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LETTER doi:10.1038/nature12000
Conformational biosensors reveal GPCR signalling
from endosomes
Roshanak Irannejad
1
, Jin C. Tomshine
1
, Jon R. Tomshine
1
, Michael Chevalier
2
, Jacob P. Mahoney
3
, Jan Steyaert
4,5
,
Søren G. F. Rasmussen
6
, Roger K. Sunahara
3
, Hana El-Samad
2
, Bo Huang
2,7
& Mark von Zastrow
1,8
A long-held tenet of molecular pharmacology is that canonical
signal transduction mediated by G-protein-coupled receptor (GPCR)
coupling to heterotrimeric G proteins is confined to the plasma
membrane. Evidence supporting this traditional view is based on
analytical methods that provide limited or no subcellular resolu-
tion
1
. It has been subsequently proposed that signalling by inter-
nalized GPCRs is restricted to G-protein-independent mechanisms
such as scaffolding by arrestins
2,3
, or GPCR activation elicits a dis-
crete form of persistent G protein signalling
4–9
, or that internalized
GPCRs can indeed contribute to the acute G-protein-mediated
response
10
. Evidence supporting these various latter hypotheses is
indirect or subject to alternative interpretation, and it remains
unknown if endosome-localized GPCRs are even present in an
active form. Here we describe the application of conformation-
specific single-domain antibodies (nanobodies) to directly probe
activation of the b
2
-adrenoceptor, a prototypical GPCR
11
, and its
cognate G protein, G
s
(ref. 12), in living mammalian cells. We show
that the adrenergic agonist isoprenaline promotes receptor and G
protein activation in the plasma membrane as expected,but also in
the early endosome membrane, and that internalized receptors con-
tribute to the overall cellular cyclic AMP response within several
minutes after agonist application. These findings provide direct
support for the hypothesis that canonical GPCR signalling occurs
from endosomes as well as the plasma membrane, and suggest a
versatile strategy for probing dynamic conformational change
in vivo
.
Ligand binding to the extracellular surface of the b
2
-adrenoceptor
(b
2
-AR) stabilizes an activating conformational change in the receptor
that promotes guanine nucleotide dissociation from the cytoplasmic
GTP-binding protein Ga
s
; this represents the critical biochemical event
initiating classical GPCR signal transduction (Fig. 1a)
13
. Activated b
2
-
ARs are substrates for phosphorylation and binding of b-arrestins,
events which inhibit interaction with G proteins and promote endocy-
tosis of receptors via clathrin-coated pits (CCPs)
14,15
.Acuteb
2
-AR Ga
s
signalling is thus traditionally thought to be restricted to the plasma
membrane
14,16,17
. However, to our knowledge, this assumption has not
been directly tested. To do so, we generated a biosensor of activatedb
2
-
AR based on a conformation-specific single-domain camelid antibody
(Nb80) used in recent structural studies
18,19
. We reasoned that this
nanobody, which selectively binds the agonist-occupied b
2
-AR and
is able to stabilize an activated receptor conformation when present
in vitro at high concentration, might act as a sensor of receptor activa-
tion when expressed at relatively low concentration in intact cells
(Fig. 1b). This proved to be the case; in cells maintained in the absence
of agonist, Nb80 fused to enhanced green fluorescent protein (Nb80–
GFP) localized to the cytoplasm and not with b
2
-ARs present in the
plasma membrane (Fig. 1c, 0 min, top; Pearson’s coefficient50.135).
Line scan analysis verified the cytoplasmic distribution of Nb80–
GFP before b
2
-AR activation (Fig. 1d, top) as expected because the
cytoplasmic concentration of Nb80–GFP achieved in our experiments
(approximately 20 nM) was considerably lower than the equilibrium
dissociation constant estimated in vitro for Nb80 binding to purified
b
2
-ARs in the absence of agonist (0.76 60.14 mM; Supplementary Fig.
1a–d). After application of the adrenergic agonist isoprenaline (10 mM),
Nb80–GFP was rapidly recruited to the plasma membrane and co-
localized there with b
2
-ARs (Fig. 1c, middle; Pearson’s coefficient 5
0.625). Line scan analysis verified robust Nb80–GFP recruitment to
the plasma membrane and concomitant depletion from the cytoplasm
(Fig. 1d, middle), consistent with the much higher affinity of Nb80 for
isoprenaline-activated b
2
-ARs (2.9 60.5 nM; Supplementary Fig. 1d).
Agonist-induced membrane recruitment of Nb80–GFP was specific
because the D1 dopamine receptor (DRD1), which is also G
s
-coupled
but does not bind Nb80 in vitro (data not shown), failed to recruit
Nb80–GFP to the plasma membrane in response to dopamine (10 mM)
application (Supplementary Fig. 2). Furthermore, b
2
-AR–cyan fluor-
escent protein (CFP) and Nb80–yellow fluorescent protein (YFP) gene-
rated a pronounced fluorescence (Fo¨rster) resonance energy transfer
(FRET) signal after isoprenaline application whereas DRD1–CFP did
not (Supplementary Fig. 3a, b).
b
2
-AR internalization began 1 to 2min after Nb80–GFP recruit-
ment to the plasma membrane, indicated by the emergence of surface-
labelled b
2
-AR in peripheral cytoplasmic vesicles. Nb80–GFP did not
co-localize with b
2
-AR-containing endocytic vesicles upon first
appearance (Fig. 1c, middle, arrow in merged image points to an
example) but was recruited at later time points (Fig. 1c, bottom,
Pearson’s coefficient 50.702; examples are indicated by arrowheads).
Endosome recruitment of Nb80–GFP was evident by line scan analysis
(Fig. 1d, bottom; line scans are from the representative individual
examples with further quantification in legend) and localized to
EEA1-marked early endosomes (Pearson’s coefficient 50.846;
Supplementary Fig. 4) through which b
2
-ARs iteratively cycle in the
presence of agonist
20
.b
2
-AR-containing endosomes were initially
devoid of Nb80–GFP and later acquired Nb80–GFP during their
movement (Supplementary Videos 1 and 2). Interaction at endosomes
was verifiedby b
2
-AR–CFP andNb80–YFP normalizedFRET (nFRET)
(Supplementary Fig. 3c). These results suggest that b
2
-AR activation
initiates a precisely choreographed series of events: Nb80–GFP is first
recruited from the cytoplasm to the plasma membrane, then b
2
-ARs
internalize devoid of Nb80–GFP, followed by a second phase of Nb80–
GFP recruitment to the internalized b
2
-ARs.
Nb80–GFP recruitment to endosomes required b
2
-ARs because a
phosphorylation-deficient mutant version of the b
2
-AR (b
2
-AR-3S,
with three serine mutations) that couples to Ga
s
but is impaired in
agonist-induced endocytosis
21
recruited Nb80–GFP to the plasma mem-
brane but produced much less recruitment to endosomes (Fig. 1e, top).
Nb80–GFP co-localized with b
2
-AR-3S after agonist-induced activation
(Pearson’s coefficient 50.674) but this was largely restricted to the
1
Department of Psychiatry, University of California, San Francisco, California 94158, USA.
2
Department of Biochemistry & Biophysics, University of California, San Francisco, California 94158, USA.
3
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
4
Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, B-1050 Brussels,
Belgium.
5
Structural Biology Research Centre, VIB, B-1050 Brussels, Belgium.
6
Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, 2200 Copenhagen N,
Denmark.
7
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, USA.
8
Department of Cellular & Molecular Pharmacology, University of California, San
Francisco, California 94158, USA.
534 | NATURE | VOL 495 | 28 MARCH 2013
Macmillan Publishers Limited. All rights reserved
©2013
20 min Iso
20 min Iso
+
5 min CGP
ab
cd
ef
Normalized uorescence intensity
20 min Iso
+
20 min CGP
β2-AR-3S
β2-AR
Merged
Merged
Iso
0 min
3.5 min
20 min
Nb80–GFP
Nb80–GFP
Distance
(p
ixels
)
)
g
1
Agonist
binding
2
Receptor
activation
3
G protein
activation
4
cAMP
production
RR* R*
αα αα
ββ β
γγ γ
AC
GTP ATP
cAMP
GDPGTP
GDP
GDP
R*R
Blosensor of
receptor activation
Nb80
GFP
Nb80–GFP
0 min
3.5 min
20 min
120
110
100
90
80
70
60
50
120
110
100
90
80
70
60
50
120
110
100
90
80
70
60
50
Normalized uorescence intensity
0
40
80
120
160
200
20
60
100
140
180
220
Distance (pixels)
120
110
100
90
80
70
60
50
120
110
100
90
80
70
60
50
120
110
100
90
80
70
60
50
0
20
40
60
80
100
120
140
400
300
200
100
0 250 500 750 1,000 1,250 1,500
Time (s)
Cytoplasmic Nb80 (% of initial)
+ CGP
Figure 1
|
Nb80–GFP detects activatedb
2
-ARs in
the plasma membrane and endosomes. a,The
main events in b
2
-AR cAMP signalling include
agonist binding (step 1), conformational activation
of the receptor (step 2) that is coupled to
conformational activation of G
s
(step 3) that
produces guanine nucleotide exchange on G
s
and
subsequent activation of adenylyl cyclase (AC)
(step 4). b, Scheme for detecting conformational
activation of b
2
-AR with Nb80–GFP.
c, Representative Nb80–GFP (green) and b
2
-AR
(red) localization at the indicated time (left) after
10 mM isoprenaline addition (.30 Nb80–GFP
positive endosomes per cell observed at 20 min;
n529 cells, 10 experiments). d, Representative
individual Nb80–GFP line scans (shown at the
same magnification as panel c). e, Representative
Nb80–GFP (green) and b
2
-AR-3S (red)
localization after 20 min of isoprenaline treatment
(top) followed by reversal with 50 mM CGP-12177
for the indicated times (6.4 Nb80–GFP positive
endosomes per cell; n540 cells, 3 experiments).
f, Representative individual Nb80–GFP line scans.
g, Recovery of cytoplasmic Nb80–GFP
fluorescence (black) or bleaching control of the
plasma membrane b
2
-AR-3S (grey)
(mean 6s.e.m., n55 experiments). Scale bars,
10 mm.
Surface Nb80–GFP
(% of maximum)
390
345
β2-AR β2-AR
e
β2-AR
Merged
Merged
Nb80–mRuby
Nb80–GFP Nb80–GFP
372 375 378 381 384 387
273
c
b
a
Time (s) 321 324 327 330 d318
Iso 0 min 2 min 2.5 min 10 min
Nb80–GFP
β2-AR
Merged
100
75
50
25
0
0 2 2.5 10
β2-AR
Nb80–GFP
Time (s)
Time
Time
Time (min)
Figure 2
|
Nb80–GFP accumulates on b
2
-AR-
containing endosomes after their formation.
a,b
2
-AR (red) and Nb80–GFP (green) at the
indicated times after isoprenaline addition. Scale
bar, 10 mm. b, Average Nb80–GFP fluorescence
measured in the TIRF illumination field at the
indicated times (mean 6s.e.m., n57 cells).
c, TIRF image series showing b
2
-AR (red) and
Nb80–GFP (green) in sequential frames.
d, Kymograph of an individual b
2
-AR-containing
endosome (red, Alexa555) showing Nb80–GFP
(green) acquisition over 4 min. e, Kymograph of
b
2
-AR (green, Alexa488) and Nb80–mRuby (red)
over 6 min.
LETTER RESEARCH
28 MARCH 2013 | VOL 495 | NATURE | 535
Macmillan Publishers Limited. All rights reserved
©2013
plasma membrane (line scan analysis in Fig. 1f is representative; further
quantification in the figurelegend). Nb80–GFP membrane recruitment
was also reversible because the biosensor returned to a cytoplasmic
distribution after addition of the competitive antagonist CGP-12177
(Fig. 1e, f, middle and bottom rows; Pearson’s coefficient50.106),
verified by recovery of Nb80–GFP fluorescence intensity in the cyto-
plasm (Fig. 1g, see also Supplementary Video 3). Thus, Nb80–GFP
detected activated b
2
-ARs both in the plasma membrane and endo-
somes after acute agonist application.
Discrete Nb80–GFP recruitment phases were clearly resolved by total
internal reflection fluorescence (TIRF) microscopy that selectively detects
events occurring in the plasma membrane and extending approximately
100 nm into the peripheral cytoplasm
22
.First,within2minafteragonist
application, Nb80–GFP was progressively recruited to the plasma mem-
brane in a diffuse distribution (Fig. 2a, compare first and second columns
fromleft;quantificationinFig.2b).Nb80–GFPdidnotco-localizewith
b
2
-ARs when they clustered in relatively static punctae characteristic of
receptor-containing CCPs
23
(Fig. 2a, third column). Second, over a period
of several additional minutes, Nb80–GFP was recruited to a discrete
population of highly mobile punctae (Fig. 2a, fourth column) represent-
ing peripheral b
2
-AR-containing endocytic vesicles
23
.
Sequential TIRF imaging emphasized the distinction between rela-
tively static b
2
-AR puncta not co-localizing with Nb80–GFP (Fig. 2c,
arrowhead indicates an example) and mobile endosomes that did
(Fig. 2c, arrow indicates a representative example; many are visible
in Supplementary Video 4). Later recruitment of Nb80–GFP occurred
rather suddenly, typically within approximately 5 s (Supplementary
Video 5), as also evident in kymographs of individual endosome trajec-
tories (Fig. 2d). Ruling out potential artefacts of wavelength-dependent
differences in TIRF microscopy illumination depth, later recruitment of
the biosensor to endosomes was similarly observed when the excitation
wavelengths used to detect receptor and biosensor were reversed (Fig. 2e).
Nb80–GFP did not detectably concentrate in CCPs labelled with
either of two independent markers, the adaptor protein b-arrestin-2
(Fig. 3a and Supplementary Video 6) or the coat protein component
clathrin light chain (Fig. 3b and Supplementary Video 7). Represen-
tative examples are shown and this was verified across multiple cells
and experiments (Pearson’s coefficient 50.365 and 0.319, respect-
ively, with numbers of replicates specified in the legend). In contrast,
and as expected based on previous work, extensive co-localization
of b
2
-AR with b-arrestin was observed under the same conditions
(Pearson’s coefficient 50.677). Separation of Nb80–GFP localization
from that of either b-arrestin or clathrin was clear in line scans(bottom
panels of Fig. 3a, b were derived from the images shown and are
representative, further quantification is in the legend).
A simple interpretation is that Nb80–GFP associates with activated
b
2
-ARs in the plasma membrane but then dissociates before receptors
cluster in CCPs and internalize. This was surprising because b
2
-AR
clusteringin CCPs occurs much more rapidly
23
than the overallreversal
rate of Nb80–GFP recruitment observed after agonist washout in
intact cells (Fig. 1g), or the kinetics of Nb80 dissociation from purified
b
2
-ARs in vitro (Supplementary Fig. 1b, e). One possibility is that
Nb80–GFP dissociation is accelerated during the clustering process,
by mechanisms such as receptor phosphorylation or steric exclusion
mediated by b-arrestins or other CCP-associated components. An
alternative possibility is that the fraction of b
2
-ARs that have bound
Nb80–GFP in the plasma membrane by the time of the clustering
reaction are unable to enter CCPs, and only those b
2
-ARs not initially
bound to Nb80–GFP in the plasma membrane are able to cluster in
CCPs and subsequently internalize. In either case, the data clearlyindi-
cate that Nb80–GFP associateswith b
2
-ARs after endocytosis, and after
uncoating of the endocytic vesicle has occurred (Fig. 3c). Accordingly,
Nb80–GFP recruitment to b
2
-AR-containing endosomes cannot repre-
sent an artefact of persistent nanobody binding from the plasma
membrane; instead, this observation reveals that b
2
-ARs present in
early endosomes are in an activated conformation.
Normalized uorescence
intensity
Distance (pixels)
Iso β-arrestin Nb80–GFP MergedIso
0 min
5 min
10 min
β-arrestin
Nb80
50
40
30
20
10
00 10 20 30
Normalized uorescence
intensity
Clathrin
Nb80
Nb80–GFP MergedIso Clathrin
0 min
5 min
10 min
0
20
40
60
80
0 10 20 30 40
Distance (pixels)
TIRF illumination eld (~100 nm)
RR*
Nb80
GFP R*
Arrestin
Clathrin/AP-2 coat
Recruitment phase 1
Recruitment
p
hase 2
R*
R*
Arrestin Nb80
GFP
Arrestin
Endosome
Plasma membrane
a
b
c
Figure 3
|
Nb80–GFP does not accumulate in clathrin-coated pits or
vesicles. a, Representative TIRF microscopy frames showing Nb80–GFP
(green) and b-arrestin-2–mCherry (red) before and after agonist addition.
Fluorescence intensity profiles are shown below for the indicated region and
path; representative of n539 cells, 5 experiments and 4,849 punctae.
b, Equivalent analysis comparing Nb80–GFP (green) to clathrin light chain-
dsRed (red); representative of n526 cells, 3 experiments and 3,965 punctae.
Arrowheads indicate examples of Nb80–GFP labelled endosomes. Scale bars,
5mm. c, Model for two phases of Nb80–GFP recruitment by the activated b
2
-
AR, first at the plasma membrane and then at endosomes.
RESEARCH LETTER
536 | NATURE | VOL 495 | 28 MARCH 2013
Macmillan Publishers Limited. All rights reserved
©2013
Because endosomes contain activated b
2
-ARs, we next asked if
receptors engage their cognate G protein from this compartment.
Heterotrimeric G proteins and adenylyl cyclase can be observed in
endosomes as well as at the plasma membrane, supporting the concept
of endosome-based G protein signalling
4,6,10,24
. To directly investigate
the subcellular location of G protein activation, we developed a dis-
tinct biosensor based on another nanobody, Nb37, which specifically
recognizes the guanine-nucleotide-free form of Ga
s
representing the
catalytic intermediate of G protein activation (Fig. 4a)
25
. We hoped
that, because Nb37 binds a surface of the alpha-helical domain that is
accessible only in the nucleotide-free form, we would be able to detect
production of this critical butfleeting activation intermediate in living
cells. This was indeed the case because Nb37–GFP localized in the
cytoplasm of untreated cells and was rapidly recruited from the cyto-
plasm to the plasma membrane in response to isoprenaline application
(Fig. 4b, yellow arrowhead, Pearson coefficient50.627). Membrane
recruitment of Nb37–GFP was agonist-dependent even in cells over-
expressing Ga
s
and, notably, the cytoplasmic concentration of Nb37–
GFP achieved in our experiments (also approximately 20nM) was
substantially lower than that producing detectable inhibition of G
protein activation in vitro (Supplementary Fig. 5). Together, these
observations suggest that Nb37–GFP indeed functions as a specific
biosensor of G
s
activation under the experimental conditions used.
Nb37–GFP was recruited not only to the plasma membrane (Fig. 4b,
yellow arrowhead) but also to b
2
-AR-containing endosomes. Notably,
endosome recruitment of Nb37–GFP occurred after the appearance of
b
2
-ARs in the endosome. Such later recruitment was evident in dual
label confocal image series (Fig. 4b shows an example: white arrowhead
indicates recently internalized b
2
-AR, white arrows indicate Nb37–GFP
recruitment, Pearson coefficient 50.710; see also Supplementary Video
8). Nb37–GFP recruitment was specifically dependent on receptor
activation because a mutant b
2
-AR that is defective in G-protein coup-
ling (b
2
-AR-Cys 341 Gly)
26
did not produce detectable recruitment
(Supplementary Fig. 6), whereas the distinct G
s
-coupled DRD1
recruited Nb37–GFP to both the plasma membrane and endo-
somes (data not shown). Nb37–GFP localized uniformly on b
2
-AR-
containing endosomes in cells overexpressing Ga
s
(Fig. 4b and
Supplementary Video 8) but was recruited non-uniformly in cells
expressing endogenous levels (Fig. 4c, arrowhead indicates an
example). Moreover, Nb37–GFP recruitment to endosomes had a
scintillating appearance, fluctuating rapidly when viewed in live image
series (Supplementary Video 9), suggesting that G protein activation
detected by Nb37–GFP occurs dynamically from limited regions of the
endosome membrane.
We then asked if active b
2
-AR G
s
coupling from endosomes con-
tributes to the cellular response, focusing on cAMP as a classical
second messenger carrying the downstream signal, using a previously
described real-time assay of cAMP accumulation in living cells and
normalizing to the signal produced by receptor-independent activa-
tion of adenylyl cyclase with forskolin (5 mM)
27
. Isoprenaline produced
a rapid increase in cytoplasmic cAMP accumulation, reaching a maxi-
mum within approximately 10min (Fig. 4d, blue line). Dyngo-4a, a
dynamin inhibitor that blocks b
2
-AR endocytosis by inhibiting CCP
function
28,29
, did not detectably affect the earliest portion of the
forskolin-normalized cAMP accumulation curve but significantly
reduced its later rise (Fig. 4d, green line, grey dots indicate Pvalues),
consistent with the two phases of endosome-localized activation
detected by the nanobody-derived biosensors. A similar inhibition
was observed in cells expressing a mutant b
2
-AR with a carboxy-
terminal alanine residue added that prevents efficient recycling
β2-AR
β2-AR Merged
c
e
Nb37–GFP
bIso
f
d
g
R
GFP
Nb37
α
0 min 6 min 6.5 min 7 min 7.5 min 8 min 8.5 min
125
100
75
50
25
0
Luminescence
(% of max Fsk)
0246810
Time (min)
0.25
0.20
0.15
0.10
0.05
0.00
P value
β2-AR-3S + 5 μM Iso
β2-AR-3S + 5 μM Iso + 30 μM Dyngo-4a
125
100
75
50
25
0
Luminescence
(% of max Fsk)
0246810
Time (min)
β2-AR β2-AR-3S
Internalization (%)
50
40
30
20
10
0
Agonist binding
G protein activation
from plasma
membrane
Arrestin binding Clustering in CCP
Recycling Endocytosis
Uncoating
G protein activation
from endosome
membrane
Signalling phase 1
Signalling phase 2
RR* R*
R*
R*
R*
αα
α
GDP ββ
β
γγ
γ
GDP GTP
Arrestin
GDP
GTP
Nb37–GFP
a
β2-AR + 5 μM Iso
β2-AR + 5 μM Iso + 30 μM Dyngo-4a e
Biosensor of G
protein activation
Figure 4
|
Internalized b
2
-ARs contribute to the
acute cAMP response. a, Scheme for detecting
conformational activation of G
s
with Nb37–GFP.
b, Confocal image frames showing Nb37–GFP
(green) and b
2
-AR (red) at the indicated time
points after isoprenaline addition (representative
of n514 cells; estimated Nb37–GFP recruitment
delay ranged from 0.7 to 2.65 min).Scale bar, 5 mm.
Yellow arrowhead indicates Nb37–GFP
recruitment to the plasma membrane. White
arrowhead indicates an endosome containing
recently internalized b
2
-AR and not associated
with Nb37–GFP, white arrows indicate Nb37–GFP
recruitment. c, Representative confocal frames
showing a discrete endosomal structure labelled
with Nb37–GFP (green) and b
2
-AR (red) at higher
magnification. Scale bar, 2 mm. Arrowhead
indicates non-uniform localization of Nb37–GFP
to the endosome. d, Forskolin-normalized b
2
-AR-
mediated cAMP response in the absence or
presence of 30 mM Dyngo-4a (mean 6s.e.m.,
n510 experiments, Pvalues in grey).
e, Isoprenaline (20 min)-induced b
2
-AR and b
2
-
AR-3S internalization measured by flow cytometry
(n54 experiments). f, Forskolin-normalized b
2
-
AR-3S-mediated cAMP response in the absence or
presence of 30 mM Dyngo-4a (n58 experiments;
P50.1192). g, Model for two phases of b
2
-AR G
s
activation, first at the plasma membrane and then
at endosomes, separated by the endocytic event.
LETTER RESEARCH
28 MARCH 2013 | VOL 495 | NATURE | 537
Macmillan Publishers Limited. All rights reserved
©2013
(b
2
-AR-Ala)
30
(Supplementary Fig. 7), suggesting that the Dyngo-
sensitive component of the cAMP response did not represent a secon-
dary consequence of resensitization by receptor recycling
17
. Dyngo-4a
did not produce the same effect on cAMP accumulation elicited by
the internalization-defective b
2
-AR-3S mutant receptor
21
(Fig. 4e, f),
further supporting the conclusion that the second signalling phase
indeed requires receptor localization in endosomes.
The present findings revise a long-held tenet of molecular phar-
macology, that acute signal transduction mediated by the canoni-
cal b
2
-AR G
s
signal transduction mechanism is plasma membrane
delimited. The results provide direct evidence supporting the hypo-
thesis that b
2
-AR endocytosis contributes to a second phase of the
acute cellular cAMP response, which represents a significant com-
ponent of the overall biochemical signal developed within several
minutes after the initial agonist application. Thus, although it remains
clear that b
2
-ARs can elicit G
s
-mediated signal transduction from the
plasma membrane, the present data reveal a discrete component of the
acute signalling response that is initiated from endosomes (Fig. 4g). It
remains unknown if b
2
-ARs are continuously bound by agonist in
endosomes, as depicted in the figure for simplicity, but conforma-
tional activation of G
s
in endosomes is both receptor- and agonist-
dependent. Unambiguous detection of endosome-based activation of
acute G-protein-linked signalling is presently limited to the b
2
-AR
G
s
system for which the critical nanobodies are available. However,
endosome-based contribution to the acute signalling response is pro-
bably widespread in the GPCR superfamily because the b
2
-AR belongs
to the largest group (family A) of GPCRs and is often considered a
prototype. We also suggest, more generally, that nanobody-based bio-
sensors represent a versatile strategy for probing other types of dyna-
mic conformational change with high spatiotemporal resolution in
living cells.
METHODS SUMMARY
Experiments were carried out using human embryonal kidney HEK293 cells
(ATCC) expressing the indicated receptor constructs and nanobodies fused to
enhanced GFP. Opticalimaging was carried out at 37 uC in DMEM not containing
phenol red and supplemented with 30 mM HEPES. Live cell cAMP accumulation
was assessed at 37 uC using pGlosensor-20F (Promega). Flow cytometry was
carried out using Alexa647 (Invitrogen)-conjugated M1 anti-Flag monoclonal
antibody (Sigma) and a FACSCalibur instrument (Becton Dickinson).
Full Methods and any associated references are available in the online version of
the paper.
Received 13 August 2012; accepted 8 February 2013.
Published online 20 March 2013.
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Supplementary Information is available in the online version of the paper.
Acknowledgements We thank B. Kobilka, P. Robinson, A. Kruse, E. Pardon, P. Temkin,
M. Puthenveedu, A. Henry,A. Marley and K. Thorn for assistance, adviceand discussion.
These studies were supported by the National Institute on Drug Abuse of the US
National Institutes of Health (DA010711and DA012864 to M.v.Z. and F32 DA029993
to J.C.T.). R.I. is supported by the American Heart Association. R.K.S. and J.P.M. are
supported by the National Institute of General Medical Sciences (GM083118 to R.K.S.
and T32 GM007767 to J.P.M.). S.G.F.R. is supported by the Lundbeck Foundation. J.S.
is supported by FWO-Vlaanderen grants (FWO551 and FWO646) and
Innoviris-Brussels (BRGEOZ132). B.H. is supported by a Packard Fellowship for
Science and Engineering.
Author Contributions R.I.constructed and validated the nanobodybiosensors, carried
out most of the cell biological experiments and analysis, contributed to overall
experimental strategy and took a lead role in writing the manuscript. J.C.T. carried out
early experiments identifying endocytic inhibitor effects on cellular cAMP signalling,
and contributed to initial project planning. J.R.T. built the luminometer system,
developed software for analysis of luminometry data, and contributed to early
experiments on cellular cAMP signalling. M.C. contributed to experimental design and
data analysis, and modelled effects of endocytic inhibitors on the cellular cAMP
response. J.P.M. contributed to the production of receptor-containing rHDL particles
and carried out in vitro studies of Nb80 binding and dissociation. J.S. developed the
nanobody reagents used as the basis for the biosensors described in this study and
advised on biosensor design and expression. S.G.F.R. contributed to developing and
screeningthe initial nanobodyreagents, and carriedout in vitro studies of Nb80binding
and dissociation in rHDL particles reconstituted with bimane-labeled receptors. R.K.S.
contributed to overall experimental interpretation, supervised J.P.M. in carrying out in
vitro studies of Nb80 binding to receptors, and performed in vitro experiments
evaluating Nb37 effects on G protein activation. H.E.-S. contributed to experimental
design anddata interpretation,and supervisedefforts to model endocytic effectson the
cellular cAMP response. B.H. contributed to overall experimental design and
interpretation, implementation of biosensorsand advised on imageanalysis. M.v.Z. was
responsible for overall project strategy, carried out some of the imaging experiments,
and drafted the manuscript together with R.I.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper. Correspondence
and requests for materials should be addressed to M.v.Z.
(Mark.VonZastrow@ucsf.edu).
RESEARCH LETTER
538 | NATURE | VOL 495 | 28 MARCH 2013
Macmillan Publishers Limited. All rights reserved
©2013
METHODS
Cell Culture, cDNA constructs and transfection. HEK293 cells were grown in
DMEM supplemented with 10% FBS (UCSF Cell Culture Facility) without anti-
biotics. Stably transfected HEK293 cell clones expressing Flag-tagged b
2
-AR-3S
were created using previously described Flag-tagged b
2
-AR
30
. A plasmid encoding
a cyclic permuted luciferase reporter construct,based on a mutated RIIbB cAMP-
binding domain from PKA(pGloSensor-20F, Promega). Nb80–eGFP and Nb37–
eGFP were created by amplifying Nb80 and Nb37 nanobody complementary
DNAs using 59-CTTGAAAAGCTTGCCGCCACCATGGGACAGGTGCAGCT
GCA-39;59-TTCAAGGGATCCATGTGATGGTGATGGTGGTGTGAGGAGA
CGGT-39and 59-CTTGAAAAGCTTGCCGCCACCATGGGACAGGTGCAGC
TGCA-39;59-TTCAAGGGATCCATGTGATGGGCTTCAGGTTCGTGATGG
TGATG-39primers, respectively, and cloning into the peGFP-N1 vector using
HindIII and BamH1. b-arrestin-2–GFP, clathrin–DsRed, EEA1–DsRed and Ga
s
–
HA were gifts from M. Caron, W. Almers, K. Mostov and P. Wedegaertner,
respectively. b-arrestin-2–mCherry was generated by subcloning b-arrestin-2 to
pmCherry (Clontech)and b
2
-AR–CFP was generated fromthe Flag-tagged b
2
-AR
construct. Transfections were performed using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instructions. Flag-tagged human b
2
-AR and b
2
-
AR-3S (Ser 355 Gly, Ser 356 Gly and Ser 364 Gly were mutated simultaneously)
constructs were labelled with Alexa555- or Alexa488-conjugated M1 anti-Flag
monoclonal antibody (Sigma) as described previously
31
.
Live-cell confocal imaging. Live cell imaging was carried out using Yokagawa
CSU22 spinning disk confocal microscope with a 3100, 1.4 numerical aperture,
oil objective and a CO
2
and 37 uC temperature-controlled incubator. A 488 nm
argon laser and a 568nm argon/krypton laser (Melles Griot) were used as light
sources for imaging GFP and Flag signals, respectively. Cells expressing both Flag-
tagged receptor and the indicated nanobody–GFP were plated onto glass cover-
slips. Receptors were surface labelled by addition of M1 anti-Flag antibody
(1:1,000, Sigma) conjugated to Alexa 555 (A10470, Invitrogen) to the media for
30 min, as described previously
32
. Indicatedagonist (isoprenaline,Sigma) or antago-
nist (CGP-12177,Tocris) were added and cells were imaged every 3 s for 20min in
DMEM without phenolred supplemented with 30 mM HEPES, pH 7.4 (UCSF Cell
Culture Facility). Time-lapse images were acquired with a Cascade II EM charge-
coupled-device (CCD) camera (Photometrics) driven by Micro-Manager 1.4
(http://www.micro-manager.org).
Live cell TIRF microscopy. TIRF imaging was carried out as described
previously
33
. Briefly, HEK293 cells co-expressing either Nb80–eGFP and
b-arrestin-2–mCherry or clathrin light chain–DsRed, were imaged in DMEM
without phenol red supplemented with 30 mM HEPES, pH 7.4 (UCSF Cell
Culture Facility). Imaging was carried out using a Nikon TE-2000E inverted
microscope with a 3100, 1.49 numerical aperture TIRF objective, equipped for
through-the-objective TIRF illumination, a 37 uC temperature-controlled stage
(Bioscience Tools) and an objective warmer (Bioptechs). A 488nm argon laser
(Melles Griot) and a 543 nm helium-neonlaser (Spectra Physics) were used as light
sources. Time-lapse sequences were acquired with a C9100-12 camera
(Hamamatsu Photonics) driven by iQ software (Andor). Cells were imaged every
3 s for 20 min.
Image analysis and statistical analysis. Images were saved as 16-bit TIFF files.
Quantitative image analysis was carried out on unprocessed images using ImageJ
software (http://rsb.info.nih.gov/ij). Co-localization analysis wasestimated by cal-
culating the Pearson’s correlation coefficient between the indicated image chan-
nels using the co-localization plug-in for ImageJ. Analysis of Nb80–GFP intensity
profile along the straight line and Nb80–GFP/b-arrestin or Nb80–GFP/clathrin
along the segmented line were carried out using the ImageJ plot profile function.
For estimating changes in Nb80–GFP surface fluorescence over time in TIRF
images, individual cells were selected manually and fluorescence values measured
over the entire stack. A blank area of the image lacking cells was used to estimate
background fluorescence. Average fluorescence intensity was measured in each
frame, background-subtracted and normalizedto the maximum value. Pvalues are
from one-tailedunpaired Student’s t-tests. For visual presentation(but not quanti-
tative analysis), image series were processed using Kalman stack filter in ImageJ.
Luminescence-based rapid cAMP assay. HEK293 cells were transfected with a
plasmid encoding a cyclic-permuted luciferase reporter construct, based on a
mutated RIIbB cAMP-binding domain from PKA (pGloSensor-20F, Promega),
which produces rapid and reversible cAMP-dependent activation of luciferase
activity in intactcells. Cells were plated in 24-well dishes containingapproximately
200,000 cells per well in 500 ml DMEM without phenol red and no serum and
equilibrated to 37 uC in a light-proof cabinet. An image of the plate was focused
on a 512 3512 pixel electron multiplying CCD sensor (Hamamatsu C9100-13),
cells were equilibrated for 1 h in the presence of 250mgml
21
luciferin (Biogold),
and sequential luminescence images were collected every 10s to obtain basal
luminescence values. The camera shutter was closed, the cabinet opened and
the indicated concentration of isoprenaline was bath applied, with gentle manual
rocking before replacing in the dark cabinet and resuming luminescence image
acquisition. In endocytic manipulation experiments, cells were pre-incubated
with 30 mM Dyngo-4a (abcam Biochemicals) for 15 min. Every 10 s, sequential
images were acquired using Micro-Manager (http://www.micro-manager.org)
and integrated luminescence intensity detected from each well wascalculated after
background subtraction and correction for vignetting using scripts written in
MATLAB (MathWorks). In each multiwell plate, and for each experimental con-
dition, a reference value of luminescence was measured in the presence of 5 mM
forskolin, a manipulation that stimulates a moderate amount of receptor-inde-
pendent activation of adenylyl cyclase. The average luminescence value—mea-
sured across duplicate wells—was normalized to the maximum luminescence
value measured in the presence of 5 mM forskolin.
FRET imaging. FRET imaging was carried out as described previously
10
. Briefly,
HEK293 cells co-expressing b
2
-AR–CFP or DRD1–CFP and Nb80–YFP were
imaged in wide field at 37 uC using a shuttered mercury arc lamp and standard
CFP excitation (ET430/243) and YFP emission (ET500/203) band pass filters
(Chroma). YFP emission was collected using a 535/30 m filter, and CFP emission
was collected through a 470/24 m filter. Corrected FRET ratios were obtained
using the following equation: NFRET 5[(I
FRET
2BG
FRET
)2(I
CFP
2BG
CFP
)3
BT
DONOR
2(I
YFP
2BG
YFP
)3DE
ACCEPTOR
]/I
CFP
.BT
DONOR
, donor bleed
through; DE
ACCEPTOR
, direct excitation of the acceptor; BG
X
, background fluor-
escence; and I
X
, integrated fluorescence intensity measured in a given channel.
Flow cytometric assay of receptor endocytosis. Surface fluorescence ofFlag–b
2
-
AR or Flag–b
2
-AR-3S expressing HEK293 cells was used to measure receptor
endocytosis. Cells were incubated with 10mM isoprenaline for 20 min at 37 uC
to drive receptor internalization to steady state and were subsequently rinsed 3
times with ice-cold PBS, then mechanically lifted and incubated with 1mgml
21
Alexa647 (Invitrogen)-conjugated M1 anti-Flag monoclonal antibody (Sigma)
at 4 uC for 1 h. Mean fluorescence intensity of 10,000 cells was measured using
a FACSCalibur instrument (Becton Dickinson). Each condition was performed
in triplicate.
Enhanced GFP calibration. Recombinant eGFP (BioVision) was used for cal-
ibrating average fluorescence intensity of the biosensors, imaged in confocal
optical sections through the cytoplasm of cells not exposed to agonist (to achieve
diffuse cytoplasmic distribution of the biosensors). eGFP was diluted in Hank’s
balanced salt solution andconfocal sections were imaged through droplets of each
using the same illumination and acquisition parameters as for imaging the bio-
sensors in cells. For each cell, a background fluorescence value was determined
by average fluorescence intensity of a blank region in the same image. The cyto-
plasmic concentration of biosensors was estimated by interpolation of the back-
ground-subtracted value using a linear least-squares fit to the standard plot.
Generation of b
2
-AR-rHDL nanoparticles. Apolipoprotein-AI (Apo-AI) was
biotinylated using NHS-PEG4-biotin (Pierce Biotechnology) at a 1:1 molar ratio.
Following a 30-min biotinylation reaction at room temperature, the sample was
dialysed to remove free biotin. Flag-tagged b
2
-AR was incorporated into recom-
binant high density lipoprotein (rHDL) particles as previously described
34,35
using
biotinylated Apo-AI. Receptor-containing particles were then purified by M1
anti-Flag immunoaffinity chromatography
36
. Particles containingpurified mono-
bromobimane-labelled b
2
-AR were generated similarly except not using biotiny-
lated HDL, with receptor labelling and fluorescence analysis carried out as
described previously
18
.
Assessing Nb80 binding to immobilized b
2
AR-rHDL. Nb80 binding to unli-
ganded and agonist-occupied b
2
-AR was measured using the OctetRED biolayer
interferometrysystem (Pall Forte
´Bio). In this assay, a targetprotein is immobilized
on the functionalized tip of a fibre optic probe that is dipped into an analyte
solution to observe analyte association to the target protein. A dissociation step
is then performed by transferring the biosensor into buffer lacking analyte.
Analyte association/dissociation is measured by monitoring changes in the inter-
ference pattern of a light beam reflected from the biosensor tip as the total mass
bound at the tip surface changes
37
. Streptavidin-coated biosensors (Pall Forte
´Bio)
were loaded with biotinylated b
2
-AR-rHDL particles for 15min at room temper-
ature and the biosensors were transferred to the OctetRED instrument. Sensors
were placed into assay buffer (20mM HEPES, pH7.7, 100mM NaCl, 1mM
EDTA, 0.02% (w/v) ascorbic acid, 0.05% (w/v) BSA) with or without 100mM
isoprenaline for 30min. To measure Nb80 association, the sensor was transferred
to assay buffer with Nb80 (at indicated concentrations) for 5 min, followed by a
30 min dissociationstep in assay buffer. Isoprenaline (100 mM) was includedin the
association and dissociation steps when measuring Nb80 binding to agonist-occu-
pied receptor. All experiments were carried out at 25 uC with the assay plate
shaking at 1,000r.p.m. Buffer-only controls were included in each experiment
to monitor for baseline drift, and nonspecific Nb80 binding was measured in a
parallel assay using sensors loaded with empty rHDL particles. Raw data were
LETTER RESEARCH
Macmillan Publishers Limited. All rights reserved
©2013
processed to remove baseline and nonspecific binding using Octet Data Ana-
lysis 7.0 software (Pall Forte
´Bio) and exported to Prism 5 (GraphPad) for curve
fitting. All association and dissociation curves were fit using a single-phase
exponential association or decay curves, respectively. Equilibrium binding
affinity of Nb80 for b
2
-AR in the presence or absence of the agonist isoprenaline
was assessed by monitoring the maximal interference shift generated by Nb80
binding (at varying Nb80 concentrations) to the probe containing b
2
-AR recon-
stituted in rHDL. The maximal shift was plotted against the Nb80 concen-
tration and fitted by nonlinear regression in Prism 5 (GraphPad) to generate
the apparent affinity.
Inhibition of bodipy-GTPcS-FL binding by Nb37. The effect of Nb37 on GTP
loading of purified G proteins was measured using 100nM bodipy-GTPcS-FL
(Invitrogen) essentially as described
25
. In this assay we used the fluorescence
emission of bodipy-GTPcS-FL (l
ex
,470 nm, l
em
,515 nm) that accompanies
binding of the labelled nucleotide to G protein
38
. Briefly, the fluorescence of
100 nM bodipy-GTPcS-FL was measured in the presence of 1 mM of theindicated
G protein using a96-well microtitre plate format on a M5fluorescence plate reader
(Molecular Devices). Nb37 was added together with bodipy-GTPc
s
-FL and the
binding reaction was initiated by the addition of G protein (1 mM) in 20 mM Tris-
HCl, pH 8.0, 3 mM MgCl
2
, 1 mM dithiothreitol in a final volume of 200 ml.
Bodipy-GTPcS-FL binding to heterotrimeric G protein included 0.1% dodecyl-
maltoside (final). Ga
s
was purified as described
39
.Ga
s
bc was purified as
described
13
. Myristoylated Ga
i
was purified as described
40
. The time scans were
limited to 240s to minimize the accumulation of hydrolysis of the product of
bodipy-GTPcS-FL, bodipy-phosphate
41
.
31. Gage, R. M., Matveeva,E. A., Whiteheart, S. W. & von Zastrow, M. Type I PDZ ligands
are sufficient to promote rapid recycling of G protein-coupled receptors
independent of binding to N-ethylmaleimide-sensitive factor. J. Biol. Chem. 280,
3305–3313 (2005).
32. Puthenveedu, M. A. et al. Sequence-dependent sorting of recycling proteins by
actin-stabilized endosomal microdomains. Cell 143, 761–773 (2010).
33. Yudowski, G. A., Puthenveedu, M. A., Henry, A. G. & von Zastrow, M. Cargo-mediated
regulation of a rapid Rab4-dependent recycling pathway. Mol. Biol. Cell 20,
2774–2784 (2009).
34. Whorton, M. R. et al. A monomeric G protein-coupled receptor isolated in a high-
density lipoprotein particle efficiently activates its G protein. Proc. Natl Acad. Sci.
USA 104, 7682–7687 (2007) .
35. Kuszak, A. J. et al. Purification and functionalreconstitution of monomericm-opioid
receptors: allosteric modulation of agonist binding by Gi
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36. Yao, X. et al. Coupling ligand structure to specific conformational switches in the
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RESEARCH LETTER
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©2013
