(Received for publication, August 14, 1996, and in revised form, October 21, 1996)
From INSERM U.401, CCIPE, 141 rue de la Cardonille,
34094 Montpellier Cedex 05, France and the ¶ Laboratoire de Chimie
Théorique, Université de Nancy I, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France
A preliminary model of the rat AT1A
angiotensin II (AII) receptor (Joseph, M. P., Maigret, B., Bonnafous
J.-C., Marie, J., and Scheraga, H. A. (1995) J. Protein
Chem. 14, 381-398) has predicted an interaction between
Asn111 located in transmembrane domain (TM) III and
Tyr292 (TM VII) in the nonactivated receptor; a disruption
of this interaction upon AII activation would allow Tyr292
to interact with the conserved Asp74 (TM II). The previous
verification that Tyr292 is essential for receptor coupling
to phospholipase C (Marie, J., Maigret, B., Joseph, M. P., Larguier,
R., Nouet, S., Lombard, C., and Bonnafous, J.-C. (1994) J. Biol. Chem. 269, 20815-20818) prompted us to check the possible
alterations in receptor properties upon Asn111 Ala
mutation. The mutated receptor (N111A) displayed: (i) strong constitutive activity, with amplification of the maximal phospholipase C response to AII; (ii) agonist behavior of the
AT2-specific ligand CGP 42112A,
[Sar1,Ile8]AII, and
[Sar1,Ala8]AII, antagonists of the wild-type
receptor; (iii) inverse agonism behavior of the non-peptide ligands DuP
753, LF 7-0156, and LF 8-0129. The results are discussed in the light
of the allosteric ternary complex models and other described examples
of constitutive activation of G protein-coupled receptors.
Constitutive activation of G protein-coupled receptors, initially
reported by Cotecchia et al. (1) for the
1-adrenergic receptor, is now well documented and has
been extended to many other members of this large family (2-21).
Moreover, mutations inducing receptor constitutive activation have been
found to be associated with human diseases (13-16). Data on the
2-adrenergic receptors have prompted their authors to
propose an extended version of the ternary complex model, based on the
existence of active and inactive receptor states (4, 22). The same
mechanistic model, either in its initial form (22) or in a refined
version (23), have provided interpretations for the "negative
antagonism" or "inverse agonism" phenomenon, evidenced for
ligands of
2-adrenergic (24, 25), B2
bradykinin (26), m5 muscarinic (21), and thyrotropin-releasing hormone
(18) receptors. As emphasized by the recent work of Cotecchia's group
on the
1B-adrenergic receptor (8), a correlation between
mechanistic considerations and molecular events associated to receptor
conformational changes is obviously required. The work reported in the
present paper is based on a previous molecular modeling study (27)
which aimed at predicting modifications of specific amino acid side
chain interactions during the process of the angiotensin II type 1 (AT1)1 receptor activation.
This preliminary model (27) postulated that an interaction between
Asn111 (TM III) and Tyr292 (TM VII) might exist
in the non-activated receptor and that activation by AII would induce
its disruption to allow Tyr292 to interact with the
conserved Asp74 (TM II). Previous experimental work from
this laboratory is consistent with an essential role of a
Tyr292-Asp74 interaction in the transduction
mechanism (28). The model prompted us to check whether the absence of
Asn111 would favor this interaction, leading to
constitutive receptor activity. In this paper we demonstrate that the
N111A mutant receptor displays a strong constitutive activity as well
as striking pharmacological changes. The results are discussed in the
light of above-mentioned current models (22, 23).
Reagents
AII, [Sar1]AII, [Sar1,Ile8]AII, and [Sar1,Ala8]AII were purchased from Bachem (Budendorf, Switzerland). CGP 42112A was provided by Drs. M. De Gasparo and S. Bottari (Ciba Geigy, Basel, Switzerland). Non-peptide antagonists LF 7-0156, LF 8-0129, and DUP 753 were synthesized by Fournier Laboratories (Daix, France). AII, [Sar1]AII, and CGP 42112A were radioiodinated as described previously in Refs. 29 and 30, respectively. myo-[2-3H]Inositol was from DuPont NEN. COS-7 cells were from the European Cell Type Collection.
Site-directed Mutagenesis and Expression
The amino acid mutation Asn111 Ala was carried
out as described previously (28). The cDNA sequences of the wild
type and N111A mutant rat AT1A receptors were subcloned in
the XbaI site of the polylinker of the eukaryotic expression
vector pCMV (31). Receptors were transiently expressed in COS-7 cells
by using the electroporation transfection method: 107 cells
were resuspended in 300 µl of electroporation buffer (50 mM K2HPO4, 20 mM
CH3COOK, 20 mM KOH, pH 7.40) and incubated for 10 min at room temperature in an electroporation cuvette (0.4-cm electrode gap, Bio-Rad) with 20 µg of pCMV carrier and different amounts of pCMV containing cDNA receptors sequences (30-300 ng range). They were submitted to an electric discharge (950 microfarads, 280 V, 50 ms), then cultured for 2 days at 37 °C in Dulbecco's modified Eagle's medium, 4.5 g/liter glucose, 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin before binding or IP
accumulation experiments.
Binding Assays
Plasma MembraneCrude membranes from COS-7 cells
transiently expressing the wild type or mutant AT1A
receptors were prepared according to Ref. 28. Protein concentrations
were measured as described in Ref. 32. 125I-AII or
125I-CGP 42112A binding assays were performed as described
previously (33). In some experiments involving GTPS treatment, the
membranes were washed three times with binding buffer (50 mM Na2HPO4, 5 mM
MgCl2, 1 mg/ml bovine serum albumin, 1 mg/ml bacitracin, pH 7.40) supplemented with 200 mM NaCl, incubated for 10 min
at 30 °C in the presence or absence of 100 µM GTP
S
before binding experiments carried out in the same medium.
125I-[Sar1]AII binding to transfected COS-7 cells grown in 6-well tissue culture clusters (5 × 105 cells/well) was effected as in Ref. 28.
Inositol Phosphate Assays
COS-7 cells expressing the wild type or N111A mutant receptors were grown in 6-well tissue culture clusters and labeled for 24 h with [2-3H]inositol (1.5 ml/well, 1 µCi/ml) in minimum essential medium deprived of serum and unlabeled inositol. Before stimulation, cells were incubated at 37 °C for 1 h in Dulbecco's phosphate-buffered saline (pH 7.40). After a 15-min LiCl treatment, cells were incubated in the presence or absence of peptide ligands for 15 min at 37 °C in Dulbecco's PBS supplemented with 10 mM LiCl, 1 mg/ml bovine serum albumin, and 1 mg/ml bacitracin. LiCl was omitted in short-time [Sar1]AII stimulation experiments (15 and 30 s). For experiments involving non-peptide ligands, cells were incubated, after the 1-h PBS incubation, in the presence or absence of non-peptide compounds for 30 min at 37 °C in Dulbecco's PBS supplemented with 10 mM LiCl and 0.1 mg/ml bacitracin without LiCl pretreatment. Pooled inositol phosphates (IPs) were extracted and measured as described previously (28, 34).
The detailed analysis of basal inositol phosphate
production, in the presence of Li+ ions, by the WT or N111A
receptors transiently expressed in COS-7 cells revealed strong
constitutive activation of the N111A mutant AT1A receptor
(Fig. 1A). A significant difference between
basal IP production by the WT and the N111A mutant receptors was
detected for expression levels of about 105 sites/cell.
This constitutive activation might be overlooked in the cases of lower
expression levels. In this respect, the electroporation technique is
well adapted to the provision of homogeneous cell transfection over a
wide range of expression levels. The maximal extent of
[Sar1]AII stimulation of IP production was greatly
potentiated in the N111A mutant receptor (Fig. 1B). This
potentiation was evident at stimulation times as short as 15 s
(Fig. 1C), which indicates that it resulted from an
intrinsic activation property of the receptor rather than
desensitization or sequestration phenomena. In addition, these
short-time experiments carried out in the absence of Li+
ions showed that the steady-state levels of IPs are similar in the WT
and mutant receptor (Fig. 1C, t = 0), the
constitutive activation requiring the addition of IP1
phosphatase inhibitors. The Kact value for the
N111A mutant receptor was not significantly altered: 2.88 ± 1.03 nM versus 1.03 ± 0.40 nM for
the WT receptor (Fig. 2) which is consistent with
Kd values for
[125I-Sar1]AII binding (1.1 ± 0.2 nM and 0.76 ± 0.10 nM for N111A and WT receptors, respectively) (35). In order to dissect the changes in
receptor coupling properties upon Asn111 Ala
replacement, we checked the effect of GTP
S, a non-hydrolyzable analog of GTP, on the affinities of the WT and mutant receptor for
125I-AII. The expected decreased in WT receptor affinity
(increase in Kd value from 1.34 ± 0.23 nM to 4.31 ± 0.65 nM) was no longer
observed for the constitutively active mutant receptor (Kd values: 2.12 ± 0.36 nM and
1.84 ± 0.11 nM in the absence and presence of
GTP
S, respectively) (Fig. 3). These data concerning
the comparison of binding affinities and Kact
values for the WT and N111A receptors (which contrast with other
examples of constitutive activation of G protein-coupled receptors (1, 3-5, 7, 18, 21)) and the effects of GTP
S treatment on agonist
binding will be discussed later in the light of published mechanistic
models (22, 23).
Changes in Pharmacological Properties of the AT1A Receptor upon Asn111
We noted
earlier (35) that the N111A mutant receptor displayed an increased
affinity for the AT2-specific ligand CGP 42112A. Direct
binding of 125I-CGP 42112A to membranes from COS-7 cells
expressing the N111A mutant receptor led to a Kd
value of 96 nM, in agreement with a previously determined
Ki value (35). Moreover, CGP 42112A displayed a
marked partial agonist activity for the N111A mutant receptor (maximal
response: 27% of the response to [Sar1]AII) (Fig.
4A) with a Kact value
of 42 nM (Fig. 4C). The other peptidic compounds
[Sar1,Ile8]AII and
[Sar1,Ala8]AII, which possess unchanged
affinities for the mutant receptor (35) and are devoid of agonist
activity for the recombinant WT receptor, also behaved as partial
agonists for the N111A mutant (Fig. 4A) (maximal activities:
52% and 27%, respectively, of the response induced by
[Sar1]AII). We previously demonstrated that
Asn111 Ala replacement induced a strong decrease in
receptor affinity for all tested non-peptide ligands: DUP 753, LF
7-0156, LF 8-0129 (35). In spite of these reduced affinities, we could
check the ability of these ligands to modulate the IP responses of the
WT and N111A mutant receptors. While the activity of the WT receptor was not significantly affected, a marked decrease in IP production was
observed upon treatment with the three non-peptide ligands (Fig.
4B). These inverse agonism properties were characterized by
Kact values of 2.4 µM, 0.40 µM, and 0.38 µM for DUP 753, LF 7-0156, and
LF 8-0129, respectively (Fig. 4C).
The data reported in this paper demonstrate striking changes in the properties of the rat AT1A receptor induced by Ala replacement of the Asn111 residue, located in the third transmembrane domain: constitutive activation of basal IP production and amplification of the response to angiotensin II; induction of inverse agonism properties of the non-peptidic ligands DUP 753, LF 7-0156, and LF 8-0129, evidenced in spite of their greatly decreased affinities for the mutated receptor (35); induction of partial agonist properties of the AT2-specific peptide ligand CGP 42112A, associated with an increased affinity, as well as [Sar1,Ile8]AII and [Sar1,Ala8]AII derivatives which are antagonists of the wild-type receptor.
We tried to correlate these observations with mechanistic
interpretations of the "allosteric ternary complex" models
previously proposed by Lefkowitz's group (22) and discussed and
refined by Kenakin (23) (Fig. 5). Classical simulation
curves (not shown) (4) easily demonstrated that the various changes
associated with Asn111 Ala replacement are
thermodynamically consistent with multiple combined variations of the
parameters describing the equilibria between the involved entities.
Appropriate choices of these parameters can explain: the constitutive
activation associated with an increase in maximal response to AII,
without significant changes in Kd and
Kact values; the lack of GTP
S effect on the
affinity of the mutant receptor for AII which is a function of the
R
R* isomerization and the affinity of R* for G; the above-mentioned
pharmacological changes, the latter providing verification that the
parameters
and/or
and or
(Fig. 5) have been affected by the
mutation.
As a consequence of the variations of one or several of these parameters, the Kd values relative to the hormone recognition by mutated receptor may vary in unpredictable directions. Previous interpretations of decreases in Kd values for mutated receptors displaying constitutive activity were based on the assumption that these parameters were unaffected by the mutations (22).
However, the model depicted in Fig. 5 is oversimplified, as earlier
discussed (23) and as emphasized by the recent evidence for the
existence of multiple activation states of the -1B adrenergic receptor (7). Obviously, more precise interpretations would imply the
experimental availability of the subtle conformational changes involved
in receptor activation processes.
In spite of the obvious limitations of models based on the structure of
bacteriorhodopsin, the preliminary model of the AT1A receptor activation (27), which prompted us to investigate a possible
role of Asn111, constitutes a first step in the prediction
of these conformational changes: AII receptor activation would cause a
disruption of an Asn111-Tyr292 interaction,
allowing Tyr292 to interact with Asp74 (Fig.
6). Recent experiments from our laboratory support a
possible role of Tyr292-Asp74 interaction in
receptor activation (28). The results are consistent with the
existence, in the wild-type receptor, of an intramolecular bond between
Asn111 and another residue; the absence of this interaction
in the N111A mutant receptor could favor conformational flexibility and
constitutive activation (Fig. 6). Although the model (27) suggests that
Tyr292 is a likely candidate to interact with
Asn111, it is difficult at the present time to exclude the
possibility that one (or several) other amino acid(s) might act as (a)
relay(s) in the cascade of conformational changes leading to receptor
coupling to a specific G protein.
Many examples of constitutive activity of G protein-coupled receptors
have been reported. It appeared possible to induce constitutive activation of numerous members of this family by mutation of amino acids located at various receptor positions (1-21). The problem of
molecular event conservation in activation processes of GPCR is raised.
It can be envisaged that some of these events are conserved, while
others are specific to some receptors or some subfamilies. A recent
work on the -1B receptor has emphasized the role of conserved polar
amino acids in receptor activation, the mutation of some of these
residues leading to constitutively active receptors. The dissection of
conformational changes underlying receptor activation and the
prediction of the molecular nature of receptor-activated states have
been attempted through the use of molecular dynamics (8). The
Asn111 residue of the AT1A receptor, the
mutation of which induces constitutive activation, is found at
homologous positions in other peptide hormone receptors:
AT2 and Xenopus angiotensin receptors,
bradykinin, opiod, interleukin 8, and somatostatin receptors for
instance. It is noticeable that mutation of Cys128 (7) in
the
-1B adrenergic receptor, which occupies a position homologous to
that of Asn111 in the AT1A receptor, also
induces constitutive activation. Tyr292 of the
AT1A receptor, which is postulated to interact with
Asn111 in the non-activated receptor (27), is not conserved
in all GPCR. More direct proof for
Asn111-Tyr292 interaction are obviously
required, even if the lack of tyrosine conservation is not necessarily
inconsistent with the model. Nevertheless, in the absence of high
resolution crystallographic data for GPCR, mutagenesis experiments
which lead to constitutive activation can support hypotheses about
receptor stabilization through interaction between specific residues
and their rearrangements upon hormonal activation. Data about rhodopsin
(10, 11) suggest that disruption of a critical salt bridge between
Lys296 (TM VII) and Glu113 (TM III) induces
constitutive activation. They provide insight into structural features
inasmuch as they can provide information on the relative spatial
positions of the transmembrane
helices and interactions between
them.