EDITORS’ PICK cro

Author’s Choice

Mini G protein probes for active G protein– coupled receptors

(GPCRs) in live cells
Received for publication, January 18, 2018, and in revised form, March 2, 2018 Published, Papers in Press, March 9, 2018, DOI 10.1074/jbc.RA118.001975
Qingwen Wan‡, Najeah Okashah‡, Asuka Inoue§1, Rony Nehmé¶2, Byron Carpenter¶3, Christopher G. Tate¶,
and Nevin A. Lambert‡4
From the ‡Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia 30912,
the §Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578 Japan, and the ¶MRC Laboratory of
Molecular Biology, Cambridge CB20QH, United Kingdom
Edited by Henrik G. Dohlman

G protein– coupled receptors (GPCRs) are key signaling pro- conclude that mG proteins are versatile tools for studying
teins that regulate nearly every aspect of cell function. Studies of GPCR activation and coupling specificity in cells and should
GPCRs have benefited greatly from the development of molec- be useful for discovering and characterizing G protein sub-
ular tools to monitor receptor activation and downstream sig- type– biased ligands.
naling. Here, we show that mini G proteins are robust probes
that can be used in a variety of assay formats to report GPCR
activity in living cells. Mini G (mG) proteins are engineered G protein– coupled receptors (GPCRs)5 signal by coupling to
GTPase domains of G␣ subunits that were developed for struc- heterotrimeric G proteins and arrestins, which in turn activate
tural studies of active-state GPCRs. Confocal imaging revealed or inhibit enzymes, kinases and other effector molecules to reg-
that mG proteins fused to fluorescent proteins were located ulate nearly every aspect of cell function (1). The past 20 years
diffusely in the cytoplasm and translocated to sites of recep- have seen the development of a wide array of genetically-en-
tor activation at the cell surface and at intracellular organ- coded optical sensors and probes to monitor nearly every step
elles. Bioluminescence resonance energy transfer (BRET) of these signaling cascades in living cells (2). An early example
assays with mG proteins fused to either a fluorescent protein of this was the development of arrestin–fluorescent protein
or luciferase reported agonist, superagonist, and inverse ago- conjugates that translocate from the cytosol to the plasma
nist activities. Variants of mG proteins (mGs, mGsi, mGsq, membrane upon activation of cell-surface GPCRs (3). There are
and mG12) corresponding to the four families of G␣ subunits now many variants of this basic approach with different report-
displayed appropriate coupling to their cognate GPCRs, ers and detection modalities, and these relatively simple tools
allowing quantitative profiling of subtype-specific coupling have been used to gain tremendous insight into the functional
to individual receptors. BRET between luciferase–mG fusion properties of arrestins and GPCRs. Similar tools have been
proteins and fluorescent markers indicated the presence of developed to monitor interactions between GPCRs and G pro-
active GPCRs at the plasma membrane, Golgi apparatus, teins (4, 5), but compared with arrestins, the properties of het-
and endosomes. Complementation assays with fragments erotrimers are less favorable for many applications in cells. Het-
of NanoLuc luciferase fused to GPCRs and mG proteins erotrimeric G proteins are membrane-associated (or tethered)
reported constitutive receptor activity and agonist-induced proteins and therefore do not change subcellular location upon
activation with up to 20-fold increases in luminescence. We binding to GPCRs. This prevents simple visualization of com-
plex formation in imaging experiments and produces back-
ground signals when receptor–G protein complexes are
This work was supported in part by National Institutes of Health Grants detected using resonance energy transfer (e.g. FRET and BRET)
GM078319 and GM109879 (to N. A. L.) and Medical Research Council,
European Research Council, and Heptares Therapeutics Grants
(4, 5). Moreover, receptor–G protein complexes in cells are
MC_U105197215 and EMPSI 339995 (to C. G. T.). C. G. T. is a shareholder short-lived. Ambient concentrations of guanine nucleotides
and member of the scientific advisory board of Heptares Therapeutics. The lead to rapid complex dissociation, thus limiting signals gener-
content is solely the responsibility of the authors and does not necessarily ated by receptor–G protein association.
represent the official views of the National Institutes of Health.
Author’s Choice—Final version open access under the terms of the Creative Here, we report that mini G (mG) proteins (6, 7) are useful G
Commons CC-BY license. protein surrogates for studies of GPCR activation in cells. Mini
This article was selected as one of our Editors’ Picks.
1
G proteins are G␣ subunits with several key modifications as
Supported by Japan Science and Technology Agency (JST) Grant
JPMJPR1331 and Japan Agency for Medical Research and Development follows: 1) a truncated N terminus, which deletes membrane
(AMED) Grant JP17gm5910013. anchors and G␤␥-binding surface; 2) deletion of the ␣-helical
2
Present address: Creoptix AG, Einsiedlerstrasse 34, CH-8820 Wädenswil, domain; 3) mutations that improve protein stability in vitro;
Switzerland.
3
Present address: Warwick Integrative Synthetic Biology Centre, University of
5
Warwick, Coventry CV4 7AL, United Kingdom. The abbreviations used are: GPCR, G protein– coupled receptor; BRET, bio-
4
To whom correspondence should be addressed: Dept. of Pharmacology luminescence resonance energy transfer; mG, mini G; NES, nuclear export
and Toxicology, Medical College of Georgia, Augusta University, sequence; DPBS, Dulbecco’s phosphate-buffered saline; Nluc, NanoLuc;
Augusta, GA 30912-2300. Tel.: 706-721-6336; Fax: 706-721-2345; E-mail: ␤2AR, ␤2-adrenergic receptor; D1R, D1 dopamine receptor; D5R, D5 dop-
[email protected]. amine receptor; A1R, A1-adenosine receptor.

7466 J. Biol. Chem. (2018) 293(19) 7466 –7473

© 2018 Wan et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.
 Mini G proteins in cells
and 4) a mutation in the C-terminal ␣5 helix that uncouples
GPCR binding from nucleotide release, thus stabilizing
receptor–mG complexes in the presence of guanine nucleo-
tides (Fig. 1A). Several mG variants also incorporate mutations
at the GPCR–G protein interface and thus maintain the recep-
tor-coupling specificity of the following four G␣ subunit fami-
lies: Gs, Gi/o, Gq/11, and G12/13. These modifications enable mini
G proteins to report receptor activation in living cells in much
the same way as arrestins and conformation-specific nanobod-
ies (8). Therefore, mG proteins will likely become broadly use-
ful tools to study GPCR activation and receptor–G protein-
coupling specificity in cells.

Results and discussion

Mini G proteins were originally engineered for high-level
expression in Escherichia coli, high stability in vitro, and effec-
tive coupling to GPCRs. To visualize mG protein expression in
mammalian cells, we fused the fluorescent protein venus (9) to
the N terminus of several mG proteins and expressed the result-
ing fusion proteins in HEK 293 cells under the control of a CMV
promoter. Confocal microscopy revealed that mG variants
shown previously to express well in E. coli (e.g. mGs_393) (6)
were located diffusely throughout the cytoplasm and nucleus,
whereas mG variants that express poorly in E. coli (e.g. mGq) (7)
formed intracellular aggregates. Therefore, for this study we
used four mG protein variants that express well in E. coli and
HEK cells, specifically mGs_393, mGsi_43 (a chimera of mGs
and mGi), mGsq_71 (a chimera of mGs and mGq), and mG12_8
(referred to hereafter as mGs, mGsi, mGsq, and mG12 for sim-
plicity) (7). Because mG proteins in the nucleus are not imme-
diately accessible to receptors at the plasma membrane, we
added an N-terminal nuclear export sequence (NES) to second-
generation mG fusion proteins, thereby restricting localization
Figure 1. Mini G proteins are recruited to active receptors at the plasma
to the cytosol (Fig. 1B). membrane. A, cartoon representation highlighting the differences between
NES–venus–mG proteins bound tightly to agonist-activated G protein heterotrimers, which diffuse within the membrane to engage
GPCRs in intact cells. For example, stimulation of ␤2-adrener- receptors (left), and mG proteins, which diffuse through the cytosol to engage
receptors (right). Mini G proteins lack membrane anchors, N-terminal G␤␥-
gic receptors fused to the fluorescent protein cerulean (␤2AR– binding surface, and the ␣-helical domain (HD). B, confocal images of HEK 293
cerulean) with a saturating concentration of the agonist isopro- cells expressing cerulean-tagged ␤2-adrenergic receptors (␤2AR-cerulean;
terenol resulted in rapid translocation of NES–venus–mGs top panels) and NES–venus–mGs (bottom panels). NES–venus–mGs is
recruited to the plasma membrane after stimulation with 10 ␮M isoprotere-
from the cytosol to the plasma membrane (Fig. 1, B and C). To nol. Scale bar, 10 ␮m. C, venus fluorescence (FV, arbitrary units (a.u.)) at the
estimate the stoichiometry of receptor–mG complexes at plasma membrane (PM) and in the cytosol plotted against time for the cells
shown in B. D, mean NES–venus–mGs fluorescence (⫾ S.E.) line profiles drawn
steady state, we used standardized confocal imaging conditions perpendicular to the plasma membrane from the extracellular (e.c.) space to
to measure cerulean and venus fluorescence at the plasma the cytosol in five cells before and after application of isoproterenol. Weak
membrane of cells expressing cerulean-␤2AR and venus–mGs accumulation of NES–venus–mGs at the plasma membrane is detectable
prior to stimulation (black arrowhead).
in the presence of 10 ␮M isoproterenol. We calibrated these
intensity measurements with a standard protein consisting of
an extracellular cerulean, a transmembrane domain, and an BRET between GPCRs and mG proteins
intracellular venus (C-TM-V). The result indicated an average BRET is widely used to monitor protein interactions in cells
venus–mGs/cerulean–␤2AR stoichiometry of ⬃1:1 (mean (10) and can also detect protein trafficking or translocation to
venus/cerulean ⫽ 1.17; 95% confidence interval 0.87–1.47; n ⫽ membrane compartments (11). To demonstrate the utility of
17). This is likely to be an overestimate due to the presence of mG proteins for BRET experiments, we cotransfected cells with
residual cytosolic venus–mGs near the plasma membrane. a fixed amount of plasmid DNA encoding ␤2-adrenergic recep-
Nevertheless, recruitment of mGs to active ␤2ARs was clearly tors fused to the Renilla luciferase Rluc8 (␤2AR–Rluc8) and
efficient, consistent with the formation of relatively stable increasing amounts of DNA encoding NES–venus–mGs, and
receptor–mG complexes. In some cells, there was detectable we quantified expression of the latter by flow cytometry. In
accumulation of mG proteins at the plasma membrane prior to unstimulated cells, there was a shallow monotonic increase in
stimulation (Fig. 1D), suggesting that mG proteins could also BRET as NES–venus–mGs expression increased, consistent
bind to ligand-free GPCRs (see below). with low- affinity binding of mGs to either ␤2ARs or the plasma

J. Biol. Chem. (2018) 293(19) 7466 –7473 7467

Mini G proteins in cells
Rluc8 receptors that are located in intracellular compartments
(e.g. the Golgi apparatus) and therefore do not have access to
extracellular isoproterenol.
Mini G proteins functionally mimic the nucleotide-empty,
GPCR-bound state of G protein heterotrimers (6), and there-
fore, mG and agonist binding to GPCRs should be mutually
cooperative. Consistent with this expectation, we found that
increasing NES–venus–mGs expression led to a 5–10-fold left-
ward shift (decreased EC50) in isoproterenol versus BRET con-
centration-response curves (Fig. 2B). As noted above, increas-
ing NES–venus–mGs expression also increased basal BRET
signals and decreased the assay’s dynamic range. Therefore,
interactions between mG proteins and GPCRs are sensitive to
mG protein abundance in a manner that is consistent with the
allosteric model of GPCR–G protein-coupling (12).
We next tested the reversibility of agonist-induced recruit-
ment of mG proteins by measuring BRET during sequential
application of an agonist and an antagonist or inverse agonist.
We found that ␤2AR–Rluc8/NES–venus–mGs complexes
were quite stable in the presence of norepinephrine and
required more than 15 min to dissociate after addition of the
inverse agonist ICI 118,551 (Fig. 2C). This is much slower than
dissociation of receptor– heterotrimer complexes in intact cells
(2), and it likely reflects stabilization of the orthosteric ligand-
binding site by the presence of a surrogate (mG protein) that
mimics a nucleotide-empty G protein (13). In contrast, musca-
Figure 2. BRET between ␤2AR–Rluc8 and NES–venus–mGs. A, net BRET is rinic acetylcholine M3R–Rluc8/NES–venus–mGsq and M4R–
plotted versus mean NES–venus–mGs fluorescence intensity per cell (FV, arbi- Rluc8/NES–venus–mGsi complexes dissociated much more
trary units (a.u.)) for control cells and cells stimulated with 10 ␮M isoprotere-
nol (iso). Cells were transfected with a constant amount of DNA encoding rapidly after addition of atropine (Fig. 2C), indicating that the
␤2AR–Rluc8, and an increasing amount of DNA encoding NES–venus–mGs. stability of agonist–receptor–mG complexes is widely variable.
Data are fitted to a two-site–specific binding equation, and data points from One practical advantage of assays that directly monitor
three independent experiments are superimposed. B, log EC50 is plotted ver-
sus mean NES–venus–mGs fluorescence intensity per cell (FV, arbitrary units) GPCR–transducer coupling as opposed to downstream signals
for five independent experiments with five different expression levels each is their ability to report both ligand potency and efficacy with-
(left). Example concentration-response curves are shown for cells expressing
low (EC50 ⫽ 116 nM), medium (EC50 ⫽ 52 nM), and high (EC50 ⫽ 22 nM) levels of out the potential confounds of spare receptors or assay read-
NES–venus–mGs (right); mean ⫾ S.E. of three independent experiments. C, outs that are not linearly related to efficacy. We found that
normalized BRET is plotted versus time for cells expressing ␤2AR-, M4R-, and
M3R-Rluc8 together with NES–venus–mGs, –mGsi, and –mGsq; acetylcholine BRET between ␤2AR–Rluc8 and NES–venus–mGs accurately
(100 ␮M; Ach), (⫺)-norepinephrine (10 ␮M), ICI 118,551 (10 ␮M), and atropine reported the difference in potency of the full agonists (⫺)-epi-
(10 ␮M) were added as indicated. Traces are the average of 4 –7 experiments. nephrine and (⫺)-norepinephrine at this receptor, as well as
weak partial agonist activity of ligands that are typically classi-
membrane. Stimulation with a saturating concentration of iso-
fied as partial agonists (pindolol) or antagonists (alprenolol)
proterenol led to a pronounced increase in energy transfer that
(14). Partial agonist activity of alprenolol is somewhat surpris-
was proportionally greater when NES–venus–mGs expression
ing but consistent with a previous report that binding of this
was lower (Fig. 2A). BRET in unstimulated cells approaches the
ligand to ␤2AR is promoted by nanobody 80 (Nb80), a G protein
BRET observed in agonist-stimulated cells when NES–venus–
surrogate that stabilizes the active state of the receptor (15).
mGs expression is high. This suggests that the former signal is
largely due to specific binding to ␤2AR–Rluc8 rather than non- Notably, the inverse agonist ICI 118,551 decreased BRET
specific membrane binding, as specific and nonspecific signals between ␤2AR–Rluc8 and NES–venus–mGs (Fig. 3A), again
would be expected to be additive. In the presence of isoprotere- supporting the notion that in unstimulated cells this signal at
nol, the BRET signals were nearly maximal when NES–venus– least partly reflects specific binding of NES–venus–mGs to
mGs expression was low, again consistent with stable, ligand-free ␤2AR–Rluc8. We also found that iperoxo induced a
high-affinity ␤2AR–mGs complexes under these conditions. greater maximum BRET signal between M3R–Rluc8 and NES–
However, a shallow increase in BRET efficiency in the presence venus–mGsq than the native ligand acetylcholine (Fig. 3B).
of agonist could still be discerned as NES–venus–mGs expres- This suggests that the former ligand acts as a superagonist at
sion increased, consistent with a superimposed (and much this receptor, similar to what has been reported for the M2
smaller) low-affinity binding component (Fig. 2A). The origin acetylcholine receptor (16). Taken together, these results sug-
of this second component is uncertain, but we speculate that it gest that mG proteins report the full range of ligand efficacy at
represents low-affinity binding of NES–venus–mGs to ␤2AR– GPCRs.

7468 J. Biol. Chem. (2018) 293(19) 7466 –7473

 Mini G proteins in cells
Mini G protein subtypes maintain appropriate coupling erotrimers from each family. The mutations incorporated into
specificity mGs were transferable to some G␣ subunits (e.g. G12) but not
Because there are four families of G␣ subunits, it was neces- others (e.g. Gq and Gi1) (7), and therefore mGsq and mGsi chi-
sary to develop mG proteins that could be surrogates for het- meras were developed wherein specificity-determining resi-
dues in mGs were replaced with residues corresponding to Gq
and Gi1. In vitro, these chimeras gain coupling to Gq– coupled
and Gi– coupled receptors, respectively, and lose coupling to
Gs– coupled receptors (7). To demonstrate the ability of mG
protein subtypes to couple to appropriate GPCRs in cell-based
assays, we profiled BRET between four NES–venus–mG vari-
ants (mGs, mGsi, mGsq, and mG12) expressed at similar levels
(53 ⫾ 21, 59 ⫾ 15, 36 ⫾ 14, and 42 ⫾ 16 arbitrary fluorescence
units, respectively; mean ⫾ S.D., n ⫽ 4) and four receptors that
collectively couple to all four G protein subtypes. We found that
mG proteins maintained appropriate coupling specificity as
defined by the primary transducer annotation in the BPS/
IUPHAR Guide to Pharmacology (Fig. 4A) (14). Importantly,
mG proteins reported not only known primary coupling inter-
actions (e.g. ␤2AR and mGs) but also known secondary cou-
pling interactions with either lower potency or maximal
response (e.g. ␤2AR and mGsi). We have observed appropriate
subtype-specific coupling of mG proteins to several additional
receptors (Fig. 4B), and we have not observed examples of clearly
inappropriate coupling, suggesting that the changes made to mGsi
and mGsq included key specificity determinants. These results
Figure 3. BRET between GPCRs and mG proteins reports the full range of
ligand efficacy. Net BRET between ␤2AR–Rluc8 and NES–venus–mGs (A) and suggest that mG proteins will be useful for quantifying the effi-
M3R–Rluc8 and NES–venus–mGsq (B) is plotted versus log concentration for ciency of coupling between GPCRs and G protein subtypes.
the indicated ligands and fitted to a four-parameter logistic equation;
mean ⫾ S.E. of four independent experiments. Data points at the far left of Indeed, when we extended this analysis to other receptors,
these panels represent vehicle controls. we noticed secondary coupling interactions that were previ-

Figure 4. Mini G protein subtypes maintain appropriate coupling specificity. A, net BRET to four different NES–venus–mG subtypes is plotted versus log
ligand concentration for ␤2-adrenergic receptors (␤2AR–Rluc8), M4 and M3 acetylcholine receptors (M4R-Rluc8 and M3R-Rluc8), and endothelin A receptors
(ETA-Rluc8). Ligands are isoproterenol (iso), acetylcholine (Ach), and endothelin-1 (ET-1); mean ⫾ S.E. of 3– 4 independent experiments. Data points at the far left
of each panel represent vehicle controls. B, heat maps representing normalized maximal BRET (which includes both constitutive and agonist-induced signals,
normalized to the best-responding mG protein) for 12 receptors (fused to Rluc8) paired with NES–venus–mG proteins. Heat maps for canonical Gs–, Gi/o–, and
Gq– coupled receptors are shown in blue, red, and black, respectively; n ⫽ 3–5 independent experiments for each receptor.

J. Biol. Chem. (2018) 293(19) 7466 –7473 7469

Mini G proteins in cells
with G␤␥–venus (Fig. 5B). For comparison, we also studied
coupling of mG proteins and empty heterotrimers to D2 dop-
amine receptors, and we found that these receptors, as
expected, coupled to both mGsi and Gi1 heterotrimers but not
mGs or Gs heterotrimers (Fig. 5A). Although caution is war-
ranted with respect to drawing conclusions based solely on mG
proteins, our results suggest that these tools will be useful for
studying G protein– coupling specificity. Our results also sug-
gest that Gs– coupled adrenergic and dopamine receptors share
the property of dual coupling to Gs and Gi/o heterotrimers, and
therefore the possibility that Gi/o heterotrimers mediate some
of the actions of D1R and D5R warrants further study.

Mini G proteins bind to active GPCRs in intracellular

compartments
Nanobodies raised against active-state GPCRs have been
used to detect active receptors in cells, including receptors
located in intracellular compartments (8, 19). Not surprisingly,
we found that mG proteins could be used in a similar manner to
indicate GPCR activation in intracellular compartments. For
example, CMV promoter-driven overexpression of cerulean-
tagged A1-adenosine receptors (cerulean–A1R) often led to
accumulation of this receptor in perinuclear compartments
that we presumptively identified as the Golgi apparatus. Stim-
ulation with adenosine recruited NES–venus–mGsi not only to
the plasma membrane but also to the Golgi apparatus (Fig. 6A).
The onset of NES–venus–mGsi recruitment to the Golgi was
delayed by ⬃5 s compared with the onset of NES–venus–mGsi
recruitment to the plasma membrane (Fig. 6B). To confirm the
Figure 5. Secondary coupling of Gs– coupled dopamine receptors to presence of active A1Rs in the Golgi apparatus, we measured
mGsi and Gi1 heterotrimers. A, recruitment of mG proteins to dopamine
receptors. Net BRET between D1R–, D5R–, or D2R–Nluc and four different BRET between the luciferase NanoLuc fused to mGsi (NES–
NES–venus–mG subtypes in response to dopamine (DA) is shown; mean ⫾ Nluc–mGsi) and venus-tagged acceptor molecules directed
S.E. of 5–7 independent experiments. B, recruitment of empty heterotrimers specifically to either the plasma membrane (venus– kras) or the
to dopamine receptors. The difference (⌬BRET) between net BRET observed
in the presence of 0.5 mM GDP alone and in the presence of apyrase and Golgi apparatus (venus– giantin) (11) in cells expressing unla-
dopamine (100 ␮M) is shown. Cells lacking endogenous G␣s, G␣q, and G␣12 beled A1Rs (Fig. 6C). Adenosine produced a concentration-
subunits expressed D1R-, D5R-, or D2R-Rluc8 and heterotrimers consisting of
G␤␥–venus and the remaining endogenous G␣ subunits (control) or overex- dependent increase in BRET to both membrane markers,
pressed G␣s or G␣i1. In some experiments cells also expressed the S1 subunit although this effect was less potent for recruitment to the Golgi
of pertussis toxin (PTX); mean ⫾ S.D. of 3– 6 independent experiments. apparatus (Fig. 6C). Taken together, these results suggest that
extracellular adenosine has rapid access to the Golgi lumen in
ously unknown or unappreciated. For example, we found that HEK 293 cells and that overexpressed A1Rs are functional in
both D1 and D5 dopamine receptors (D1R and D5R) coupled this compartment. The former observation is somewhat sur-
primarily to mGs but also secondarily to mGsi (Fig. 5A). Other prising in light of the hydrophilic character of adenosine (cal-
closely-related Gs– coupled monoamine receptors (␤2AR and culated octanol-water partition coefficient ⫽ ⫺1.1) and sug-
␤1AR) are known to couple to both Gs and Gi/o heterotrimers, gests that HEK 293 cells express nucleoside transporters
but we found only one previous report of D1R coupling to Gi capable of delivering adenosine to the Golgi lumen (19, 20).
(17) and no reports of D5R coupling to Gi. To verify D1R and Using a similar approach, we could also follow the accumula-
D5R coupling to Gi proteins, we measured BRET between lucif- tion of active ␤2ARs in early endosomes after prolonged stim-
erase-tagged receptors and “empty” (nucleotide-free) hetero- ulation with agonist (Fig. 6D) (8).
trimers consisting of G␤␥-venus and either G␣s or G␣i1. To
minimize contributions from endogenous G␣ subunits, we Mini G proteins support luciferase complementation
used HEK cells with CRISPR/Cas9-mediated deletion of G␣s, For many screening applications, split luciferase reporters
G␣q/11, and G␣12/13 subunits (18). Minimal BRET signals were are preferred because of their single wavelength output, high
generated when only the remaining endogenous G␣ subunits signal/background ratio, and high sensitivity. One recently-de-
were available to form heterotrimers with overexpressed G␤␥- veloped technology is complementation of 11-amino acid
venus (control in Fig. 5B), either with or without coexpression (SmBit) and 18-kDa (LgBit) fragments of the engineered lucif-
of pertussis toxin S1 subunit to uncouple endogenous G␣i/o erase NanoLuc (Nluc) (21). The affinity of SmBit for LgBit is
subunits. In contrast, substantial agonist-dependent BRET sig- low enough that efficient complementation in cells requires
nals were observed when either G␣s or G␣i1 were coexpressed fusion of these fragments to interacting proteins. We explored

7470 J. Biol. Chem. (2018) 293(19) 7466 –7473

 Mini G proteins in cells

Figure 6. Mini G proteins are recruited to active receptors at the Golgi apparatus. A, confocal images of cells expressing cerulean-tagged A1-adenosine
receptors (left) and NES–venus–mGsi before (center) and after (right) stimulation with 100 ␮M adenosine. Some cells retain significant cerulean–A1R in the Golgi
apparatus, and stimulation with adenosine recruits NES–venus–mGsi to this compartment (orange arrowheads) as well as to the plasma membrane (red
arrowhead). Scale bar, 10 ␮m. B, mean NES–venus–mGsi fluorescence (FV, arbitrary units; ⫾ S.E.) at the plasma membrane, Golgi apparatus, and in the cytosol
plotted against time for nine cells similar to those shown in A. Accumulation of NES–venus–mGsi at the Golgi apparatus was delayed ⬃5 s compared with the
plasma membrane. C, BRET between NES–NanoLuc–mGsi and either venus-kras (V-kras) at the plasma membrane or venus-giantin (V-giantin) at the Golgi
apparatus (GA) in response to stimulation of unlabeled A1Rs; mean ⫾ S.D. of three independent experiments. D, BRET between NES-Nluc-mGs and the early
endosome marker venus-rab5 is plotted versus time after stimulation of unlabeled ␤2AR with 10 ␮M isoproterenol; mean ⫾ S.E. of four independent
experiments.

the possibility of using this system to report the agonist-depen- venus–mG constructs. A nuclear export sequence and linker
dent interaction of mG proteins and GPCRs by fusing LgBit and (underlined) (MLQNELALKLAGLDINKTGGSG) was later
SmBit fragments to the N terminus of mG proteins and the C added to the N terminus of venus by QuikChange (Agilent
terminus of ␤2AR. Between the two possible orientations, we Technologies, Santa Clara, CA) PCR insertional mutagenesis to
found that the most efficient complementation took place produce NES–venus–mG constructs. A similar strategy was
when LgBit was fused to mG proteins (e.g. LgBit-mGs), and used to produce NES–Nluc–mG plasmids using the vector
SmBit was fused to the receptor (␤2AR–SmBit). In individual pNluc–C1. LgBit–mG constructs were made by amplifying mG
experiments, agonist stimulation produced as much as a fragments by PCR and subcloning into the vector pBiT1.1-N
20-fold increase in luminescence intensity. As was the case with (Promega Corp., Madison, WI) with XhoI and EcoRI. ␤2AR–
BRET assays, NanoLuc complementation faithfully reported SmBit was made by adding a linker and SmBit (GGSGVTGYR-
full agonist, partial agonist, and inverse agonist activity at LFEEIL) to the C terminus of the ␤2AR using QuikChange PCR.
␤2AR-SmBit (Fig. 7A). This strategy was also applicable to Additional GPCR–SmBit constructs were derived from ␤2AR–
LgBit-mGsi, -mGsq, and -mG12, as all of these fusion proteins SmBit using a BamHI site incorporated into the GGSG linker.
supported NanoLuc complementation with the promiscuous Plasmids encoding G␣ subunits were obtained from cdna.org
endothelin-A receptor, ETAR-SmBit (Fig. 7B). These results (Bloomsburg University, Bloomsburg, PA). A plasmid encoding
suggest that mG proteins should serve as useful vehicles for the S1 subunit of pertussis toxin was kindly provided by Ste-
protein complementation assays. phen R. Ikeda (NIAAA, National Institutes of Health, Rockville,
In summary, although mG proteins originated as G protein MD). Plasmids encoding cerulean–TM–venus, venus– kras,
surrogates for X-ray crystallography, their unique features venus–rab5, venus– giantin, and G␤␥–venus have been de-
present many opportunities for applications other than struc- scribed previously and were used in this study under conditions
tural biology. In cellular assays, mG proteins should be useful that are essentially the same as described previously (11,
for studying the determinants of receptor–G protein-coupling 22–25). Several different GPCR–luciferase constructs were
specificity, biased ligands, and activation of receptors at differ- made by appending either Rluc8 or Nluc directly to the receptor
ent subcellular locations. We expect that many additional uses C terminus either by QuikChange PCR or by subcloning into
will be found for mG proteins in cells and cell extracts and that pRluc8-N1 or pNluc-N1 vectors. GPCR sequences were
mG proteins will prove to be valuable tools for diverse studies of obtained either from cdna.org, as a gift from Jonathan Javitch
GPCR biology. (Columbia University, New York) or as a gift from Bryan Roth
(26) (Addgene, PRESTO-Tango kit 1000000068). All plasmid
Experimental procedures constructs were verified by Sanger sequencing.
Plasmid DNA constructs
Human codon-optimized fragments encoding mG se- Cell culture and transfection
quences (7) were synthesized as gBlocks (Integrated DNA HEK 293 cells (ATCC) were propagated in plastic flasks on
Technologies, Coralville, IA), amplified by PCR, and subcloned 6-well plates and on polylysine-coated glass coverslips accord-
into the vector pVenus-C1 using BglII and EcoRI to produce ing to the supplier’s protocol. HEK 293 cells with targeted dele-

J. Biol. Chem. (2018) 293(19) 7466 –7473 7471

Mini G proteins in cells
BRET, luminescence, and fluorescence measurements
Cells were washed with DPBS, harvested by trituration, and
transferred to opaque black or white 96-well plates containing
diluted drug solutions. For assays with nucleotide-free hetero-
trimers (Fig. 4), cells were washed with permeabilization buffer
containing 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.1 mM
K-EGTA, 20 mM NaHEPES (pH 7.2), harvested by trituration,
and permeabilized in the same buffer containing 10 ␮g ml⫺1
high purity digitonin (EMD Millipore, Burlington, MA). Meas-
urements were made from permeabilized cells supplemented
either with GDP (0.5 mM) or apyrase (2 units ml⫺1; Sigma) and
agonist. BRET and luminescence measurements were made
using a Mithras LB940 photon-counting plate reader (Berthold
Technologies GmbH, Bad Wildbad, Germany). Coelenterazine
h (5 ␮M; Nanolight, Pinetop, AZ) or furimazine (Nano-Glo;
1:1000, Promega Corp.) were added to all wells immediately
prior to making measurements with Rluc8 and Nluc (or Nluc
fragments), respectively. Raw BRET signals were calculated as
the emission intensity at 520 –545 nm divided by the emission
intensity at 475– 495 nm. Net BRET was this ratio minus the
same ratio measured from cells expressing only the BRET
donor. NES–venus–mG fluorescence in Fig. 2 was measured
using a Guava 6HT/2L flow cytometer (excitation 488 nm,
detection 525/30 nm) and reported as average fluorescence
from all positive cells.

Author contributions—Q. W., N. O., and N. A. L. investigation;

Q. W., N. O., A. I., R. N., B. C., and C. G. T. writing-review and edit-
ing; A. I., R. N., B. C., and C. G. T. resources; A. I., C. G. T., and
N. A. L. funding acquisition; N. A. L. supervision; N. A. L. writing-
original draft; N. A. L. project administration.
Figure 7. Split luciferase complementation driven by mGs. A, total lumi-
nescence (normalized) emitted by cells cotransfected with ␤2AR-SmBit and Acknowledgments—We thank Steve Ikeda, Jonathan Javitch, and
LgBit-mGs is plotted versus ligand concentration for isoproterenol (iso), (⫺)-
norepinephrine (nor), alprenolol (alp), and ICI 118,551 (ICI); mean ⫾ S.E. of Bryan Roth for providing plasmid DNA used in this study. We thank
4 –13 independent experiments. B, total luminescence (normalized) emitted Roger Sunahara for permission to use and modify his heterotrimer
by cells cotransfected with ETAR–SmBit and four different LgBit–mG proteins cartoons.
is plotted versus the concentration of endothelin-1 (ET-1); mean ⫾ S.D. of four
independent experiments.

References
tion of GNAS, GNAL, GNAQ, GNA11, GNA12, and GNA13 1. Pierce, K. L., Premont, R. T., and Lefkowitz, R. J. (2002) Seven-transmem-
(⌬Gs/⌬Gq/⌬G12 cells) were derived and authenticated as brane receptors. Nat. Rev. Mol. Cell Biol. 3, 639 – 650 CrossRef Medline
2. Lohse, M. J., Nuber, S., and Hoffmann, C. (2012) Fluorescence/biolumi-
described previously (18). Cells were transiently transfected in
nescence resonance energy transfer techniques to study G-protein-cou-
growth medium using linear polyethyleneimine (Mr 25,000; pled receptor activation and signaling. Pharmacol. Rev. 64, 299 –336
Polysciences Inc., Warrington, PA) at an nitrogen/phosphate CrossRef Medline
ratio of 20 and were used for experiments 12– 48 h later. Up to 3. Barak, L. S., Ferguson, S. S., Zhang, J., and Caron, M. G. (1997) A ␤-arres-
3 ␮g of plasmid DNA was transfected in each well of a 6-well tin/green fluorescent protein biosensor for detecting G protein-coupled
receptor activation. J. Biol. Chem. 272, 27497–27500 CrossRef Medline
plate. 4. Galés, C., Rebois, R. V., Hogue, M., Trieu, P., Breit, A., Hébert, T. E., and
Bouvier, M. (2005) Real-time monitoring of receptor and G-protein inter-
Confocal imaging actions in living cells. Nat. Methods 2, 177–184 CrossRef Medline
5. Hein, P., Frank, M., Hoffmann, C., Lohse, M. J., and Bünemann, M. (2005)
Cells grown on 25-mm round coverslips were transferred to
Dynamics of receptor/G protein coupling in living cells. EMBO J. 24,
an imaging chamber and washed with DPBS. Drug solutions 4106 – 4114 CrossRef Medline
were added directly to the chamber by pipetting. Confocal 6. Carpenter, B., and Tate, C. G. (2016) Engineering a minimal G protein to
images were acquired using a Leica (Wetzlar, Germany) SP8 facilitate crystallisation of G protein-coupled receptors in their active con-
scanning confocal microscope and a ⫻63, 1.4 NA objective. formation. Protein Eng. Des. Sel. 29, 583–594 Medline
7. Nehmé, R., Carpenter, B., Singhal, A., Strege, A., Edwards, P. C., White,
Venus was excited with a 488-nm diode laser and detected at C. F., Du, H., Grisshammer, R., and Tate, C. G. (2017) Mini-G proteins:
500 – 650 nm. Cerulean was excited with a 448-nm diode laser novel tools for studying GPCRs in their active conformation. PLoS ONE
and detected at 460 –520 nm. 12, e0175642 CrossRef Medline

7472 J. Biol. Chem. (2018) 293(19) 7466 –7473

 Mini G proteins in cells
8. Irannejad, R., Tomshine, J. C., Tomshine, J. R., Chevalier, M., Mahoney, 17. Sidhu, A., Sullivan, M., Kohout, T., Balen, P., and Fishman, P. H. (1991) D1
J. P., Steyaert, J., Rasmussen, S. G., Sunahara, R. K., El-Samad, H., Huang, dopamine receptors can interact with both stimulatory and inhibitory
B., and von Zastrow, M. (2013) Conformational biosensors reveal GPCR guanine nucleotide binding proteins. J. Neurochem. 57, 1445–1451
signalling from endosomes. Nature 495, 534 –538 CrossRef Medline CrossRef Medline
9. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, 18. Grundmann, M., Merten, N., Malfacini, D., Inoue, A., Preis, P., Simon, K.,
A. (2002) A variant of yellow fluorescent protein with fast and efficient Rüttiger, N., Ziegler, N., Benkel, T., Schmitt, N. K., Ishida, S., Müller, I.,
maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 Reher, R., Kawakami, K., Inoue, A., et al. (2018) Lack of ␤-arrestin signal-
CrossRef Medline ing in the absence of active G proteins. Nat. Commun. 9, 341 CrossRef
10. Pfleger, K. D., Seeber, R. M., and Eidne, K. A. (2006) Bioluminescence Medline
resonance energy transfer (BRET) for the real-time detection of protein- 19. Irannejad, R., Pessino, V., Mika, D., Huang, B., Wedegaertner, P. B., Conti,
protein interactions. Nat. Protoc. 1, 337–345 CrossRef Medline M., and von Zastrow, M. (2017) Functional selectivity of GPCR-directed
11. Lan, T.-H., Liu, Q., Li, C., Wu, G., and Lambert, N. A. (2012) Sensitive and drug action through location bias. Nat. Chem. Biol. 13, 799 – 806 CrossRef
high resolution localization and tracking of membrane proteins in live Medline
cells with BRET. Traffic 13, 1450 –1456 CrossRef Medline 20. Young, J. D. (2016) The SLC28 (CNT) and SLC29 (ENT) nucleoside trans-
12. De Lean, A., Stadel, J. M., and Lefkowitz, R. J. (1980) A ternary complex
porter families: a 30-year collaborative odyssey. Biochem. Soc. Trans. 44,
model explains the agonist-specific binding properties of the adenylate
869 – 876 CrossRef Medline
cyclase-coupled ␤-adrenergic receptor. J. Biol. Chem. 255, 7108 –7117
21. Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lub-
Medline
ben, T. H., Butler, B. L., Binkowski, B. F., Machleidt, T., Kirkland, T. A.,
13. DeVree, B. T., Mahoney, J. P., Vélez-Ruiz, G. A., Rasmussen, S. G., Kuszak,
Wood, M. G., Eggers, C. T., Encell, L. P., and Wood, K. V. (2016) NanoLuc
A. J., Edwald, E., Fung, J.-J., Manglik, A., Masureel, M., Du, Y., Matt, R. A.,
complementation reporter optimized for accurate measurement of pro-
Pardon, E., Steyaert, J., Kobilka, B. K., and Sunahara, R. K. (2016) Allosteric
tein interactions in cells. ACS Chem. Biol. 11, 400 – 408 CrossRef Medline
coupling from G protein to the agonist-binding pocket in GPCRs. Nature
535, 182–186 CrossRef Medline 22. Hollins, B., Kuravi, S., Digby, G. J., and Lambert, N. A. (2009) The C-ter-
14. Alexander, S. P., Christopoulos, A., Davenport, A. P., Kelly, E., Marrion, minus of GRK3 indicates rapid dissociation of G protein heterotrimers.
N. V., Peters, J. A., Faccenda, E., Harding, S. D., Pawson, A. J., Sharman, Cell. Signal. 21, 1015–1021 CrossRef Medline
J. L., Southan, C., Davies, J. A., and CGTP Collaborators. (2017) The con- 23. Martin, B. R., and Lambert, N. A. (2016) Activated G protein G␣s samples
cise guide to pharmacology 2017/18: G protein-coupled receptors. Br. J. multiple endomembrane compartments. J. Biol. Chem. 291, 20295–20302
Pharmacol. 174, S17–S129 CrossRef Medline CrossRef Medline
15. Staus, D. P., Strachan, R. T., Manglik, A., Pani, B., Kahsai, A. W., Kim, T. H., 24. Lan, T. H., Kuravi, S., and Lambert, N. A. (2011) Internalization dissociates
Wingler, L. M., Ahn, S., Chatterjee, A., Masoudi, A., Kruse, A. C., Pardon, ␤2-adrenergic receptors. PLoS ONE 6, e17361 CrossRef Medline
E., Steyaert, J., Weis, W. I., Prosser, R. S., et al. (2016) Allosteric nanobodies 25. Fonseca, J. M., and Lambert, N. A. (2009) Instability of a class A G protein-
reveal the dynamic range and diverse mechanisms of G-protein-coupled coupled receptor oligomer interface. Mol. Pharmacol. 75, 1296 –1299
receptor activation. Nature 535, 448 – 452 CrossRef Medline CrossRef Medline
16. Schrage, R., Seemann, W. K., Klöckner, J., Dallanoce, C., Racké, K., Koste- 26. Kroeze, W. K., Sassano, M. F., Huang, X.-P., Lansu, K., McCorvy, J. D.,
nis, E., De Amici, M., Holzgrabe, U., and Mohr, K. (2013) Agonists with Giguère, P. M., Sciaky, N., and Roth, B. L. (2015) PRESTO-Tango as
supraphysiological efficacy at the muscarinic M2 ACh receptor. Br. J. an open-source resource for interrogation of the druggable human
Pharmacol. 169, 357–370 CrossRef Medline GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 CrossRef Medline

J. Biol. Chem. (2018) 293(19) 7466 –7473 7473