, Ali Salahpour, Eric Joly, Sandrine Hilairet, Dan Chelsky, Michael Dennis, Michel Bouvier •
Université de Montréal1
28 Mar 2000-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: The data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein–protein interactions.
Abstract: Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein–protein interaction assay in whole cells, we unambiguously demonstrate that the human β2-adrenergic receptor (β2AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between β2AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between β2AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the β2AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the β2AR and the regulatory protein β-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein–protein interactions.

698 citations

Journal Article•DOI•
Oligomerization of G-protein-coupled transmitter receptors

[...]

Michel Bouvier1•
Université de Montréal1
01 Apr 2001-Nature Reviews Neuroscience
TL;DR: The evidence supporting the existence of G-protein-coupled-receptor dimerization is reviewed and its functional importance is discussed to have important implications for the development and screening of new drugs.
Abstract: Examples of G-protein-coupled receptors that can be biochemically detected in homo- or heteromeric complexes are emerging at an accelerated rate. Biophysical approaches have confirmed the existence of several such complexes in living cells and there is strong evidence to support the idea that dimerization is important in different aspects of receptor biogenesis and function. While the existence of G-protein-coupled-receptor homodimers raises fundamental questions about the molecular mechanisms involved in transmitter recognition and signal transduction, the formation of heterodimers raises fascinating combinatorial possibilities that could underlie an unexpected level of pharmacological diversity, and contribute to cross-talk regulation between transmission systems. Because G-protein-coupled receptors are major pharmacological targets, the existence of dimers could have important implications for the development and screening of new drugs. Here, we review the evidence supporting the existence of G-protein-coupled-receptor dimerization and discuss its functional importance.

672 citations

"Visualization of class A GPCR oligo..." refers background in this paper

  • ...Third, GPCRs signaling can be modulated within the plasma membrane by forming dimers and/or higher-order oligomers [1-5]....

    [...]

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Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

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Wanling Song, Anna L. Duncan, Mark S.P. Sansom
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03 Nov 2015-Biophysical Journal
Sarah L. Latty, James H. Felce, Laura Weimann, Steven F. Lee, Simon J. Davis, David Klenerman 
Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure.

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12 Sep 2017-Cell Reports
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Frequently Asked Questions (20)
Q1. What contributions have the authors mentioned in the paper "Visualization of class a gpcr oligomerization by image-based fluorescence fluctuation spectroscopy" ?

The authors combine two image correlation spectroscopy methods based on molecular brightness, i. e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, the authors provide a comparison between two labelling strategies, namely Cterminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Their data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. From a methodological point of view, their study suggests three key aspects. Second, their methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots ( such as vesicles ). Taken together, the authors suggest that their combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels. These authors contributed equally to this work. CC-BY-NC-ND 4. 0 International license available under a was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Third, their data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86. 

The fundamental advantage of spatial-temporal brightness analysis over SMT is that a spatially resolved view of the plasma membrane allows discarding from the analysis those regions where receptor aggregation phenomena different than oligomerization, such as recruitment by ‘endocytic machinery’, have occurred [21, 22]. 

G protein-coupled receptors (GPCRs) constitute the largest class of membrane-bound receptors with >800 members expressed in humans. 

Time-based image fluorescence fluctuation spectroscopy methods, which rely on the statistical analysis of many pixels of an image, have allowed to characterize the oligomerization state of the GPI-anchored membrane receptor uPar [17] and the ErbB [18] observing the agonist-dependent formation of dimers and oligomers. 

The reason that the expected gain in brightness was not observed is because the benzylguanine moiety conjugated to the Atto488 acts as a potent quencher, reducing the apparent brightness of the dye of a factor of seven [31]. 

The imaging mode was XYT and 50 frames were taken with a scanner speed of 400 Hz using the following parameters: pinhole-size: 67.93/ zoom-factor: 30.3x/resolution 256x256 pixels. 

Since the first bioluminescence resonance energy transfer (BRET) investigation on the oligomerization behavior of β2-adrenergic receptors (β2-ARs) [8], a large number of studies have addressed the oligomerization state of GPCRs with fluorescence approaches [4]. 

As far as GPCRs are concerned, another important phenomenon that may affect the apparent oligomerization is receptor internalization. 

The first experimental approach, temporal brightness analysis, is an image based version of the Photon Counting Histogram method [23], which was recently used to characterize the oligomerization state of many Class A GPCRs, including the β2-AR and M2R used in their study [15]. 

One of the most promising and accurate approach developed over the last few years appears to be single molecule tracking (SMT) [10]. 

This is an interesting, often overlooked feature of SNAP-dyes, since, depending on the molecular structure of the dye, strong quenching may occur after benzylguanine conjugation. 

The second approach to generate a dimeric control was to C-terminally tag GPCRs with two EYFP molecules separated by a single α-helical spacer to limit selfassociation of EYFP molecules (see Materials and Methods and Supplementary Figure 4e). 

The brightness measured for this β1-AR-2xEYFP construct is εtβ1-2x=1.21 ± 0.01 and εsβ1-2x=3.01 ± 0.04, in full agreement with the values measured for CD28 and twice the values measured for β1-AR-EYFP. 

The molecular concentration of EYFP-tagged receptors was calculated by dividing the mol (calculated by multiplying the number of molecules with the Avogadro number) by the focal volume. 

The presence of such aggregates may affect the correct brightness readout (e.g. an endosome will appear –in brightness- as a large oligomer (Figure 1a), and moving vesicles can generate extra variance over space and time). 

By progressively enlarging the area of interest, the measured brightness increases as endosomes and other bright features of the plasma membrane are included in the intensity histogram (Supplementary Figure 7). 

As receptor monomers and dimers may have distinct functions on cell signaling, it is important to rigorously assess the dimerization behavior in intact cells with appropriate methods. 

In conclusion, the authors have analyzed the basal oligomerization state of three prototypical GPCRs, observing a largely monomeric state for two of them, M2Rs and β1-ARs, and a mixture of monomers and oligomers for β2-ARs. 

The SNAP-tag labels displayed an overall excellent photostability (negligible photobleaching over the 50-100 frames used for imaging, under their experimental conditions), making them ideal choices for temporal brightness measurements where photobleaching may be a problem. 

7. Whorton, M.R., et al., A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein.