Functional Differences between Full and Partial Agonists ...

0022-3565/01/2973-1218 –1226$3.00 Vol. 297, No. 3
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 3638/907253
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 297:1218–1226, 2001 Printed in U.S.A.

Functional Differences between Full and Partial Agonists:
Evidence for Ligand-Specific Receptor Conformations

ROLAND SEIFERT,1 KATHARINA WENZEL-SEIFERT,1 ULRIK GETHER,2 and BRIAN K. KOBILKA

Howard Hughes Medical Institute (R.S., K.W.-S., U.G., B.K.K.), Division of Cardiovascular Medicine (B.K.K.), Stanford University Medical
School, Stanford, California

Received December 7, 2000; accepted March 8, 2001 This paper is available online at http://jpet.aspetjournals.org

ABSTRACT cle: stabilizing the ternary complex (high-affinity, GTP-sensitive Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016

The interaction of an agonist-bound G-protein-coupled recep- agonist binding), and steady-state GTPase activity. We ob-
tor (GPCR) with its cognate G-protein initiates a sequence of tained results for the wild-type ␤2AR and a constitutively active
experimentally quantifiable changes in both the GPCR and mutant of the ␤2AR (␤2ARCAM) using fusion proteins between
G-protein. These include the release of GDP from G␣, the the GPCRs and Gs␣ to facilitate GPCR/G-protein interactions.
formation of a ternary complex between the nucleotide-free There was no correlation between efficacy of ligands in acti-
G-protein and the GPCR, which has a high affinity for agonist,
followed by the binding of GTP to G␣, the dissociation of the vating GTPase and their ability to stabilize the ternary complex
GPCR/G-protein complex, and the hydrolysis of GTP. The ef- at ␤2ARCAM. Our results suggest that the GPCR state that
ficacy of an agonist is a measure of its ability to activate this optimally promotes the GDP release and GTP binding is differ-
cascade. It has been proposed that efficacy reflects the ability
of the agonist to stabilize the active state of the GPCR. We ent from the GPCR state that stabilizes the ternary complex. By
examined a series of ␤2-adrenoceptor (␤2AR) agonists (weak
partial agonists to full agonists) for their efficacy at promoting strongly stabilizing the ternary complex, certain partial agonists
two different steps of the G-protein activation/deactivation cy-
may reduce the rate of G-protein turnover relative to a full

agonist.

Many hormones and neurotransmitters exert their effects Kobilka, 1992; Seifert et al., 1998b). G-protein deactivation is
through GPCRs that activate heterotrimeric G-proteins and, accomplished by the GTPase activity of G␣ (Gilman, 1987).
thereby, change the activity of effector systems (Gilman,
1987; Kobilka, 1992). Binding of an agonist to a GPCR in- Several models have been developed to conceptualize the
duces distinct conformational changes in the receptor protein as yet incompletely understood mechanisms of GPCR activa-
that enable GPCR to promote GDP release from G␣ (Wess, tion (for a historical perspective, see Kenakin, 1996a;
1997; Gether and Kobilka, 1998). The agonist-occupied Colquhoun, 1998). The ternary complex model of GPCR ac-
GPCR forms a high-affinity ternary complex with guanine tivation states that there is a correlation between the efficacy
nucleotide-free G␣ (Kent et al., 1980; Kobilka, 1992; Seifert et of agonists at stabilizing the ternary complex and at promot-
al., 1998a,b). Upon binding of GTP or the hydrolysis-resis- ing multiple G-protein activation/deactivation cycles (Kent et
tant GTP analog GTP␥S to G␣, the high-affinity ternary al., 1980). The ternary complex model has been elaborated
complex is disrupted, and both G␣ and G␤␥ can regulate the into the extended ternary complex model to explain the find-
activity of effector systems (Kent et al., 1980; Gilman, 1987; ing that GPCRs can activate G-proteins even in the absence
of agonist and that certain receptor ligands, namely, inverse
R.S. and K.W.-S. were the recipients of a research fellowship of the Deut- agonists, suppress G-protein activation by agonist-free
sche Forschungsgemeinschaft. GPCRs (Costa and Herz, 1989; Lefkowitz et al., 1993; Gether
and Kobilka, 1998). The agonist-independent GPCR activity
1 Present address: Department of Pharmacology and Toxicology, The Uni- is referred to as “constitutive activity”. The extended ternary
versity of Kansas, 5064 Malott Hall, Lawrence, KS 66045. complex model assumes that agonists stabilize GPCR in the
active (R*) state, while inverse agonists stabilize the inactive
2 Present address: Department of Cellular Physiology, Institute of Medical (R) state. It has been proposed that for constitutively active
Physiology 12.5, The Panum Institute, University of Copenhagen, Blegdams-
vej 3, DK-2200 Copenhagen N, Denmark.

ABBREVIATIONS: GPCR, G-protein-coupled receptor; GTP␥S, guanosine 5Ј-O-(3-thiotriphosphate); CAM, constitutively active mutant; ␤2AR,
␤2-adrenoceptor; ␤2ARGs␣, fusion protein consisting of the ␤2-adrenoceptor and Gs␣; ␤2ARCAM, constitutively active mutant of the ␤2-adreno-
ceptor; ␤2ARCAMGs␣, fusion protein consisting of the constitutively active mutant of the ␤2-adrenoceptor and Gs␣; DHA, dihydroalprenolol; ISO,
isoproterenol; SAL, salbutamol; DOB, dobutamine; EPH, (Ϫ)-ephedrine; DCI, dichloroisoproterenol; ICI, ICI 118,55 ([erythro-DL-1(7-methylindan-
4-yloxy)-3-isopropylaminobutan-2-ol]).

1218

Multiple Receptor Conformations 1219

mutant (CAM) GPCRs, the equilibrium between R and R* is Construction of ␤2ARCAMGs␣ Fusion Protein DNAs. The fu- Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016
shifted toward R* as a result of diminished structural con- sion proteins used in our present study contained the long splice
straints that control spontaneous R to R* transition. Exper-
imentally, this results in increased agonist affinity and po- variant of Gs␣. The construction of ␤2ARGs␣ DNA was described
tency and increased efficacy of partial agonists at CAM- recently (Seifert et al., 1998a,b). For construction of ␤2ARCAMGs␣
GPCRs compared with wild-type GPCRs (Samama et al., DNA, the KpnI/EcoRV-fragment of the ␤2AR was excised from
1993). In addition, the relative inhibitory effects of inverse pGEM-3Z-␤2ARGs␣ and replaced by the corresponding KpnI/EcoRV-
agonists at CAM-GPCRs are larger than at wild-type GPCRs fragment of ␤2ARCAM DNA. The KpnI/EcoRV fragment of ␤2ARCAM
(Samama et al., 1994; Gether and Kobilka, 1998). DNA differs from the corresponding ␤2AR DNA fragment by muta-
tions that encode for four discrete amino acid substitutions in the
Of interest, an increasing number of experimental results
cannot readily be explained by the extended ternary complex third intracellular loop of the receptor (Samama et al., 1993). The
model. As an example, certain ␤2AR ligands behave like
“protean drugs”, i.e., they act as agonist in one setting, but as ␤2ARCAMGs␣ DNA was cloned into the baculovirus expression vector
inverse agonist in another (Chidiac et al., 1994). Addition- pVL 1392.
ally, the efficacy and potency of ␤2AR agonists is strongly
dependent on the specific purine nucleotide present for G- Cell Culture and Membrane Preparation. Recombinant bacu-
protein activation and the specific G-protein to which the
␤2AR couples (Seifert et al., 1999a; Wenzel-Seifert and Seif- loviruses were generated in Sf9 cells using the BaculoGOLD trans-
ert, 2000). Moreover, the agonist-free and agonist-occupied
␤2AR differ from each other with respect to their ability to fection kit (Pharmingen, San Diego, CA). After initial transfection,
activate different cellular effectors (Zhou et al., 1999). These
and other experimental findings (Keith et al., 1996; Blake et recombinant baculoviruses were isolated by plaque purification.
al., 1997; Krumins and Barber, 1997; Zuscik et al., 1998;
Thomas et al., 2000) could be reconciled by a multistate High-titer virus stocks were generated by three sequential amplifi-
model of GPCR activation in which ligands stabilize unique
and ligand-specific GPCR conformations, which enable cations of plaque-purified virus colonies. Culture of Sf9 cells and
GPCR to modulate its cognate G-protein(s) in a ligand-spe-
cific manner (Kenakin, 1996a,b; Tucek, 1997; Gether and membrane preparation were performed as described recently (Seifert
Kobilka, 1998).
et al., 1998a,b).
The aim of this study was to examine the hypothesis that [3H]DHA Binding. Membranes were thawed and sedimented by
the functional properties of partial agonists are due to their
ability to stabilize distinct conformational states in GPCRs. a 15-min centrifugation at 4°C and 15,000g to remove residual en-
Therefore, we characterized functional differences between
full agonists and partial agonists using fusion proteins be- dogenous guanine nucleotides as far as possible and resuspended in
tween the wild-type ␤2AR and Gs␣ (␤2ARGs␣) and ␤2ARCAM
(Samama et al., 1993; Gether et al., 1997) and Gs␣ (␤2AR- binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl,
CAMGs␣) expressed in Sf9 insect cells. We and others have pH 7.4). Expression levels of fusion proteins were determined by
shown that fusion proteins are a very sensitive experimental
system for studying receptor/G-protein interactions (Seifert incubating Sf9 membranes (15–25 ␮g of protein per tube) in the
et al., 1999b; Milligan, 2000). Because of the defined 1:1 presence of [3H]DHA at a concentration of 10 nM. Nonspecific bind-
stoichiometry of GPCR to G␣, fusion proteins eliminate bias ing was determined in the presence of [3H]DHA (10 nM) plus 10 ␮M
in the analysis of ligand potencies and efficacies caused by (Ϯ)-alprenolol. The total volume of the binding reaction was 500 ␮l.
varying ratios of receptor to G-protein (Hoyer and Boddeke, Incubations were performed for 90 min at 25°C and shaking at 250
1993; Kenakin, 1997). In addition, fusion proteins allow pre-
cise determination of agonist and inverse agonist efficacy in rpm. Competition binding experiments were carried out with Sf9
an expression level-independent manner by measurement of
steady-state GTP hydrolysis and ternary complex formation membranes expressing ␤2ARGs␣ or ␤2ARCAMGs␣ (15– 40 ␮g of pro-
(Seifert et al., 1999b; Milligan, 2000). The results of our tein per tube), 1 nM [3H]DHA and agonists at various concentrations
present study suggest that agonists have different efficacies
with respect to their ability to promote different steps of the with or without GTP␥S (10 ␮M). Ligand-competition studies were
G-protein activation/deactivation cycle and provide evidence performed in triplicates. Bound [3H]DHA was separated from free
for the existence of multiple ligand-specific conformational [3H]DHA by filtration through GF/C filters using a 48-well harvester
GPCR states. Finally, our results suggest that one possible
mechanism for partial agonism is strong stabilization of the (model M-48R; Brandel, Gaithersburg, MD), followed by three
ternary complex, thereby reducing G-protein turnover.
washes with 2 ml of binding buffer (4°C). Filter-bound radioactivity
Experimental Procedures
was determined by liquid scintillation counting using Cytoscint cock-
Materials. The DNA encoding ␤2ARCAM was kindly donated by
Dr. R. J. Lefkowitz (Duke University, Durham, NC) (Samama et al., tail from ICN (Irvine, CA). The experimental conditions chosen en-
1993). Sources of other materials have been described elsewhere sured that not more than 10% of the total amount of [3H]DHA added
(Seifert et al., 1998a,b).
to binding tubes was bound to filters.

Steady-State GTPase Activity. Membranes were thawed and

sedimented by a 15-min centrifugation at 4°C and 15,000g to remove

residual endogenous guanine nucleotides as far as possible and re-

suspended in 10 mM Tris/HCl, pH 7.4. Assay tubes contained Sf9

membranes expressing ␤2ARGs␣ or ␤2ARCAMGs␣ (10 ␮g of protein per
tube), 1.0 mM MgCl2, 0.1 mM EDTA, 0.1 mM ATP, 1 mM adenylyl
imidodiphosphate, 100 nM GTP, 5 mM creatine phosphate, 40 ␮g of
creatine kinase, 0.2% (w/v) bovine serum albumin in 50 mM Tris/

HCl, pH 7.4, and ␤2AR ligands at various concentrations. Reaction
mixtures (80 ␮l) were incubated for 3 min at 25°C before the addition
of 20 ␮l of [␥-32P]GTP (0.2– 0.5 ␮Ci/tube). Reactions were conducted
for 20 min at 25°C. Reactions were terminated by the addition of 900

␮l of a slurry consisting of 5% (w/v) activated charcoal and 50 mM
NaH2PO4, pH 2.0. Charcoal absorbs nucleotides but not Pi. Charcoal-
quenched reaction mixtures were centrifuged for 15 min at room

temperature at 15,000g. Seven hundred microliters of the superna-

tant fluid of reaction mixtures was carefully removed to avoid any
aspiration of charcoal, and 32Pi was determined by liquid scintilla-
tion counting. Enzyme activities were corrected for spontaneous
degradation of [␥-32P]GTP. Spontaneous [␥-32P]GTP degradation
was determined in tubes containing all of the above-described com-

ponents plus a very high concentration of unlabeled GTP (1 mM)
that, by competition with [␥-32P]GTP, prevents [␥-32P]GTP hydroly-
sis by enzymatic activities present in Sf9 membranes. Spontaneous
[␥-32P]GTP degradation was Ͻ1% of the total amount of radioactivity
added. The experimental conditions chosen ensured that not more

1220 Seifert et al.

than 10% of the total amount of [␥-32P]GTP added was converted to
32Pi.

Miscellaneous. Protein was determined using the Bio-Rad DC
protein assay kit (Bio-Rad, Hercules, CA). Immunoblotting studies
were performed as described (Seifert et al., 1998a,b). Ligand compe-
tition curves and concentration-response curves were analyzed by
nonlinear regression, using the Prism program (GraphPad, San Di-
ego, CA).

Results Fig. 1. Effects of (Ϫ)-ISO and ICI on GTPase activity in Sf9 membranes Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016
expressing ␤2ARGs␣ and ␤2ARCAMGs␣. GTPase activity in membranes
Expression of ␤2ARGs␣ and ␤2ARCAMGs␣ in Sf9 Mem- expressing ␤2ARGs␣ (6.5 pmol/mg) (A) or ␤2ARCAMGs␣ (2.5 pmol/mg) (B)
branes. Similar to nonfused ␤2AR and ␤2ARCAM (Gether et was determined as described under Experimental Procedures. Reaction
al., 1997), the maximum expression level of ␤2ARCAMGs␣ in mixtures contained (Ϫ)-ISO or ICI at the concentrations indicated on the
Sf9 cells was ϳ2.5-fold lower than the expression level of abscissa. The dashed lines are extrapolations of basal GTPase activities
␤2ARGs␣ (␤2ARGs␣, 7.2 Ϯ 2.1 pmol/mg; ␤2ARCAMGs␣, 3.2 Ϯ to illustrate the relative contributions of (Ϫ)-ISO and ICI at the ligand-
1.2 pmol/mg). However, the different expression levels of the regulated enzyme activity. To reliably quantitate the inhibitory effect of
ICI on GTPase in membranes expressing ␤2ARGs␣, this construct was
fusion proteins were not of relevance for our studies since the expressed at a higher level than ␤2ARCAMGs␣. Therefore, the absolute
GTPase activities in A are higher than in B. To facilitate comparison of
efficacies of ligands at stabilizing the ternary complex and at the two constructs, the scales of the ordinates in A and B are different.
Data shown are the means Ϯ S.D. of three independent experiments
promoting GTP hydrolysis are expression level-independent performed in duplicate.

(Seifert et al., 1999; Milligan, 2000). Immunoblotting studies Fig. 2. Competition by (Ϫ)-ISO, SAL, and DOB of [3H]DHA binding in Sf9
membranes expressing ␤2ARGs␣ or ␤2ARCAMGs␣: effect of GTP␥S.
with the M1 antibody recognizing the N-terminal FLAG [3H]DHA binding in Sf9 membranes was performed as described under
Experimental Procedures. Reaction mixtures contained Sf9 membranes
epitope of ␤2AR and ␤2ARCAM (Gether et al., 1995, 1997) and (15– 40 ␮g of protein per tube) expressing ␤2ARGs␣ (3.3–7.5 pmol/mg)
anti-Gs␣ Ig confirmed that ␤2ARGs␣ and ␤2ARCAMGs␣ ex- (A–C) or ␤2ARCAMGs␣ (2.5– 4.9 pmol/mg) (D–F), 1 nM [3H]DHA, and
pressed in Sf9 membranes were structurally intact (data not ligands at the concentrations indicated on the abscissa. Reaction mix-
tures additionally contained distilled water (control) or GTP␥S (10 ␮M).
shown). Data shown are the means Ϯ S.D. of four to seven independent experi-
ments performed in triplicate.
Agonist and Inverse Agonist Regulation of Steady-
shift the ICI-competition curve in membranes expressing
State GTPase Activity in Sf9 Membranes Expressing ␤2ARGs␣.

␤2ARGs␣ and ␤2ARCAMGs␣. An important hallmark of con- The ligand binding properties of ␤2ARCAMGs␣ differed con-
stitutive GPCR activation is the increased relative inhibitory siderably from the ligand binding properties of ␤2ARGs␣. The
high-affinity Ki values of (Ϫ)-ISO, (ϩ)-ISO, SAL, and DOB at
effect of inverse agonist at CAM-GPCR compared with wild- ␤2ARCAMGs␣ were about 4 to 10 times lower than at
␤2ARGs␣. While there was no large change in the fraction of
type GPCR (Lefkowitz et al., 1993; Gether and Kobilka, ␤2ARCAM displaying high agonist affinity when liganded to
(Ϫ)-ISO and SAL compared with ␤2ARGs␣, the fraction of
1998). In addition, the agonist-affinity of CAM-GPCRs is receptors displaying high agonist-affinity upon binding of
DOB and (ϩ)-ISO in ␤2ARCAMGs␣ was substantially higher
increased relative to wild type-GPCRs, while the inverse than in ␤2ARGs␣. In contrast to ␤2ARGs␣, where distinct
high-affinity binding sites for EPH and DCI could not be
agonist-affinity is decreased. The full agonist (Ϫ)-ISO was distinguished, highly effective formation of GTP␥S-sensitive
more potent at activating GTP hydrolysis at ␤2ARCAMGs␣
(EC50 ϭ 2.4 Ϯ 1.1 nM) than at ␤2ARGs␣, (EC50 ϭ 13 Ϯ 3 nM),
while the inverse agonist ICI was less potent at ␤2ARCAMGs␣
(IC50 ϭ 8.5 Ϯ 2.5 nM) than at ␤2ARGs␣ (IC50 ϭ 2.8 Ϯ 1.0 nM)
(Fig. 1). For ␤2ARGs␣, 17% of the ligand-regulated GTPase
activity (the difference between maximum (Ϫ)-ISO-stimu-
lated and minimum ICI-inhibited GTP hydrolysis) was at-

tributable to the inverse agonist, while for ␤2ARCAMGs␣, 48%
of the ligand-regulated GTPase activity was attributable to

ICI.

Ligand Binding Properties of ␤2ARGs␣ and ␤2AR-
CAMGs␣. Sf9 membranes expressing ␤2ARGs␣ and ␤2ARCAMGs␣
bound the ␤2AR antagonist [3H]DHA in a monophasic and sat-
urable manner and with similar Kd values (␤2ARGs␣, 0.36 Ϯ
0.03 nM; ␤2ARCAMGs␣, 0.24 Ϯ 0.05 nM). We also studied the
agonist and inverse agonist binding properties of ␤2ARGs␣
and ␤2ARCAMGs␣. The ligand competition curves are shown
in Figs. 2– 4, and Table 1 summarizes the nonlinear regres-

sion analysis of the binding data. In membranes expressing

␤2ARGs␣, strong agonists [(Ϫ)-ISO, (ϩ)-ISO, SAL, and DOB]
efficiently stabilized the ternary complex as is shown by the

GTP␥S-sensitive high-affinity agonist binding. With these
ligands, ϳ40% of the ␤2ARs displayed high agonist affinity.
For the partial agonist EPH, distinct high-affinity binding

sites at ␤2ARGs␣ could not be discriminated, but GTP␥S
could still shift the EPH competition curve ϳ5-fold to the
right. The binding of another partial agonist, DCI, to

␤2ARGs␣, was virtually GTP␥S insensitive. GTP␥S did not

Multiple Receptor Conformations 1221

Fig. 3. Competition by EPH, DCI, and ICI of [3H]DHA binding in Sf9 onist efficacy constitutes a major problem since efficacy is Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016
membranes expressing ␤2ARGs␣ or ␤2ARCAMGs␣: effect of GTP␥S. influenced by numerous variables such as the expression
[3H]DHA binding in Sf9 membranes was performed as described under level of GPCR and the availability of G-protein and effector
Experimental Procedures. Reaction mixtures contained Sf9 membranes system (Hoyer and Boddeke 1993; Kenakin 1996a, 1997). The
(15– 40 ␮g of protein per tube) expressing ␤2ARGs␣ (3.3–7.5 pmol/mg) fusion protein approach offers a unique possibility to assess
(A–C) or ␤2ARCAMGs␣ (2.5– 4.9 pmol/mg) (D–F), 1 nM [3H]DHA, and agonist efficacy in an expression level-independent manner
ligands at the concentrations indicated on the abscissa. Reaction mix- by measuring steady-state GTPase activity (Seifert et al.,
tures additionally contained distilled water (control) or GTP␥S (10 ␮M). 1999; Milligan, 2000). At ␤2ARGs␣, ligands activated GTP
Data shown are the means Ϯ S.D. of four to seven independent experi- hydrolysis in the order of efficacy (Ϫ)-ISO ϳ SAL Ն (ϩ)-
ments performed in triplicate. ISO Ͼ DOB Ͼ EPH Ͼ DCI (Figs. 5 and 6; Table 2). In
accordance with data obtained for effector system activation
Fig. 4. Competition by (Ϫ)-ISO and (ϩ)-ISO of [3H]DHA binding in Sf9 by nonfused ␤2AR and ␤2ARCAM (Samama et al., 1993), the
membranes expressing ␤2ARGs␣ or ␤2ARCAMGs␣: effect of GTP␥S. potencies of agonists for GTPase activation at ␤2ARCAMGs␣
[3H]DHA binding in Sf9 membranes was performed under Experimental were higher than at ␤2ARGs␣. There was a small but signif-
Procedures. Reaction mixtures contained Sf9 membranes (15– 40 ␮g of icant increase in the efficacy of the partial agonists DOB,
protein per tube) expressing ␤2ARGs␣ (3.3–7.5 pmol/mg) (A and B) or EPH, and DCI at activating GTPase in membranes express-
␤2ARCAMGs␣ (2.5– 4.9 pmol/mg) (C and D), 1 nM [3H]DHA, and ligands at ing ␤2ARCAMGs␣ compared with membranes expressing
the concentrations indicated on the abscissa. Reaction mixtures addition- ␤2ARGs␣. For SAL and (ϩ)-ISO, the increase in efficacy at
ally contained distilled water (control) or GTP␥S (10 ␮M). Data shown are ␤2ARCAMGs␣ versus ␤2ARGs␣ was not significant.
the means Ϯ S.D. of four to seven independent experiments performed in
triplicate. Discussion

ternary complexes with EPH and DCI was observed at ␤2AR- Sf9 Cell Membranes Expressing GPCR-G␣ Fusion
CAMGs␣. In agreement with data obtained for nonfused ␤2AR Proteins as Model System for the Analysis of Ligand-
and ␤2ARCAM (Samama et al., 1994), the affinity of ICI for Receptor Interactions. In the present study, we expressed
␤2ARCAMGs␣ was about 2-fold lower than for ␤2ARGs␣. a wild-type GPCR and a CAM-GPCR fused to G␣ in Sf9 cells
GTP␥S increased the affinity of ICI for ␤2ARCAMGs␣ by ϳ2.5- to study ligand-receptor interactions. Compared with con-
fold. ventional coexpression systems, the fusion protein approach
offers several advantages. Specifically, the defined 1:1 stoi-
Efficacies of Partial Agonists on Steady-State GT- chiometry of GPCR and G␣ eliminates bias in the analysis of
Pase Activity in Sf9 Membranes Expressing ␤2ARGs␣ ligand potencies and efficacies because of varying GPCR/G␣
and ␤2ARCAMGs␣. The precise determination of partial ag- ratio (Seifert et al., 199b; Milligan, 2000). In addition, the
physical proximity of GPCR and G␣ ensures efficient inter-
action of the proteins. Fusion proteins allow for the sensitive
analysis of ligand potencies and efficacies in an expression
level-independent manner directly at the G-protein level by
measuring steady-state GTP hydrolysis. This is a very im-
portant point because nonfused ␤2ARCAM expresses at con-
siderably lower levels than ␤2AR (Samama et al., 1993;
Gether et al., 1997), resulting in different GPCR/G-protein
ratios. Finally, for GPCRs mediating adenylyl cyclase activa-
tion such as the ␤2AR, the number of effector molecules
limits signal output (Alousi et al., 1991), introducing addi-
tional bias into the analysis of ligand potencies and efficacies.

GPCR-G␣ fusion proteins are not naturally occurring so
that caution must be exerted when extrapolating conclusions
obtained with fused proteins to the in vivo situation. Perhaps
most evident, in native membranes there is a large excess of
Gs molecules relative to ␤2AR molecules (Ransna¨ s and Insel,
1988). However, the mere excess of G-protein relative to
GPCR does not automatically imply that one GPCR molecule
activates multiple G␣ molecules. Rather, recent data from
various GPCR/G-protein combinations, including the
␤2AR/Gs pair indicate that even in coexpression systems with
a high G-protein to GPCR ratio, GPCRs activate G-proteins
only linearly (Seifert et al., 1998a; Wenzel-Seifert et al., 1999;
Wenzel-Seifert and Seifert, 2000). Thus, GPCR-G␣ fusion
proteins may mimic the close association of GPCRs and G-
proteins in vivo (Seifert et al., 1999b).

Another issue that needs to be considered is the GTP
concentration in our experiments. Ternary complex forma-
tion in membranes was studied in washed membranes, i.e.,

1222 Seifert et al.

TABLE 1

Ligand binding properties of ␤2ARGs␣ and ␤2ARCAMGs␣ expressed in Sf9 membranes: effect of GTP␥S

[3H]DHA binding was determined as described under Experimental Procedures in Sf9 membranes expressing ␤2ARGs␣ (3.3–7.5 pmol/mg) or ␤2ARCAMGs␣ (2.5– 4.9 pmol/mg).
The ligand competition binding curves shown in Figs. 2, 5, and 7 were analyzed by nonlinear regression for best fit to single-site or two-site binding. Kh and Kl designate the
dissociation constants for high- and low-affinity agonist binding, respectively. %Rh indicates the percentage of receptors displaying high agonist affinity. The corresponding
values obtained in the presence of GTP␥S (10 ␮M) are designated KhGTP␥S, KlGTP␥S, and %RhGTP␥S, respectively. Dissociation constants for ICI are listed below Kl and KlGTP␥S,
respectively. Data shown represent the means Ϯ S.D. of four to seven independent experiments performed in triplicate.

Ligand Kh Kl Rh KhGTP␥S KlGTP␥S RhGTP␥S Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016
% %
␤2ARGs␣ 1.0 Ϯ 0.6 nM 2.0 Ϯ 1.0 nM
(Ϫ)-ISO 17 Ϯ 4 44.3 Ϯ 3.8 1.9 Ϯ 0.9 65.5 Ϯ 8.9
(ϩ)-ISO 21 Ϯ 5 100 Ϯ 38 38.9 Ϯ 4.4 190 Ϯ 20 16.3 Ϯ 3.3
SAL 62 Ϯ 23 3400 Ϯ 310 37.7 Ϯ 4.4 4300 Ϯ 500
DOB 2000 Ϯ 450 45.0 Ϯ 5.1 2900 Ϯ 650
EPH 0.2 Ϯ 0.1 2300 Ϯ 600 2800 Ϯ 660
DCI 3.8 Ϯ 1.0 3000 Ϯ 1100 35.4 Ϯ 4.0 15000 Ϯ 2000
ICI 2.2 Ϯ 1.1 56.1 Ϯ 5.3
16 Ϯ 4 96 Ϯ 17 43.6 Ϯ 5.1 140 Ϯ 20
␤2ARCAMGS␣ 32 Ϯ 12 1.7 Ϯ 0.3 80.0 Ϯ 6.8 1.9 Ϯ 0.4
(Ϫ)-ISO 12 Ϯ 4 56.0 Ϯ 8.9
(ϩ)-ISO 3.8 Ϯ 1.3 48.8 Ϯ 4.9 13 Ϯ 2.2
SAL 75 Ϯ 13 135 Ϯ 21
DOB 76 Ϯ 16 160 Ϯ 20
EPH 1400 Ϯ 430 500 Ϯ 170
DCI 1800 Ϯ 400 2900 Ϯ 600
ICI 240 Ϯ 80 470 Ϯ 120
3.4 Ϯ 0.4 1.4 Ϯ 0.4

Fig. 5. Concentration-response curves for the stimulatory effects of (Ϫ)- Fig. 6. Concentration-response curves for the stimulatory effects of (Ϫ)-
ISO, SAL, DOB, EPH, and DCI on steady-state GTPase activity in mem- ISO and (ϩ)-ISO on steady-state GTPase activity in membranes express-
branes expressing ␤2ARGs␣ and ␤2ARCAMGs␣. GTPase activity in mem- ing ␤2ARGs␣ and ␤2ARCAMGs␣. GTPase activity in membranes expressing
branes expressing ␤2ARGs␣ (3.3–7.5 pmol/mg) (A) or ␤2ARCAMGs␣ (2.5– ␤2ARGs␣ (3.3–7.5 pmol/mg) (A) or ␤2ARCAMGs␣ (2.5– 4.9 pmol/mg) (B) was
4.9 pmol/mg) (B) was determined as described under Experimental determined as described under Experimental Procedures. Reaction mix-

Procedures. Reaction mixtures contained ligands at the concentrations tures contained ligands at the concentrations indicated on the abscissa.

indicated on the abscissa. For each construct, the stimulatory effect of For each construct, the stimulatory effect of (Ϫ)-ISO (10 ␮M) on GTP
(Ϫ)-ISO (10 ␮M) on GTP hydrolysis was set 100%, and all data points hydrolysis was set 100%, and all data points were referred to this stim-
were referred to this stimulation. Data shown are the means Ϯ S.D. of
three to six independent experiments performed in duplicate. ulation. Data shown are the means Ϯ S.D. of three to six independent
experiments performed in duplicate.

in the virtual absence of GTP. GTPase studies were con- most enzymes work at substrate concentrations around the
ducted with a substrate concentration of 100 nM (under Km value. These data suggest that even in vivo G-proteins
Experimental Procedures). It could be argued that such stud- may operate under nonsaturating GTP concentrations, al-
ies are not of relevance for the in vivo situation because the lowing for at least some ternary complex formation and effi-
bulk intracellular GTP concentration is in the high micromo- cient regulation of G-protein turnover by modulating the
lar range (Otero, 1990; Jinnah et al., 1993). In addition, GTP concentration in the G-protein vicinity.
adenylyl cyclase assays in membranes have been routinely
performed with GTP concentrations in the high micromolar Can the extended ternary complex model explain
range (Samama et al., 1993; Chidiac et al., 1994; Gether et the functional properties of ␤2ARGs␣ and ␤2AR-
al., 1995). However, the concentration of GTP available to CAMGs␣? Several of our results are in agreement with the
G-proteins in vivo has not been determined. First, the access extended ternary complex model, which proposes that
of GTP to G-proteins is restricted (Otero, 1990; Wieland and GPCRs exist in an equilibrium between an inactive R state
Jakobs, 1992; Klinker et al., 1996). Second, there is a remark- and an active R* state (Lefkowitz et al., 1993; Gether and
able discrepancy between the high intracellular GTP concen- Kobilka 1998). The model proposes that in CAM-GPCRs, R to
tration and the low Km of G-proteins for GTP (ϳ0.1 ␮M) R* isomerization occurs more readily than in wild-type
(Seifert et al., 1998a; Wenzel-Seifert et al., 1999), and in vivo, GPCRs, but there is no fundamental difference in the R and
R* states of CAM-GPCRs. Thus, the model predicts that the

Multiple Receptor Conformations 1223

TABLE 2 well than a fusion protein of the ␤2AR and the long splice
variant of Gs␣ (Seifert et al., 1998a,b). Given the fact that
Efficacies and potencies of ␤2AR ligands at the GTPase of ␤2ARGs␣ and ␤2ARCAM expresses much less well than ␤2AR (see Results;
␤2ARCAMGs␣ Gether et al., 1997), we predicted that the expression levels of

GTPase activity was measured as described under Experimental Procedures in Sf9 a fusion protein of ␤2ARCAM and the short splice variant of
membranes expressing various ␤2ARGs␣ and ␤2ARCAMGs␣. Reaction mixtures con- Gs␣ would be too low for sensitive quantitative analysis of
tained ␤2AR ligands at 0.1 nM–1mM as appropriate to obtain saturated concentra- ternary complex formation and particularly GTPase activa-
tion-response curves. Ligand efficacies and potencies (EC50 values) were obtained by
nonlinear regression analysis of the concentration-response curves shown in Figs. 3 tion.
and 4. The efficacy of (Ϫ)-ISO was set 1.00, and the effects of other ligands are
referred to this effect. Data shown are the means Ϯ S.D. of three to seven indepen- Certain effects of agonists on the functional properties of
dent experiments performed in duplicate. The data shown for ␤2ARGs␣ were previ-
ously reported in Seifert et al. (1999a). Data for ␤2ARGs␣ were compared versus ␤2ARGs␣ and ␤2ARCAMGs␣ are less readily explained by the
␤2ARCAMGs␣ using the t test. extended ternary complex model. Specifically, the extended

Parameter ␤2ARGs␣ ␤2ARCAMGs␣ ternary complex model also predicts that there is a correla-

Efficacy 1.00 1.00 tion between the efficacy of agonists at stabilizing the ter- Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016
(Ϫ)-ISO 0.91 Ϯ 0.03 0.96 Ϯ 0.04n.s.
(ϩ)-ISO 0.95 Ϯ 0.02 0.99 Ϯ 0.03n.s. nary complex and their efficacy at promoting multiple G-
SAL 0.76 Ϯ 0.04 0.82 Ϯ 0.04*
DOB 0.66 Ϯ 0.05 0.76 Ϯ 0.06* protein activation/deactivation cycles. This concept is based
EPH 0.49 Ϯ 0.08 0.58 Ϯ 0.02*
DCI on the observed highly significant correlation between the
13 Ϯ 3 2.4 Ϯ 1.1*
EC50 (nM) 150 Ϯ 15 27 Ϯ 12* efficacy of agonists at stabilizing the ternary complex and the
(Ϫ)-ISO 21 Ϯ 10*
(ϩ)-ISO 93 Ϯ 10 19 Ϯ 9* efficacy of agonists at promoting effector system activation
SAL 90 Ϯ 19 544 Ϯ 150*
DOB 560 Ϯ 180 17 Ϯ 4* (Kent et al., 1980). We studied the outcome of multiple G-
EPH 29 Ϯ 4
DCI protein activation/deactivation cycles more directly by as-

* p Ͻ 0.05; n.s. not significant. sessing steady-state GTPase activity. In agreement with the

relative inhibitory effects of inverse agonists at CAM-GPCRs results of a previous study (Kent et al., 1980), we observed

are higher than at wild type-GPCRs. The increased relative significant correlations between the efficacy of agonists at

inhibitory effect of ICI at ␤2ARCAMGs␣ compared with stabilizing the ternary complex (as determined by the per-
␤2ARGs␣ is in agreement with this model (Fig. 1). The ex-
tended ternary complex model also predicts that the inverse centage of high-affinity agonist-binding sites and the ratio

agonist affinity and potency at CAM-GPCR is lower than at KlGTP␥S/Kh) and the efficacy of agonists at activating GTPase
for ␤2ARGs␣ (Fig. 7, A and C). However, no such correlation
wild-type GPCR (Samama et al., 1994). Again, our findings was observed for ␤2ARCAMGs␣ (Fig. 7, B and D). Most strik-
ingly, at ␤2ARCAMGS␣, EPH, DCI, and particularly DOB
with fusion proteins are in agreement with the model (Figs. exhibited a higher percentage of high-affinity binding sites

1 and 3). Another postulate of the extended ternary complex than (Ϫ)-ISO. In contrast, all of these ligands had lower
efficacies than (Ϫ)-ISO at stimulating GTPase. In addition,
model is that guanine nucleotides increase inverse agonist (Ϫ)-ISO and (ϩ)-ISO, which differ from each other only in the
chirality of the ␤-carbon hydroxyl group of the ethylamine
affinity of GPCR, presumably by uncoupling of GPCR from side chain of the phenyl ring, differed considerably from each

G-protein and, thereby, stabilizing the R state (Barker et al., other in their ability to stabilize the ternary complex at

1994; Leeb-Lundberg et al., 1994). In agreement with this ␤2ARCAMGs␣ (Fig. 4). However, the efficacies of (Ϫ)-ISO and
(ϩ)-ISO at activating GTPase in membranes expressing
postulate, we found an increase in inverse agonist-affinity of ␤2ARCAMGs␣ were similar (Fig. 6; Table 2).

␤2ARCAMGs␣ by GTP␥S (Fig. 3; Table 1). Dissociation in the efficacy of agonists at stabilizing the
An additional prediction of the extended ternary complex
ternary complex and promoting multiple cycles of G-protein
model is that CAM-GPCRs possess a higher agonist affinity
activation/deactivation were observed previously. First,
and potency than wild-type GPCRs (Lefkowitz et al., 1993;
when the ␤2AR couples to either the long or the short splice
Samama et al., 1993). Our data with ␤2ARGs␣ and ␤2AR- variant of Gs␣, SAL and DOB are similarly efficient at sta-
CAMGs␣ show that this property of GPCRs is preserved in bilizing the ternary complex. However, with respect to GT-
fusion proteins (Tables 1 and 2). Moreover, the extended
Pase activation, the two ligands are more effective at the
ternary complex model predicts that the efficacies of partial
␤2AR coupled to the long splice variant of Gs␣ compared with
agonists are higher at CAM-GPCR than at wild-type GPCR the ␤2AR coupled to the short splice variant of Gs␣ (Seifert et
al., 1998b). Second, at the ␤2AR and ␤2ARCAM expressed in
(Samama et al., 1993). This was also the case for ␤2ARGs␣ Chinese hamster ovary cells, DOB is more effective than SAL
and ␤2ARCAMGs␣, but the differences were small (Table 2).
The explanation for these only small differences in agonist at stabilizing the ternary complex, but not at activating ad-

efficacies between the two fusion proteins presumably is that enylyl cyclase (Samama et al., 1993). Third, at certain mu-

we studied coupling of the ␤2AR and ␤2ARCAM to the long tants of the ␤2AR (Hausdorff et al., 1990), histamine H2
splice variant of Gs␣ and that this G-protein confers to the receptor (Smit et al., 1996), prostaglandin EP3 receptor (Irie
wild-type ␤2AR properties of high constitutive activity in et al., 1994), 5-hydroxytryptamine2A receptor (Roth et al.,
terms of partial agonist efficacy (Seifert et al., 1998b). It is 1997), and M4 muscarinic acetylcholine receptor (Van Kop-
pen et al., 1994) the ability of agonist to promote multiple
likely that the differences in partial agonist efficacies be-
G-protein activation/deactivation cycles is severely reduced
tween ␤2AR and ␤2ARCAM would have been more evident if
coupling of these GPCRs to the short splice variant of Gs␣ had compared with their wild-type counterparts, but the ability of
been analyzed. However, we did not construct a fusion pro-
mutant GPCRs to form ternary complexes is normal. Non-
tein of ␤2ARCAM and the short splice variant of Gs␣ because
a fusion protein of the ␤2AR and this G-protein expresses less signaling ternary complexes were also reported for the ␣1B-
adrenergic receptor (Chen et al., 2000), cannabinoid CB1

1224 Seifert et al.

Fig. 8. Relation between the ratio EC50/Kh and efficacy of agonists at Downloaded from jpet.aspetjournals.org at ASPET Journals on February 28, 2016
activating GTPase in Sf9 membranes expressing ␤2ARGs␣ and ␤2ARCAMGs␣.
The ratios of the EC50 for GTPase activation (Figs. 5 and 6; Table 2) and Kh
values (Figs. 2 and 3; Table 1) for various agonists at ␤2ARGs␣ and ␤2AR-
CAMGs␣ were plotted against the respective efficacies of these ligands at
activating GTPase (Figs. 5 and 6; Table 2). Data were analyzed by linear
regression analysis. A, r2 ϭ 0.653; p ϭ 0.192. B, r2 ϭ 0.82; p ϭ 0.014. The
dotted lines indicate the 95% confidence interval of the regression line.

GPCR by a full agonist is less likely to lead to a complete
G-protein activation/deactivation cycle than is GPCR occu-
pancy by a partial agonist. A possible mechanism for this
hypothesis is illustrated in Fig. 9. This model proposes that
the ternary complex for partial agonists is more stable, rel-
ative to the other GPCR and G-protein states (PR, G, GGDP,
GGTP), than is the ternary complex for full agonists, relative

Fig. 7. Relation between the efficacy of agonists at stabilizing the ternary
complex and the ligand efficacy at activating GTPase in Sf9 membranes
expressing ␤2ARGs␣ and ␤2ARCAMGs␣. The efficacies of ligands at stabi-
lizing the ternary complex in membranes expressing ␤2ARGs␣ and ␤2ARCAMGs␣
(Figs. 2–4; Table 1) were plotted against the respective efficacies of these ligands
at activating GTPase (Figs. 5 and 6; Table 2). Data were analyzed by linear
regression analysis. In A and B, ternary complex formation is expressed as the
percentage of receptors displaying high agonist affinity (Rh, %). A, r2 ϭ 0.71; p ϭ
0.035. B, r2 ϭ 0.08; p, ϭ 0.586 (slope not significantly different from zero). In C
and D, ternary complex formation is expressed as the ratio KlGTP␥S/Kh. For EPH
and DCI, distinct Kh values at ␤2ARGs␣ could not be determined. Therefore, we
used the values listed under Kl in Table 1 to calculate the ratio. C, r2 ϭ 0.86; p ϭ
0.007. D, r2 ϭ 0.04; p ϭ 0.716 (slope not significantly different from zero). The
dotted lines indicate the 95% confidence interval of the regression lines.

receptor (Bouaboula et al., 1997), and the MOR-1 opioid Fig. 9. Model of the different effects of full and partial agonists on the
receptor (Brown and Pasternak, 1998). Based on these obser-
vations encompassing multiple GPCRs coupled to G-proteins G-protein activation/deactivation cycle. The arrows symbolize the rela-
from different families it can be concluded that the GPCR
conformation that optimally promotes multiple G-protein ac- tive rate constants for interconversion of the various GPCR and G-protein
tivation/deactivation cycles is distinct from the GPCR confor-
mation that promotes high-affinity interactions between states. G, guanine nucleotide-free G-protein; GGDP, GDP-liganded G-
GPCR and G-protein. These observations suggest that one protein; GGTP, GTP-liganded G-protein; F, full agonist; P, partial agonist;
possible mechanism of partial agonism is stabilization of the R, G-protein-coupled receptor.
ternary complex relative to GTP binding and G-protein dis-
sociation. A highly stable ternary complex would be less
likely to bind GTP, thereby reducing G-protein turnover.

Mechanisms of Action of Full and Partial Agonists.
Of interest, the ratio of EC50 for GTPase activation to Kh for
agonists at ␤2ARCAMGs␣ shows a significant correlation with
the efficacy of agonists at activating GTPase (Fig. 8). The
data obtained for ␤2ARGs␣ show a similar trend as for ␤2AR-
CAMGs␣ but do not reach significance. It is surprising that the
ratio of EC50 to Kh is significantly higher for full agonists
than partial agonists. This suggests that occupancy of a

Multiple Receptor Conformations 1225

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We thank Dr. Terry Kenakin (Glaxo-Wellcome, Research Triangle Kobilka BK (1992) Adrenergic receptors as models for G protein-coupled receptors.
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