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R
GPCR–Ga fusion proteins:
molecular analysis of
receptor–G-protein
coupling
Roland Seifert, Katharina Wenzel-Seifert and
Brian K. Kobilka
The efficiency of interactions between G-proteincoupled receptors (GPCRs) and heterotrimeric guanine
nucleotide-binding proteins (G proteins) is greatly
influenced by the absolute and relative densities of
these proteins in the plasma membrane. The study of
these interactions has been facilitated by the use of
GPCR–Ga fusion proteins, which are formed by the
fusion of GPCR to Ga. These fusion proteins ensure a
defined 1:1 stoichiometry of GPCR to Ga and force the
physical proximity of the signalling partners. Thus,
fusion of GPCR to Ga enhances coupling efficiency can
be used to study aspects of receptor–G-protein coupling
that could not otherwise be examined by co-expressing
GPCRs and G proteins as separate proteins. The results
of studies that have made use of GPCR–Ga fusion
proteins will be discussed in this article, along with the
strengths and limitations of this approach.
G-protein-coupled receptors (GPCRs) interact reversibly
with G proteins to regulate the activity of effector systems.
This review deals with the novel GPCR–Ga fusion protein
technique (see Table 1)1–18. (The reader is also referred to the
many excellent reviews for general information on GPCRs
and G proteins19–22.) The efficiency of GPCR–G-protein
coupling depends on the ratio of GPCRs to G proteins and
on the absolute concentrations of each23. In addition, segregation of receptors and G proteins into different membrane
microdomains constrains signal output24. The precise determination of the efficacy of partial agonists constitutes
a major and yet unresolved pharmacological problem and
is very sensitive to variations in the concentrations of GPCR
and G protein23,25,26. Intriguingly, the efficacy of a given agonist depends on which specific parameter of the G-protein
activation–deactivation cycle is assessed, i.e. an agonist
actually possesses multiple efficacies8,27. From these considerations it is evident that there is a need for a generally
applicable and sensitive system for analysing interactions
between GPCRs and G proteins under defined experimental conditions. However, it is not easy to achieve precisely defined GPCR:G-protein ratios in the mammalian
and insect cell-expression systems that are currently
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available. Moreover, for some G proteins it is difficult to
analyse GPCR–Ga-coupling at the G-protein level, i.e. by
measuring guanosine 59-O-(3-thiotriphosphate) (GTPgS)
binding and GTP hydrolysis.
Basic properties of GPCR–Ga fusion proteins
Construction of fusion protein DNAs and
structural properties of fusion proteins
Fusion proteins are generated by linking the GPCR
C-terminus, which is located intracellularly, to the Nterminus of Ga (Refs 1–6, 9–13, 18). This is achieved by
fusing the open reading frames of the two proteins using
DNA restriction enzyme or polymerase chain reaction
(PCR)-based techniques, or both. Figure 1 illustrates the
two-dimensional topology of GPCR–Ga fusion proteins
in the plasma membrane. In most GPCRs, the second and
third intracellular loops are crucial for G-protein coupling21,28–30, although the first intracellular loop and the Cterminus can also be involved31–33. With respect to Ga, the
extreme C-terminus is essential for receptor coupling28,34.
Thus, the GPCR C-terminus must bend backward to the
membrane and GPCR core in order to allow interaction of
the non-constrained C-terminus of Ga with the cytosolic
domains of the GPCR.
The most salient properties of GPCR–Ga fusion proteins
are: (1) the defined 1:1 stoichiometry of the signalling partners; (2) the close physical proximity of the signalling
partners; and (3) the tight tethering of Ga to the membrane.
The importance of tethering Ga to the membrane for signalling has been documented for both GPCR–Gia and
GPCR–Gsa fusion proteins. Indeed, fusion prevents the
release of Gsa into the cytosol following Gsa activation by
GTP or GTPase-resistant GTP analogues5,7,35–37. Removal
of the acylation sites in Gia renders the G protein cytosolic
and prevents its interaction with the GPCR. However,
tethering of acylation-deficient Gia to the membrane by
means of fusion to the GPCR restores signalling11.
The GPCR moiety of some fusion proteins has been
tagged with epitopes that can be detected with highly sensitive monoclonal antibodies (Fig. 1)4–8. Because the expression level of fusion proteins can be unequivocally
determined by antagonist saturation binding5,9,13, fusion
proteins provide a valuable standard for the precise estimation of expression levels of other fused or non-fused
GPCRs, e.g. orphan GPCRs, that could not be determined
otherwise.
Combination of different GPCRs with different
G-protein a-subunits in fusion proteins
The fusion protein technique has been applied successfully to a number of mammalian GPCRs, i.e. the b2adrenoceptor (b2AR)1–8, a2A-adrenoceptor (a2AAR)9–15,
adenosine A1 receptor16, 5-HT1A receptor17 and a-factor receptor (Ste2) from yeast18. With respect to Ga, the short
(GsaS) and long (GsaL) splice variants of Gsa (Refs 1–8), the
Gi/Go-proteins Gia1, Gia2, Gia3 and Goa1 (Refs 9–17) and
the yeast G-protein Gpa (Ref. 18) have been fused to, and
shown to functionally interact with, GPCR partners.
0165-6147/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(99)01368-1
TiPS – September 1999 (Vol. 20)
R. Seifert,
Associate Professor,
Department of
Pharmacology and
Toxicology,
The University of
Kansas, 5001 Malott
Hall, Lawrence,
KS 66045, USA,
Email:
[email protected].
ukans.edu
K. Wenzel-Seifert,
Research Associate,
Higuchi Biosciences
Center,
The University of
Kansas, 5003 Malott
Hall, Lawrence,
KS 66045, USA,
Email:
[email protected].
ukans.edu
and
B. K. Kobilka,
Associate
Investigator,
Howard Hughes
Medical Institute,
Stanford University
Medical School,
CA 94305, USA.
Email: Kobilka@cmgr.
stanford.edu
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Table 1. Summary of GPCR–Ga fusion protein studies
GPCR
Ga
Structural properties of
fusion protein
Expression system
Most important findings of study
Refs
b2AR
GsaS
S49 cyc– lymphoma cells
GsaS
b2AR
GsaS
See Ref. 1
b2AR
GsaS or GsaL N-terminally, the b2AR is
FLAG epitope-tagged.
C-terminally, the receptor
is His6-tagged
Sf9 insect cells
b2AR
GsaL
See Ref. 4
Sf9 insect cells
b2AR
GsaL
Sf9 insect cells
b2AR
GsaL
b2AR
GsaL
See Ref. 4. In b2AR(D26)–
GsaL and b2AR(D70)–
GsaL, 26 and 70 residues,
respectively, of the b2AR
C-terminus were deleted
See Ref. 4. Between the
b2AR and GsaL, a
thrombin cleavage site
(TS) was introduced.
Thrombin can cleave
~70% of the b2AR–
TS–GsaL molecules in
membranes
See Ref. 4
a2AAR
Gia1
COS7 cells
a2AAR
Gia1
A PTX-resistant mutant of
Gia1 (C351G)Gia1 was
used
See Ref. 9
a2AAR
Gia1
b2AR–Gsa fusion protein is more efficient than non-fused
b2AR expressed in S49 wild-type cells at supporting
ternary complex formation and activating AC
b2AR–Gsa-expressing cells are resistant to homologous
desensitization. Agonist treatment of b2AR–Gsa-expressing
tumour cells inhibits their proliferation in the cell culture
Injection of b2AR–Gsa-expressing tumour cells into syngeneic
mice protects the animals against growth of the respective
wild-type tumour cells
The b2AR coupled to GsaL, but not the b2AR coupled to GsaS
possesses the properties of a constitutively active GPCR.
These differences can be explained by the lower GDPaffinity of GsaL compared to the GDP-affinity of GsaS, i.e.
GsaL is more often GDP-free than GsaS and, therefore, more
often available to stabilize the active R* state of the GPCR
GPCR–Ga coupling in a b2AR–GsaL fusion protein is much
more efficient than in a system consisting of non-fused b2AR
plus a large molar excess of GsaL as assessed by ternary
complex formation, GTPgS binding and GTPase- and AC
activity. The fusion allowed calculation of the agonistregulated GTP turnover of Gsa
GPCR–Ga coupling is unimpaired in b2AR(D26)–GsaL and
b2AR(D70)–GsaL as assessed by ternary complex formation
and GTPgS binding. However, deletions impair the GTPase
activity of Gsa and enhance the ability of the fusion protein
to activate AC
Non-cleaved b2AR–TS–GsaL ensures efficient GPCR–Ga
coupling as does b2AR–GsaL. Upon thrombin cleavage,
ternary complex formation and GTPgS binding are preserved,
whereas AC activation and GTP hydrolysis are impaired. Thus,
there is pre-coupling of the b2AR and cleaved GsaL, allowing
for one round of the G-protein cycle. Thereafter, GsaL
dissociates form the receptor, with subsequently impaired
coupling
There are differences in the efficacies of guanine-, inosineand xanthine nucleotides at disrupting the ternary complex
and at activating AC. These differences can be explained
by differences in the kinetics of nucleotide interaction with
GsaL and/or the stabilization of different active states of GsaL.
The purine nucleotides could be used as experimental probes
to unmask the existence of multiple active GPCR states
The a2AAR–(C351G)–Gia1 fusion protein is efficient at
supporting agonist-stimulated GTP hydrolysis and can be
used to calculate GTP turnover of the fused G protein
The a2AAR–(C351G)Gia1 fusion protein can be used to
determine the efficacies of partial agonists in an expression
level-independent manner by measuring GTPase activity
Acylation-deficient fused Gia1 does not interact with coexpressed a2AAR because of the cytosolic localization of
the mutant Gia1. However, fusion of acylation-deficient
Gia1 mutants to the a2AAR restores GPCR–Ga-coupling
by tethering the G protein to the membrane
1
b2AR
Deletion of the 5
C-terminal amino
acids of the receptor
See Ref. 1
a2AAR
Gia1
a2AAR
Gia1
384
Myristoylation-deficient
PTX-resistant Gia1,
palmitoylation-deficient
Gia1 and combined
acylation-deficient Gia1
was used
See Ref. 9
See Ref. 9. PTX-insensitive
(C351G)Gia1 mutant is
compared with wild-type
Gia1
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S49 cyc– lymphoma cells;
carB carcinoma cells
carB carcinoma cells;
C57/PDV tumour cells
Sf9 insect cells
Sf9 insect cells
COS7 cells
COS7 cells
Rat 1 fibroblasts
COS7 cells
2
3
4
5
6
7
8
9
10
11
Unlike in COS7 cells, there is substantial cross-talk
12
between the fused a2AAR and endogenous Gi proteins in
Rat 1 cells as assessed by PTX-sensitive GTP hydrolysis
mediated via the PTX-insensitive fusion protein. Crosstalk does not occur with endogenous Gs proteins
The maximum agonist-stimulated GTP turnover of
13
(C351G)Gia1 is more than 50% lower than the GTP turnover
of wild-type Gia1. Agonist stimulates the GTPase of wild-type
Gia1 ~tenfold more potently than the GTPase of (C351G)Gia1
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Table 1. (cont.)
GPCR
Ga
Structural properties of
fusion protein
Expression system
a2AAR
Gia1
See Ref. 9
Rat 1 fibroblasts
a2AAR
Gia1
A1R
Gia1-3 and
Goa1
5-HT1AR
Gao1
Ste2
Gpa1
Most important findings of study
Refs
The paper extends the observations made in Ref. 12, i.e. the
a2AAR fused to (C351G)Gia1 can couple to endogenous Gi
proteins to mediate AC inhibition. However, the fused a2A
AR, unlike non-fused a2AAR cannot couple to endogenous Gs
proteins. These findings can be explained by blockade of the
Gs-interaction sites of the a2AAR or differential
compartmentalization of Gi- and Gs proteins
See Ref. 9. The PTXCOS7 cells
Compared to the originally described a2AAR–(C351G)Gia1
insensitive (C351G)Gia1
fusion protein, partial agonists have higher efficacies for
and (C351I)Gia1 mutants
the a2AAR–(C351I)Gia1 fusion protein. These data show that
are compared with wildhydrophobicity of the C-terminus of Ga plays a key role in
type Gia1
mediating efficient GPCR–Ga coupling
PTX-resistant mutants of
HEK293 cells
The A1AR couples more efficiently to fused Gi- and GoGia1–3 and Goa1 were
proteins than to co-expressed Gi and Go proteins. There is no
used
evidence for preferential interaction of the A1AR with any
of the G proteins studied
PTX-resistant mutants of
COS7 cells
The efficacy of the agonist 8-hydroxy-2-(di-nGoa1 were used
propylamino)tetralin depends on the hydrophobicity of the
amino acid at position 351 of Goa1
The 62 C-terminal amino
Gpa1- and Ste2/Gpa1Ste2-Gpa1 is efficient at transducing signals in the
acids of Ste2 were deleted.
deficient Saccharomyces Ste2/Gpa1-deficient cells. Chimeric Gpa1–Gsa restores
In one fusion protein, the C- cerevisiae
signal transduction in Gpa1-deficient cells only when fused
terminal portion of Gpa1 was
to Ste2. Thus, the function of the G-protein C-terminus is
replaced by the corresponding
mainly to bring Gpa1 in close vicinity to Ste2
domain of mammalian Gsa
14
15
16
17
18
Abbreviations: a2AAR, a2A-adrenoceptor; A1R, adenosine A1 receptor; AC, adenylate cyclase; b2AR, b2-adrenoceptor; (C351G)Gia1 and (C351I)Gia1, pertussis toxin-insensitive mutants
of the subtype 1 of the inhibitory G-protein a-subunit of adenylate cyclase; Ga, non-specified G-protein a-subunit; Gia1–3, subtypes 1–3 of the inhibitory G-protein a-subunit of
adenylate cyclase; Goa1, a-subunit of a G-protein abundant in brain and neuroendocrine cells; Gpa1, G-protein a-subunit from Saccharomyces cerevisiae; GsaL, long splice variant
of the stimulatory G-protein a-subunit of adenylate cyclase; GsaS, short splice variant of the stimulatory G-protein a-subunit of adenylate cyclase; GTPgS, guanosine 59-O-(3-thiotriphosphate); 5-HT1AR, 5-HT1A receptor; PTX, pertussis toxin; Ste2, a-factor receptor of Saccharomyces cerevisiae; ternary complex, complex of the agonist-occupied receptor and
nucleotide-free G-protein heterotrimer which possesses a high agonist affinity.
These data show that the fusion protein approach can be
applied to many GPCRs and G-protein a-subunits.
Because of the defined stoichiometry of signalling partners, fusion proteins are attractive systems to compare the
coupling of a given GPCR to various G-protein a-subunits.
The A1 receptor couples equally well to Gia1, Gia2, Gia3 and
Gao1 (Ref. 16). This non-specificity in coupling is not an
artefact of the fusion as similar data were obtained with
non-fused A1 receptor co-expressed with the different
Gi/Go-proteins16. Unexpectedly, marked differences have
been observed for the coupling of the b2AR to GsaS and
GsaL. Specifically, the b2AR fused to GsaL shows the properties of constitutive activity, whereas the b2AR fused to
GsaS does not1,4,5. These differences can be explained by the
fact that GsaL is more often guanine nucleotide-free than
GsaS and is, therefore, more often available to stabilize the
active (R*) state of the b2AR (Refs 4, 38). Future studies
using non-fused proteins expressed at precisely defined
stoichiometries are required to determine whether the
differences in the coupling of the b2AR to Gsa splice variants in the fused state are of physiological relevance.
Guanine nucleotide exchange and effector coupling
Because of the 1:1 stoichiometry of GPCR to Ga in fusion
proteins, GPCR antagonist saturation binding not only
provides an exact measure of GPCR-expression level but
also of Ga expression level. This unique property of fusion
proteins has allowed, for the first time, measurements of
GPCR-regulated molar GTP turnover by a G protein in a
membrane system5,9,13. Previously, such measurements
could only be performed using systems consisting of purified and lipid vesicle-reconstituted proteins39,40. The GTP
turnover numbers and Km values for GTP hydrolysis obtained with fusion proteins agree favourably with the values obtained with purified proteins in reconstituted systems5,9,13,39,40. The fixed GPCR:Ga stoichiometry has also
been applied successfully to the determination of the efficacies of partial agonists and inverse agonists in an expression level-independent manner by measuring steady-state
GTPase activity4,8,10. In addition, GPCR–Ga fusion proteins
are efficient at promoting GTPgS binding4,10,17. However,
compared to the kinetics of GTP hydrolysis4,8,10, the kinetics of GTPgS binding to fusion proteins have so far
been incompletely characterized4,10,17.
Although GPCRs can efficiently interact with their fused
Ga partner1,4,5,10,17, this is not necessarily true for the
effector-coupling of fused Ga. Fused Gsa efficiently activates adenylate cyclase and the yeast G protein Gpa1
activates downstream signalling1,4–6,18, but fused Gia fails
to regulate downstream effectors12,14. Possibly, fusion of
TiPS – September 1999 (Vol. 20)
385
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FLAG epitope
N
Acylation sites
GPCR
b
N
Plasma
membrane
g
C
C
His6 tag
Ga
trends in Pharmacological Sciences
Fig. 1. Assumed two-dimensional topology of GPCR–Ga fusion proteins in
the plasma membrane. The GPCR portion of the fusion protein is shown in
red, the G-protein a-subunit in purple. N and C in red and purple designate
the locations of the N- and C-termini of GPCR and Ga, respectively. GPCRs
are C-terminally palmitoylated and G-protein a-subunits are N-terminally
myristoylated or palmitoylated, or both. Acylation tethers the proteins to the
membrane. G-protein bg-subunits (blue) can interact with GPCR–Ga fusion
proteins, but it is unknown whether bg-subunits are required for fusionprotein function. The FLAG- and His6-epitopes allow immunological detection of fusion proteins with monoclonal antibodies. Abbreviations: Ga, asubunit of a non-specified heterotrimeric guanine nucleotide-binding
protein; GPCR, G-protein-coupled receptor.
Gia to a GPCR shields the effector-regulating domains of
the G protein.
GPCR–Gsa fusion proteins
Evidence for highly efficient coupling
Strosberg’s group1 was the first to construct and express
a GPCR–Ga fusion protein. In their seminal paper, Bertin
et al.1 showed that a fusion protein of the b2AR and GsaS
was more efficient at stabilizing high-affinity agonistbinding and stimulating adenylate cyclase when expressed in Gsa-deficient S49 cyc2 lymphoma cells than
non-fused b2AR expressed in S49 wild-type cells. These
data were tantalizing in view of the fact that, in S49 wildtype cells, there is an ~100-fold molar excess of Gsa relative to b2AR (Ref. 41), whereas in the fusion protein, there
is only a 1:1 stoichiometry of the signalling partners.
Another interesting property of the b2AR–Gsa fusion
protein is its resistance to homologous desensitization,
which may endow fusion proteins with the capacity for
efficient, long-term signalling2,3.
Fusion proteins of the b2AR and GsaL and GsaS, respectively, have been expressed in Sf9 insect cells4,5. In Sf9
cells, b2AR-Gsa can be expressed at levels up to ten times
higher than in S49 cyc2 cells. Moreover, coupling of the
non-fused b2AR to endogenous Gsa-like G proteins of
insect cells is poor and, therefore, offers an excellent
background for studying GPCR–Gsa fusion proteins4,5.
There is no evidence for cross-talk of the fused b2AR to
Gsa-like G proteins of the insect cells4,5,8 and no evidence
386
TiPS – September 1999 (Vol. 20)
for alteration of the functional properties of the fused
b2AR and Gsa.
The adenylate cyclase and agonist-binding data obtained
by Bertin et al.1 have been confirmed by Seifert and colleagues. Moreover, they found that there is highly efficient
ligand-regulation of GTPgS binding and GTPase activity
in Sf9 membranes expressing b2AR-GsaL (Ref. 5). In contrast, detection of ligand-regulation of GTPase and GTPgS
binding by non-fused b2AR and Gsa can be very difficult
and could require special techniques such as immunoprecipitation of activated G proteins or elimination of the
activity of more abundant G proteins5,42–44.
Further insight into the functional role of the tether between the receptor and Gsa was obtained from a fusion protein with a thrombin cleavage site between the b2AR and
GsaL (b2AR-TS-GsaL). Thrombin-cleavage of b2AR-TS-GsaL
did not alter ternary complex formation and GTPgS binding but strongly reduced adenylate cyclase- and GTPase
activation7. Thus, proteolytic cleavage of the link between
receptor and G protein did not interfere with non-covalent
interactions that were originally promoted by the fusion.
However, adenylate cyclase- and GTPase activation required repeated cycles of interaction between receptor and
G protein, and are, therefore, very sensitive to proteolytic
disruption of the tether between GPCR and Gs.
Comparison of b2AR–Gs-coupling in non-fused
and fused systems
In typical systems expressing non-fused b2AR and Gsa,
there is a large molar excess of G protein relative to GPCR
but, nonetheless, the b2AR interacts with only a small
fraction of the available Gsa molecules (Fig. 2a)5,41. This
interaction is, however, sufficient to transfer a considerable fraction of the b2ARs into a state of high agonistaffinity and to induce significant adenylate cyclase activation. Because only few Gsa molecules are engaged in
coupling, the extent of agonist-stimulated GDP–GTP exchange relative to the basal activity of the much larger
pool of unstimulated G proteins (of all classes) might not
be sufficient to permit the use of GTPase- and GTPgS binding assays to monitor GPCR–G-protein interactions. Of
particular importance is the fact that, once Gsa is activated,
it can dissociate from the membrane into the cytosol35–37.
Thus, the b2AR has first to find a new Gsa partner before
being able to activate another G-protein cycle. Dissociation
of Gsa from the membrane can also explain the poor efficiency of cleaved b2AR-TS-GsaL at activating adenylate
cyclase and GTPase7.
Fusion ensures close physical proximity of the coupling
partners and induces pre-coupling (Fig. 2b). Pre-coupling
is preserved upon thrombin cleavage of b2AR-TS-GsaL
and allows the fusion protein to undergo one G-protein
cycle7. Because of the tether in b2AR-Gsa, the majority if not
all Gsa molecules are engaged in coupling, giving rise not
only to efficient ternary complex formation, but also to
efficient stimulation of GDP–GTP exchange as assessed
by activation of GTPgS binding and GTPase. Thus, at any
given time, more G-protein cycles occur in the fused sys-
R
tem than in membranes expressing b2AR and Gsa as separate proteins, despite much lower absolute Gsa levels in
membranes expressing b2AR-Gsa. Accordingly, b2AR-Gsa
activates adenylate cyclase more efficiently than the b2AR
and Gsa expressed as separate proteins. The comparison
of b2AR/Gsa interactions in the fused, cleaved and nonfused state demonstrates the importance of physical proximity of GPCR and Ga for their efficient coupling and
indicates that release of Gsa from the membrane limits
maximal output in the signalling cascade. Moreover, the
fusion of b2AR to Gsa allows for analysis of the steps of the
G-protein cycle that previously were readily accessible
only in experiments with purified proteins39.
a
What limits high-affinity agonist binding?
One of the earliest steps in GPCR–G-protein coupling
is the formation of the ternary complex, but the factors
that determine its formation and its functional importance
are only incompletely understood27,47,48. In the visual system, ternary complex formation is stable as long as the G
protein remains guanine nucleotide-free48. Because fusion
proteins promote highly efficient GPCR–G-protein coupling and because each GPCR has its own G-protein
partner (Figs 1 and 2b), one might have expected that at
equilibrium, virtually all fusion protein molecules should
accumulate in the state of high agonist-affinity. This, however, is not the case, neither for b2AR-Gsa nor A1R-Gia
(Refs 1, 2, 4, 5, 16). There are several possible explanations
for the incomplete ternary complex formation in GPCR–Ga
fusion proteins. Unlike ternary complex formation of
rhodopsin with transducin, ternary complex formation
of the b2AR–Gs and A1R–Gi/Go fusion proteins might
be instable. It is also possible that even multiple wash
procedures of membranes cannot efficiently remove all
guanine nucleotide endogenously bound to Ga. The difficulty in completely removing tightly bound guanine
nucleotides from G proteins in membranes is well documented49. Finally, it is possible that not all of the fused
Ga is functional7.
Another puzzling observation is that the b2AR ligands
(2)-ephedrine and dichloroisoproterenol are only poorly
effective with regard to ternary complex formation at
b2AR–Gsa but these ligands are, nonetheless, strong partial agonists with respect to GTPase- and adenylate cyclase
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b2AR + Gsa
W
b2AR–Gsa
ISO
ISO
1
1
AC
AC
as
as
as
as
GDP
GDP
GDP
GDP
GTP
GTP
2
2
ISO
G-protein bg-subunits
An elusive issue is the role of G-protein bg-complexes in
fusion-protein function. Although it is clear that GPCR–Ga
fusion proteins can interact with bg-subunits (Fig. 1)1,5,9, it
is not clear whether bg-complexes are required for fusionprotein function. Indeed, ternary complex formation in
Sf9 membranes expressing various non-fused GPCRs and
G-protein a-subunits is enhanced by mammalian bgcomplexes5,45,46, but in membranes expressing b2AR-GsaL,
mammalian b1g2-complex is without effect5. A possible interpretation of these results is that the role of bg-subunits
is to induce optimal positioning of Ga relative to GPCR
and that the fusion mimics exactly that bg-function.
E
ISO
ISO
AC
AC
as
as
as
as
GTP
GDP
GDP
GTP
ISO
GDP
3
GDP
3
AC
as
AC
as
as
GDP
GDP
as
GDP
Pi
GDP
Pi
trends in Pharmacological Sciences
Fig. 2. b2-adrenoceptor (b2AR)–Gsa-adenylate cyclase interactions with non-fused b2AR plus
Gsa and with b2AR–Gsa fusion protein. a: Signalling in a system consisting of non-fused b2AR
plus Gsa. 1. There is a vast excess of Gsa relative to b2AR. Note that for the sake of clarity, only
few Gsa molecules are shown here. Most of the Gsa molecules do not participate in coupling to
the b2AR and are ‘dormant’. However, the b2AR efficiently interacts with a minority of the expressed
Gsa molecules to form a ternary complex and to catalyse GDP–GTP exchange. 2. The GTP-liganded
Gsa molecules efficiently activate adenylate cyclase. 3. The ability of Gsa to activate adenylate
cyclase is abrogated by the intrinsic GTPase activity of Gsa and Gsa dissociation from the membrane.
Therefore, the b2AR has first to find a new Gsa molecule before being able to promote another
cycle of Gsa- and adenylate cyclase activation. The search for a new Gsa partner requires a certain
time and, thereby, reduces signal efficiency. Since only few Gsa molecules are active, it is difficult
to detect GDP–GTP exchange events. b: Signalling with b2AR–Gsa fusion protein. 1. Fusion induces
pre-coupling so that virtually all of the expressed Gsa molecules can couple to the fused b2AR partner. 2. Fusion of Gsa to the b2AR ensures efficient coupling of the G protein to adenylate cyclase.
3. At this step, there is an important difference between the fused and non-fused signalling system. While fusion allows for G-protein deactivation by GTP hydrolysis to take place, fused Gsa
cannot dissociate from the membrane but rather swings back to the b2AR and is immediately
available for another G-protein cycle. Since in the fused system more G proteins participate in
cycling than in the non-fused systems and because cycling of any given fused Gsa occurs more
often than of non-fused Gsa, GDP–GTP exchange events, i.e. GTPgS binding and GTPase activation
can readily be detected with b2AR–Gsa. Abbreviations: AC, adenylate cyclase; as, a-subunit of the
stimulatory G protein of adenylate cyclase; ISO, isoproterenol.
activation4,8. These findings indicate that the efficiency of
ternary complex formation neither predicts nor limits the
efficiency of GDP–GTP exchange. Thus, certain partial agonists must be particularly efficient at promoting downstream steps of the G-protein cycle, i.e. GTP binding and,
possibly, GTP hydrolysis8.
GPCR–Gia/Goa fusion proteins
Structural properties and applications
The a2A-AR, A1R and 5-HT1A receptor were fused to
pertussis toxin (PTX)-resistant mutants of various Gia/Goa
proteins and expressed in mammalian cell lines (COS-7,
Rat 1, HEK293)9–13,15–17. The mutations in Gia/Goa proteins were introduced to differentiate between the PTXsensitive GTPase activity of endogenous Gi-proteins of
the host cells and the PTX-insensitive GTPase activity of
the fusion protein9,10,12,13,15–17. However, the introduction
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of a point mutation at the extreme C-terminus of Gia/Goaproteins is problematic. Specifically, such a mutation reduces agonist potency by ~tenfold and agonist-stimulated
GTP turnover by more than 50% (Ref. 13). Also, the hydrophobicity of the amino acid substituted for the natural
cysteine has a profound impact on the efficacy of partial
agonists15,17. These data support the idea that the structure of the C-terminus of G-protein a-subunits is crucial
for the coupling of G proteins to GPCRs (Refs 28, 34).
GPCR–Gia/Goa fusion proteins have been used predominantly to determine the kinetics of GTP hydrolysis
and the efficacies of agonists by measuring GTP hydrolysis or GTPgS binding9,10,12,13,15–17. Receptor–G-protein coupling in GPCR–Gia/Goa fusion proteins is more efficient
than in co-expression systems16, although the difference
between fused and non-fused systems is much less pronounced than for b2AR/Gs-coupling4,5. An explanation
for the different properties of GPCR–Gsa and GPCR–Gia
fusion proteins versus their respective co-expression systems could be the fact that Gia, unlike Gsa, is not released
from the membrane following activation11.
Cross-talk of GPCR–Gia/Goa fusion proteins with
G proteins of the host cell
When expressed in COS7 cells and HEK293 cells, the
fused GPCR interacts predominantly or exclusively with its
fused Gia/Goa partner as assessed by the PTX-resistance
of the fusion protein function9,13,16,17. In marked contrast,
the fused GPCR interacts very efficiently with endogenous
Gi-proteins when expressed in Rat 1 fibroblasts as shown
by the substantial inhibitory effect of PTX on agoniststimulated GTPase or agonist-inhibited adenylate cyclase
in membranes expressing a fusion protein of the a2AAR
and a PTX resistant form of Gia1 (Refs 12, 14). The cell
type-dependent cross-talk of fused GPCRs to endogenous Gi-proteins is poorly understood, but it could reflect
the relative abundance of Gi proteins and receptors in
different cells types.
Cross-talk with endogenous G proteins and the fact
that mutations in the extreme C-terminus of Gia/Goaproteins have profound effects on GPCR–G-protein coupling call for systems devoid of these drawbacks. Based
on the lack of expression of mammalian-type Gi-proteins,
the poor coupling of Gi/Go-protein-linked GPCRs to endogenous insect G-proteins and the highly efficient coupling upon co-expression of GPCRs with mammalian
Gi/Go-proteins45,46,50,51, Sf9 cells should provide an excellent system for analysing GPCR–Gia/Goa fusion proteins
without the need to introduce mutations in the C-terminus
of Gia/Goa. Moreover, the downregulation of endogenous G proteins in Sf9 cells during the infection with baculovirus should minimize the problems with cross-talk52.
Role of the length of the GPCR C-terminus for
fusion-protein function
In fusion proteins, the GPCR C-terminus serves as
tether between the GPCR core and Ga. The length of the
C-terminus of different GPCRs is extremely variable53,54.
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The C-termini of the a2AAR and b2AR comprise 25 and 72
amino acids, respectively55,56. This marked difference in
the length of the C-terminus could therefore have a significant impact on GPCR–G protein- and G protein–effector
coupling in fusion proteins. To address this question, the
properties of fusion proteins in which 26 [b2AR(D26)–GsaL]
or 70 [b2AR(D70)–GsaL] residues of the b2AR C-terminus
had been deleted were examined. An important prerequisite for these studies was the previous finding that the
b2AR C-terminus per se is not important for coupling to
Gsa (Ref. 56). Deletions in the b2AR C-terminus left intact
ternary complex formation and GTPgS binding, which
indicates that, despite the shortened tether, GPCR and
Ga can still adopt the correct orientation with each other.
However, deletions strongly reduced steady-state GTP
hydrolysis, which suggests that tight tethering of Gsa to
b2AR induces a conformational change in the G protein,
which slows down the deactivation step of the G-protein
cycle. There is additional experimental evidence for
regulation of Gs-deactivation by the b2AR. As a result of
slower GTP hydrolysis, the time that GsaL stays in the
active GTP-liganded form is prolonged. Accordingly,
b2AR(D26)–GsaL and b2AR(D70)–GsaL are more efficient
than b2AR–GsaL at activating adenylate cyclase6.
An important implication of these data is that it is not
necessary for Gsa to diffuse away from the b2AR to reach
an adenylate cyclase molecule. Rather, it appears that the
signalling proteins are packed together very closely, perhaps as a quaternary complex between agonist, b2AR, Gsa
and adenylate cyclase. The existence of organized supramolecular signalling complexes was already proposed in
earlier studies57,58. Thus, although fusion of a GPCR to
Ga is artificial, it may nonetheless represent a model for
a highly rigid and compartmentalized signalling system
in vivo6.
The data obtained with b2AR(D26)–GsaL and
b2AR(D70)–GsaL also have implications for future fusionprotein studies. Even GPCRs with an extremely short Cterminus54 can presumably be used to generate a functional fusion protein, but what the maximum allowable
length of a GPCR C-terminus is, is currently unknown.
The finding that adenylate cyclase- and GTPase activation
in uncleaved b2AR-TS–GsaL (which has an additional 27
amino acids in the linker) is considerably weaker than in
b2AR-GsaL could be related to the higher mobility of Gsa
in b2AR-TS–GsaL as compared to b2AR–GsaL (Refs 5, 7).
Additionally, caution should be excercised when fusion
protein studies are aimed at comparing the ability of two
different GPCRs to couple to a given G protein. One can
presumably only directly compare those receptors that
have the same or a very similar length of the C-terminus.
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edited by Sir John Vane and
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£50.00 (hardback) (x + 204 pages)
ISBN 0 953403904
With a potential market of close to
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B
Clinical Significance
and Potential of
Selective COX-2
Inhibitors
I
only eight years for the first highly
selective COX-2 inhibitor to be introduced to the market. What are often
termed as COX-2-preferential inhibitors (e.g. etodolac, nimesulide) actually pre-date the confirmation of the
existence of this isoform of prostaglandin synthase. The essence of the
COX-2 theory is that this isoform
is responsible for prostaglandin
synthesis at sites of inflammation,
whereas COX-1 is responsible for
prostaglandin synthesis in the context of homeostatic functions. The
latter would include gastrointestinal
mucosal defence, platelet aggregation and renal blood flow. In
recent years, a few holes have been
punched in the COX-2 hypothesis
but, generally speaking, it has survived the tests of time and clinical
trials. The ultimate test of this
hypothesis is now under way. Celecoxib (Monsanto) was introduced to
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Acknowledgements
When working at
Stanford University,
K. Wenzel-Seifert and
R. Seifert were supported
by a research fellowship
of the Deutsche
Forschungsgemeinschaft.
The authors would like to
thank Drs E. Sanders-Bush,
V. T. Lam, U. Gether and
T. W. Lee for their
collaboration in the fusion
protein project. The
authors are also thankful
to Drs M. L. Michaelis,
E. K. Michaelis and
R. Dobrowsky (Department
of Pharmacology and
Toxicology, The University
of Kansas) and the
reviewers of the
manuscript for many
helpful suggestions.
K
the US market in the early part of this
year and Vioxx (Merck) is the second
highly selective COX-2 inhibitor to
reach the consumer.
Vane and Botting’s recent volume
entitled Clinical Significance and
Potential of Selective COX-2 Inhibitors
consists of contributions from speakers at two conferences on this topic
held in late 1997 and early 1998. It provides a very good overview of some
key elements of the COX-2 hypothesis. For example, the potential role
of COX-2 in diseases such as arthritis, Alzheimer’s disease and colon
cancer are very well reviewed. The
roles of COX-2 versus COX-1 in the
gastrointestinal tract and kidney are
also covered in some detail. Indeed,
a weakness of the book is that four
chapters on the subject of NSAIDinduced gastrointestinal damage is
probably three too many, particularly
when these chapters do not devote
0165-6147/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(99)01354-1
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