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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 resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. 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 β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (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 contribute 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.
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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).Nb80GFPdidnotco-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
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©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
2
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26732–26741 (2009).
36. Yao, X. et al. Coupling ligand structure to specific conformational switches in the
b
2
-adrenoceptor. Nature Chem. Biol. 2, 417–422 (2006).
37. Abdiche, Y., Malashock, D., Pinkerton, A. & Pons, J. Determining kinetics and
affinities of protein interactions using a parallel real-time label-free biosensor, the
Octet. Anal. Biochem. 377, 209–217 (2008).
38. McEwen, D. P., Gee, K. R., Kang, H. C. & Neubig, R. R. Fluorescent BODIPY-GTP
analogs: real-time measurement of nucleotide binding to G proteins. Anal.
Biochem. 291, 109–117 (2001).
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adenylyl cyclase activator G
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40. Lee, E., Linder, M. E. & Gilman, A. G. Expression of G-protein asubunits in
Escherichia coli.Methods Enzymol. 237, 146–164 (1994).
41. Jameson, E. E. et al. Real-time detection of basal and stimulated G protein
GTPase activity using fluorescent GTP analogues. J. Biol. Chem. 280, 7712–7719
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RESEARCH LETTER
Macmillan Publishers Limited. All rights reserved
©2013
... Receptor endocytosis might contribute to distinct cAMP signaling profiles for different GPCRs. For example, inhibiting endocytosis partially decreases cAMP production by the activated Gαs-coupled dopamine receptor D1 (DRD1) and the β2adrenergic receptor (B2AR) (Kotowski et al., 2011;Irannejad et al., 2013). In the case of DRD1, inhibiting endocytosis measurably decreases cAMP as early as one to two minutes after agonist addition (Kotowski et al., 2011). ...
... In the case of DRD1, inhibiting endocytosis measurably decreases cAMP as early as one to two minutes after agonist addition (Kotowski et al., 2011). In the case of B2AR, inhibiting endocytosis decreases cAMP production mainly at later time points, greater than five minutes after agonist treatment (Irannejad et al., 2013). This later time may reflect the time required for B2AR sorting to the specific endosomal domain from which it initiates endosomal G protein signaling , as we discuss in more detail in section 5.2. ...
... Several generated nanobodies specifically bind the active conformation of a specific GPCR or family of GPCRs (Manglik et al., 2017). Additionally, another nanobody, referred to as Nb37, recognizes a nucleotide-free Gαs conformation as a readout of GDP exchange by the Gαs subunit of the activated G protein (Westfield et al., 2011;Irannejad et al., 2013). Nanobodies have greatly aided in vitro and in vivo studies of GPCR biology (Manglik et al., 2017). ...
Thesis
G protein-coupled receptors (GPCRs) transduce diverse signals, including light, ions, hormones, and neurotransmitters, into equally diverse cellular responses. These cellular responses underlie complex physiological processes, including sensation, learning and memory, cardiac function, and immune function. Understanding the variables which contribute to GPCR signaling diversity at a cellular level is essential to understanding the role of GPCRs in physiology and disease. The subcellular location from which GPCR signaling occurs is an increasingly recognized variable which contributes to signaling diversity. I have used the delta opioid receptor (DOR) as a prototype GPCR to investigate mechanisms regulating GPCR localization and the effects of subcellular location on GPCR function. DOR is an ideal and therapeutically relevant prototype GPCR to study these questions. In neuronal cells, DOR localizes to multiple membrane compartments, including the plasma membrane and the Golgi apparatus. Relocation of DOR from intracellular sites to the plasma membrane is associated with enhanced pain-relieving effects of DOR agonists, which highlights the therapeutically relevant link between DOR localization and function. I first investigated the mechanisms which regulate DOR localization to the Golgi in a rat neuroendocrine cell line which shares common mechanisms with primary neurons in regulation of DOR trafficking. Through systematic mutagenesis of the DOR C-terminal primary amino acid sequence and high-resolution imaging, we identified conserved dual RXR amino acid motifs which are required for signal-regulated retention of DOR in the Golgi. Using biochemical approaches, we showed that these RXR motifs also mediate interaction with the coatomer protein I (COPI) complex. These data support a model in which DOR retention in the Golgi is mediated by active retrograde trafficking within the biosynthetic pathway. I next explored the effect of subcellular location on DOR activation. GPCR activation and coupling to effectors is driven by conformational changes in the receptor upon agonist binding. We used fluorescently tagged biosensors which recognize these conformational changes and high-resolution imaging to visualize DOR activation in different subcellular locations. We found that DOR in the plasma membrane and the Golgi differentially recruit two active conformation biosensors in response to the same agonist. These results indicate that subcellular location drives distinct engagement of effectors and suggest the exciting possibility that subcellular location may alter GPCR conformational landscapes upon ligand binding. I also determined the effect of subcellular location on DOR signaling using biosensors for second messenger signaling molecules cAMP and calcium. We found that DOR activation in both the plasma membrane and the Golgi inhibits cAMP production, suggesting that DOR couples to inhibitory G proteins regardless of compartment-specific effects on effector engagement or conformational landscapes. In a rat neuroendocrine cell line, DOR activation at the plasma membrane modulates calcium release from intracellular stores in a Gi/o, Gq/11, and phospholipase C- dependent manner. Modulation of calcium is specific to DOR signaling from the plasma membrane and is not observed upon DOR activation in the Golgi. These data suggest that DOR subcellular location influences the signaling profile of active receptors. Together this work adds to our understanding of how GPCR subcellular localization is regulated and how subcellular location can drive distinct GPCR activation and signaling. In the future, this mechanistic understanding could be applied to tune localization of therapeutically relevant GPCRs like DOR or to target GPCRs in specific subcellular compartments for desired therapeutic effects.
... These tools have also led to new approaches for drug discovery by enabling GPCR agonist fragment screening followed by SBDD. Finally, these approaches have led to the development of VHHs as biosensors to investigate GPCR signaling (Irannejad et al., 2013;Staus et al., 2014;Staus et al., 2016;Stoeber et al., 2018). ...
... May 2022 | Volume 9 | Article 863099 (or the receptor:transducer complex) upon ligand incubation. Fluorescent (Irannejad et al., 2013) and bioluminescence resonance energy transfer-based read-outs have been deployed (Che et al., 2020). The transmembrane movement assay, is based on fluorescence emission spectra using monobromobimane labeled GPCR and monitors the intramolecular change in distance between critical transmembrane α-helices (Rasmussen et al., 2011a). ...
... Cbs are excellent biosensors; the unique single-domain nature of a VHH allows for functional expression in the reducing cytoplasmic environment as a fusion with a fluorescent protein moiety, allowing for sub-cellular tracking. Enhanced GFP-fused Nb80 was used to probe the ADRB2 active state conformer in living mammalian cells (Irannejad et al., 2013). ADRB2 activation by the agonist isoprenaline was observed not only at the plasma membrane but also on the early endosome membrane. ...
Article
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The human genome encodes 850 G protein-coupled receptors (GPCRs), half of which are considered potential drug targets. GPCRs transduce extracellular stimuli into a plethora of vital physiological processes. Consequently, GPCRs are an attractive drug target class. This is underlined by the fact that approximately 40% of marketed drugs modulate GPCRs. Intriguingly 60% of non-olfactory GPCRs have no drugs or candidates in clinical development, highlighting the continued potential of GPCRs as drug targets. The discovery of small molecules targeting these GPCRs by conventional high throughput screening (HTS) campaigns is challenging. Although the definition of success varies per company, the success rate of HTS for GPCRs is low compared to other target families ( Fujioka and Omori, 2012 ; Dragovich et al., 2022 ). Beyond this, GPCR structure determination can be difficult, which often precludes the application of structure-based drug design approaches to arising HTS hits. GPCR structural studies entail the resource-demanding purification of native receptors, which can be challenging as they are inherently unstable when extracted from the lipid matrix. Moreover, GPCRs are flexible molecules that adopt distinct conformations, some of which need to be stabilized if they are to be structurally resolved. The complexity of targeting distinct therapeutically relevant GPCR conformations during the early discovery stages contributes to the high attrition rates for GPCR drug discovery programs. Multiple strategies have been explored in an attempt to stabilize GPCRs in distinct conformations to better understand their pharmacology. This review will focus on the use of camelid-derived immunoglobulin single variable domains (VHHs) that stabilize disease-relevant pharmacological states (termed ConfoBodies by the authors) of GPCRs, as well as GPCR:signal transducer complexes, to accelerate drug discovery. These VHHs are powerful tools for supporting in vitro screening, deconvolution of complex GPCR pharmacology, and structural biology purposes. In order to demonstrate the potential impact of ConfoBodies on translational research, examples are presented of their role in active state screening campaigns and structure-informed rational design to identify de novo chemical space and, subsequently, how such matter can be elaborated into more potent and selective drug candidates with intended pharmacology.
... As such, efforts at modulating DA signaling as a therapeutic strategy for various pathophysiological conditions have only taken into consideration the consequences of signaling by plasma membrane-localized DA receptors (Jin et al., 2003;Panchalingam and Undie, 2001;Undie et al., 1994). However, evidence from the past decade suggests that for some GPCRs endocytosis might in fact activate a second phase of acute or prolonged Gαs-mediated cAMP response from the endosomes (Calebiro and Koszegi, 2019;Calebiro et al., 2010;Feinstein et al., 2013;Ferrandon et al., 2009;Irannejad et al., 2013;Irannejad et al., 2015;Irannejad and von Zastrow, 2014;Kotowski et al., 2011;Lobingier and von Zastrow, 2019;Stoeber et al., 2018;Thomsen et al., 2018). Recent studies further support this notion by providing evidence that cAMP generation by activated receptors at the endosome is necessary to regulate transcriptional responses that are distinct from those elicited by activation of the plasma membrane receptor pool (Bowman et al., 2016;Godbole et al., 2017;Jean-Alphonse et al., 2014;Jensen et al., 2017;Peng et al., 2021;Tsvetanova and von Zastrow, 2014). ...
... We have previously shown that a single-domain camelid antibody, nanobody 80 (Nb80), originally developed to stabilize an active conformation of beta 2 adrenergic receptor (β2AR) for crystallography purposes , can be repurposed as a conformational biosensor to detect activated β2AR and β1AR in living cells (Irannejad et al., 2017;Irannejad et al., 2013). Through directed evolution on Nb80, a high-affinity nanobody (Nb6B9) was generated that stabilizes the active conformation of epinephrine-bound β2AR (Ring et al., 2013). ...
... To investigate whether activated D1DRs couple to G proteins to elicit a G-protein-mediated response at the Golgi, we took advantage of another nanobody-based biosensor, Nb37-GFP. We previously used Nb37-GFP to detect transiently active β1AR/Gs and β2AR/Gs complexes at the Golgi and endosomes, respectively (Irannejad et al., 2017;Irannejad et al., 2013). Nb37-GFP was recruited to the plasma membrane and the Golgi upon stimulation with DA, suggesting that the D1DR Golgi pool couples to G protein and activates it (Figure 1-figure supplement 4b and c). ...
Article
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Dopamine is a key catecholamine in the brain and the kidney, where it is involved in a number of physiological functions such as locomotion, cognition, emotion, endocrine regulation and renal function. As a membrane impermeant hormone and neurotransmitter, dopamine is thought to signal by binding and activating dopamine receptors, members of the G protein couple receptor (GPCR) family, only on the plasma membrane. Here, using novel nanobody-based biosensors, we demonstrate for the first time that the dopamine D1 receptor (D1DR), the primary mediator of dopaminergic signaling in the brain and kidney, not only functions on the plasma membrane but becomes activated at the Golgi apparatus in the presence of its ligand. We present evidence that activation of the Golgi pool of D1DR is dependent on Organic Cation Transporter 2 (OCT2), a dopamine transporter, providing an explanation for how the membrane impermeant dopamine accesses subcellular pools of D1DR. We further demonstrate that dopamine activates Golgi-D1DR in murine striatal medium spiny neurons (MSN) and this activity depends on OCT2 function. We also introduce a new approach to selectively interrogate compartmentalized D1DR signaling by inhibiting Gas coupling, using a nanobody-based chemical recruitment system. Using this strategy, we show that Golgi-localized D1DRs regulate cAMP production and mediate local protein kinase A activation. Together, our data suggest that spatially compartmentalized signaling hubs are previously unappreciated regulatory aspects of D1DR signaling. Our data provide further evidence for the role of transporters in regulating subcellular GPCR activity.
... This new toolkit comprises sensors selective for modulatory ligands, reporters of GPCR and G protein activation, abilities to eavesdrop on numerous second-messenger systems and measure ion channel function. Here we can cite just a small sampling of an impressive decade's landmarks and reviews (Banghart and Sabatini, 2012;Irannejad et al., 2013Irannejad et al., , 2014Tsvetanova and von Zastrow, 2014;Spangler and Bruchas, 2017;Banghart et al., 2018;Patriarchi et al., 2019;Zeng et al., 2019;Ravotto et al., 2020;Sabatini and Tian, 2020;Smith et al., 2020;Stoeber et al., 2020;Unger et al., 2020;Jullie et al., 2021;Labouesse and Patriarchi, 2021;Redolfi et al., 2021;Tjahjono et al., 2021), while offering also a small sampling of progress emerging just at the time of this writing (Condon et al., 2021;Copits et al., 2021;Melzer et al., 2021;Wan et al., 2021;Duffet et al., 2022;Qian et al., 2022;Wu et al., 2022). ...
Article
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Adaptive neuronal circuit function requires a continual adjustment of synaptic network parameters known as “neuromodulation.” This process is now understood to be based primarily on the binding of myriad secreted “modulatory” ligands such as dopamine, serotonin and the neuropeptides to G protein-coupled receptors (GPCRs) that, in turn, regulate the function of the ion channels that establish synaptic weights and membrane excitability. Many of the basic molecular mechanisms of neuromodulation are now known, but the organization of neuromodulation at a network level is still an enigma. New single-cell RNA sequencing data and transcriptomic neurotaxonomies now offer bright new lights to shine on this critical “dark matter” of neuroscience. Here we leverage these advances to explore the cell-type-specific expression of genes encoding GPCRs, modulatory ligands, ion channels and intervening signal transduction molecules in mouse hippocampus area CA1, with the goal of revealing broad outlines of this well-studied brain structure’s neuromodulatory network architecture.
... Further, Nbs have been shown to be excellent tools for the generation of intrabodies targeting intracellular structures (reviewed in (Soetens et al., 2020;Wagner and Rothbauer 2020). Nb-based intrabodies have been used as imaging probes (Rothbauer et al., 2006;Traenkle and Rothbauer 2017), biosensors (Irannejad et al., 2013;Stoeber et al., 2018;Cao et al., 2019) and even as intracellular modulators of signaling pathways and cellular targets (van Impe et al., 2013;Gulati et al., 2018;Singh et al., 2018) (reviewed in (Wagner and Rothbauer 2020). Moreover Nbs have been shown to be well-suited for in vivo imaging approaches as they show superior properties with regard to tissue distribution, rapid tumor accumulation, tumor penetration and fast clearance from the blood circulation Chanier and Chames 2019). ...
Article
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Cell-free systems are well-established platforms for the rapid synthesis, screening, engineering and modification of all kinds of recombinant proteins ranging from membrane proteins to soluble proteins, enzymes and even toxins. Also within the antibody field the cell-free technology has gained considerable attention with respect to the clinical research pipeline including antibody discovery and production. Besides the classical full-length monoclonal antibodies (mAbs), so-called "nanobodies" (Nbs) have come into focus. A Nb is the smallest naturally-derived functional antibody fragment known and represents the variable domain (V H H,~15 kDa) of a camelid heavy-chain-only antibody (HCAb). Based on their nanoscale and their special structure, Nbs display striking advantages concerning their production, but also their characteristics as binders, such as high stability, diversity, improved tissue penetration and reaching of cavity-like epitopes. The classical way to produce Nbs depends on the use of living cells as production host. Though cell-based production is well-established, it is still time-consuming, laborious and hardly amenable for high-throughput applications. Here, we present for the first time to our knowledge the synthesis of functional Nbs in a standardized mammalian cell-free system based on Chinese hamster ovary (CHO) cell lysates. Cell-free reactions were shown to be time-efficient and easy-to-handle allowing for the "on demand" synthesis of Nbs. Taken together, we complement available methods and demonstrate a promising new system for Nb selection and validation.
... After characterisation of GLP-1R responses in the adult beta cell β-arrestin 2 KO mouse model, we next generated an in vitro beta cell model for a more detailed examination of the molecular mechanisms associated with the changes observed in receptor trafficking and signalling in vivo and in primary islets. We first verified the pattern of recruitment of β-arrestin 2 to the plasma membrane following GLP-1R stimulation in our chosen model, namely the INS-1 832/3 rat insulinoma cell line (Extended Data Fig. 5a Gs proteins following GPCR stimulation (Irannejad et al., 2013). In agreement with our previously detected acute cAMP defect in beta cell β-arrestin 2 KO islets, this experiment revealed a small but significant reduction in Emax, with no change in logEC50, for exendin-4-induced GLP-1R signalling at the plasma membrane in β-arrestin 2 KD compared with control cells, with no changes associated with the degree of endosomal signalling (Fig. 6b, c). ...
Preprint
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The GLP-1R is a GPCR from the glucagon receptor family with important roles in the regulation of beta cell function and feeding behaviours. After ligand-stimulated G protein binding, active GLP-1Rs are rapidly desensitised by GRKs, followed by recruitment of β-arrestins, scaffolding proteins that terminate G protein interaction through steric hindrance but also act as independent signalling mediators. GLP-1R agonists (GLP-1RAs) are well-established therapeutics in type 2 diabetes and obesity that are nevertheless associated with dose-related gastrointestinal side effects affecting ∼50% of patients. Exploiting the power of ligand-directed signalling bias with modified β-arrestin engagement is a promising approach to favour therapeutically beneficial over harmful effects of GLP-1RAs. Although GLP-1R interacts with both β-arrestin isoforms 1 and 2 with similar affinities, expression of the latter is greatly enhanced in beta cells, making this the most functionally relevant isoform. To increase our understanding of the molecular consequences of β-arrestin 2 activity in beta cell GLP-1R function we have assessed in vivo glycaemic responses to the pharmacological GLP-1RA exendin-4 in an adult beta cell-specific β-arrestin 2 KO mouse model. KO mice displayed worse exendin-4 responses acutely that then improved after glucose rechallenge 6 hours post-agonist injection, an effect mirrored by differences in plasma insulin levels and in ex vivo islet calcium and insulin secretion responses. Similar effects were observed for semaglutide and tirzepatide, two clinically relevant GLP-1RAs, but not for the less β-arrestin 2-reliant biased agonist exendin-phe1. Acute exendin-4-induced cAMP was impaired but cAMP responses to GLP-1 following overnight exendin-4 exposure tended to improve in KO versus control islets. Acute signalling defects were attributed to the concerted effect of the phosphodiesterase PDE4 and β-arrestin 1, as beta cell β-arrestin 2 KO islets regained cAMP responsivity with either β-arrestin 1 knockdown or PDE4 inhibition. Cell-cell connectivity was preserved in beta cell β-arrestin 2 KO but lost in control islets imaged in vivo following implantation in mouse eyes. While islet GLP-1R internalisation was not affected by β-arrestin 2 KO, both recycling and lysosomal targeting were significantly impaired, with the receptor instead redirected to the TGN. Trafficking results were replicated in INS-1 832/3 β-arrestin 2 knock-down cells, where we also detected increased levels of exendin-4-induced TGN signalling as well as reduced GLP-1R ubiquitination and recruitment of the E3 ubiquitin ligase NEDD4, suggesting a role for this post-translational modification in β-arrestin 2-dependent GLP-1R trafficking. The present study represents the first in-depth in vivo and ex vivo analysis of the effects of beta cell β-arrestin 2 gene ablation on acute versus sustained pharmacological GLP-1R responses.
... Conformational differences in -arrestin may thus explain, in part, the inability of CXCL9 and CXCL10 to induce the formation of G i :-arrestin complexes; however, delineating finer structural details will be necessary to test this hypothesis. Such conformational changes in -arrestin2 likely represent a receptor-unbound conformation (39,40) and may also correlate with spatially distinct pools of GPCR signaling (41,42). ...
Article
G protein–coupled receptors (GPCRs) are the largest family of cell surface receptors and signal through the proximal effectors, G proteins and β-arrestins, to influence nearly every biological process. The G protein and β-arrestin signaling pathways have largely been considered separable; however, direct interactions between Gα proteins and β-arrestins have been described that appear to be part of a distinct GPCR signaling pathway. Within these complexes, Gα i/o , but not other Gα protein subtypes, directly interacts with β-arrestin, regardless of the canonical Gα protein that is coupled to the GPCR. Here, we report that the endogenous biased chemokine agonists of CXCR3 (CXCL9, CXCL10, and CXCL11), together with two small-molecule biased agonists, differentially formed Gα i :β-arrestin complexes. Formation of the Gα i :β-arrestin complexes did not correlate well with either G protein activation or β-arrestin recruitment. β-arrestin biosensors demonstrated that ligands that promoted Gα i :β-arrestin complex formation generated similar β-arrestin conformations. We also found that Gα i :β-arrestin complexes did not couple to the mitogen-activated protein kinase ERK, as is observed with other receptors such as the V2 vasopressin receptor, but did couple with the clathrin adaptor protein AP-2, which suggests context-dependent signaling by these complexes. These findings reinforce the notion that Gα i :β-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of the spectrum of biased agonism.
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The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β(2)-adrenoceptor (β(2)AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.
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G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.
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The crystal structure of Gs α, the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was determined at 2.5 Å in a complex with guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Gs α is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gs α·GTPγS with that of Gi α·GTPγS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gs α. The βγ binding surface of Gs α is largely conserved in sequence and structure to that of Gi α, whereas differences in the surface formed by the carboxyl-terminal helix and the α4-β6 loop may mediate receptor specificity.
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Fluorescence and bioluminescence resonance energy transfer (FRET and BRET) techniques allow the sensitive monitoring of distances between two labels at the nanometer scale. Depending on the placement of the labels, this permits the analysis of conformational changes within a single protein (for example of a receptor) or the monitoring of protein-protein interactions (for example, between receptors and G-protein subunits). Over the past decade, numerous such techniques have been developed to monitor the activation and signaling of G-protein-coupled receptors (GPCRs) in both the purified, reconstituted state and in intact cells. These techniques span the entire spectrum from ligand binding to the receptors down to intracellular second messengers. They allow the determination and the visualization of signaling processes with high temporal and spatial resolution. With these techniques, it has been demonstrated that GPCR signals may show spatial and temporal patterning. In particular, evidence has been provided for spatial compartmentalization of GPCRs and their signals in intact cells and for distinct physiological consequences of such spatial patterning. We review here the FRET and BRET technologies that have been developed for G-protein-coupled receptors and their signaling proteins (G-proteins, effectors) and the concepts that result from such experiments.
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D(1) dopamine receptors are primary mediators of dopaminergic signaling in the CNS. These receptors internalize rapidly following agonist-induced activation, but the functional significance of this process is unknown. We investigated D(1) receptor endocytosis and signaling in HEK293 cells and cultured striatal neurons using real-time fluorescence imaging and cAMP biosensor technology. Agonist-induced activation of D(1) receptors promoted endocytosis of receptors with a time course overlapping that of acute cAMP accumulation. Inhibiting receptor endocytosis blunted acute D(1) receptor-mediated signaling in both dissociated cells and striatal slice preparations. Although endocytic inhibition markedly attenuated acute cAMP accumulation, inhibiting the subsequent recycling of receptors had no effect. Further, D(1) receptors localized in close proximity to endomembrane-associated trimeric G protein and adenylyl cyclase immediately after endocytosis. Together, these results suggest a previously unanticipated role of endocytosis, and the early endocytic pathway, in supporting rapid dopaminergic neurotransmission.
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Remarkable progress has been made in the field of G protein-coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis.