(Received for publication, March 20, 1995; and in revised form, October 6, 1995)
From the
Type 1 angiotensin receptors (AT) are G-protein
coupled receptors, mediating the physiological actions of the
vasoactive peptide angiotensin II. In this study, the roles of 7 amino
acids of the rat AT
receptor in ligand binding and
signaling were investigated by performing functional assays of
individual receptor mutants expressed in COS and Chinese hamster ovary
cells. Substitutions of polar residues in the third transmembrane
domain with Ala indicate that Ser
, Ser
, and
Ser
are not essential for maintenance of the angiotensin
II binding site. Replacement of Asn
or Ser
does not alter the binding affinity for peptidic analogs, but
modifies the ability of the receptor to interact with AT
(DuP753)- or AT
(CGP42112A)-specific ligands. These 2
residues are probably involved in determining the binding specificity
for these analogs. The absence of G-protein coupling to the Ser
mutant suggests that this residue, in addition to previously
identified residues, Asp
and Tyr
,
participates in the receptor activation mechanism.
Finally,
Lys (third helix) and Lys
(fifth helix)
mutants do not bind angiotensin II or different analogs. Co-expression
of these two deficient receptors permitted the restoration of a normal
binding site. This effect was not due to homologous recombination of
the cDNAs but to protein trans-complementation.
The physiological actions of the vasoactive octapeptide hormone
angiotensin II (AngII) in the cardiovascular, endocrine, and neuronal
systems are mediated by membrane-bond receptors. Two pharmacologically
distinct AngII receptors have been identified: AT and
AT
(1, 2) . AT
receptors bind
biphenylimidazole antagonists such as DuP753 with high affinity and
pentapeptide analogs such as CGP42112A with low affinity, whereas
AT
receptors have the reverse affinities for these
compounds. Cloning of both receptor types has revealed that they belong
to the seven transmembrane domain receptor
family(3, 4, 5, 6) . Two closely
related AT
isoforms (AT
and AT
)
have been identified in rat and mouse species(7, 8) .
AT
receptors have been shown to be coupled to G-proteins
and to activate phospholipase C (PLC)(9, 10) . This
results in inositol trisphosphate (IP
) generation, which
then causes an increase in intracellular calcium concentrations, and
diacylglycerol formation, which leads to protein kinase C
activation(11, 12) .
The molecular location of the
ligand binding domain of the G-protein-coupled receptors has been
intensively investigated using genetic, biochemical, and biophysical
approaches. The binding site for small molecules such as the bioamine
neurotransmitters involves polar residues of the transmembrane domains
(TM)(13) . In contrast, the binding site for large hormones
such as the pituitary glycoproteins is located in the large
amino-terminal extracellular domain of the corresponding
receptors(13) . The ligand binding domains of peptide
receptors, such as AT, have been more recently
investigated. Peptide binding by AT
receptors is dependent
on the presence of four extracellular cysteines (14) as well as
on several additional residues located in the extracellular
domains(15) . Whereas the binding of non-peptidic ligands is
unaffected by these extracellular mutations, numerous polar residues of
the hydrophobic transmembrane segments are determinant in binding of
non-peptidic
antagonists(16, 17, 18, 19) . Two of
these residues, Asp
and Tyr
, also play an
essential role in the coupling of AT
to
PLC(16, 18) .
Biochemical analysis (20) and
molecular modeling studies (21) ()indicate a major
role for the third and fifth transmembrane segments (TM-III and TM-V)
in AngII binding. To define the functional roles of polar residues in
these transmembrane domains, substitutions of different polar residues
into Ala (K102A, S105A, S107A, S109A, N111A, S115A, and K199A) were
therefore created in the rat AT
receptor (Fig. 1).
The pharmacological profiles of the mutated receptors for peptidic and
non-peptidic agonists and antagonists, as well as their signaling
properties were analyzed.
Figure 1:
Bidimensional representation of the rat
AT receptor amino acid sequence. Gray circles indicate the residues replaced by alanine using site-directed
mutagenesis. Black circles indicate the deleted residues. The
amino-terminal sequence Met
-Ala
-Leu
. . . Tyr
-Ile
-Phe
was
replaced by Met-Ala-Cys-Tyr-Ile-Phe. The sequence of the second
extracellular loop, Ile
-His
-Arg
. . . Ser
-Thr
-Leu
was
replaced by Ile-His-Arg-Cys-Ser-Thr-Leu. Numbering is according to (3) .
Some of the mutants were defective for the
binding of AngII and its analogs. Since the mutations were located in
different domains of the AT receptor, we investigated
whether the recently described mechanism of intermolecular
complementation could be observed for this peptide hormone receptor.
This phenomenon was described for different mutants of the
-adrenergic receptors as well of the M
and M
muscarinic receptors when they were
co-expressed, suggesting that molecular association was occurring
between complementary transmembrane domains of two different defective
receptors(22, 23, 24) . The ligand binding
and signaling properties of four binding defective AT
mutants were therefore analyzed after their co-transfections in
COS cells, in different paired combinations.
K102A, S107A, K199A, and
(168-188) were generated in the M13mp19 construct. Four
oligonucleotides were synthesized on a PCR-Mate (Applied Biosystems ):
5`-CAC CTA TGT GCC ATC GCT TCG-3` for replacement of Lys
;
5`-GCT TCG GCC GCC GTG AGC TTC-3` for replacement of Ser
;
5`-GGC CTT ACC GCC AAT ATT CTG-3` for replacement of Lys
;
5`-G CCA GCT GTC ATC CAC CGA TGC TCG ACG CTC CCC ATA GGG CTG-3`
deleting the sequence coding for the second extracellular loop. An
uracil-containing M13mp19-AT
DNA was used as template in
an in vitro mutagenesis reaction using the synthetic
oligonucleotides (Muta-Gene D kit, Bio-Rad). Transformation into a
strain with a functional uracil N-glycosylase allows selection
against the paternal unmutated strand.
The other mutations were
performed by deletion of a restriction fragment and replacement with an
appropriate linker. The (3-25) mutant was obtained by
deleting the 360-base pair HindIII-BspHI fragment
corresponding to the amino-terminal extracellular region of AT
and replacing with double-stranded linker corresponding to sense
oligonucleotide 5`-AG CTT ACC ATG GCC TGC TAC ATA TTT GTC-3`.
The
S105A, S109A, N111A, and S115A mutants were constructed using four
double-stranded linkers corresponding to sense oligonucleotides: 5`-CG
GCC GCT TCC GTG AGC TTC AAC CTC TA-3`, replacing Ser;
5`-CG AGC GCC TCC GTG GCC TTC AAC CTC TA-3`, both replacing Ser
and changing a Eco47III restriction site to a BglI site for screening purpose; 5`-CG TCC GCT TCC GTG AGC TTC
GCC CTC TA-3`, replacing Asn
as well as suppressing a Eco47III restriction site; 5`-C GCG GCC GTG TTC CTT CTC AC-3`,
replacing Ser
. These linkers were inserted, respectively,
in the NruI-MluI (for Ser
,
Ser
, and Asn
) and MluI-PmlI (for Ser
) single restriction
sites of the synthetic cDNA.
The other AT mutants were
constructed using the same strategy. These constructions were performed
in order to tag the amino (Ins[Nter]) and/or carboxyl
terminus (Ins[Cter]) of the AT
coding sequence
and were used here as a control to verify the absence of homologous
recombination between two co-transfected AT
cDNAs. The two
inserted sequences, corresponding to the sense oligonucleotides (FLAG
sequence, SIS, Eastman Kodak Co.): 5`-AG CTT ACC ATG GAC TAC AAA GAC
GAT GCC GAT AAG GCC CTT AAC TCT TC-3` (5` sequence) and 5`-C GAA GTG
GAG GAC GAT GAC GAT AAA GAC TAC AAA GAC GAT GAC GAT AAA TGA CGG ACC
GT-3` (3` sequence) were, respectively, inserted in the HindIII-EagI and BstBI-XbaI
restriction sites of the synthetic cDNA. To produce the construction
Ins[Nter-Cter] containing the double insertion, the 560-base
pair EcoRI-XbaI fragment of Ins[Cter] was
inserted into the expression vector containing HindIII-EcoRI fragment of Ins[Nter].
The
mutated sequences were verified by dideoxy sequencing using Sequenase
version 2 (United States Biochemical Corp.). D74N was constructed as
described previously(16) . All the mutated AT cDNAs were subcloned into the expression vector
pECE(26) .
CHO K1 cells were maintained in Ham's F-12
medium (Boehringer Mannheim) supplemented with 10% fetal calf serum
plus 0.5 mM glutamine, 100 U/ml penicillin and 100 µg/ml
streptomycin. The CHO.AT clone has been previously
described(16, 28) . To establish the CHO.S107A,
CHO.N111A and CHO.S115A clones, CHO cells were co-transfected with 10
µg of the corresponding plasmid and 2 µg of the selection
marker pSV2neo using the calcium phosphate precipitation
method(29) . Transfected cells were selected by their
resistance to 750 µg/ml G418 (Life Technologies, Inc.) and cloned
by limiting dilution.
Figure 4:
Messenger RNA detection by RT-PCR. A, Schematic representation of the Ins[Nter-Cter]
cDNA containing the two insertion sequences. The two rectangles correspond to the inserted heterologous sequences, and the continuous line corresponds to the AT cDNA.
Locations of the PCR primers 1, 2, 3, and 4 are indicated by arrows. B, RT-PCR experiments were performed on mRNAs
isolated from COS cells transfected with Ins[Nter],
Ins[Cter] or Ins[Nter-Cter] or co-transfected with
Ins[Nter] and Ins[Cter]. The PCR product amplified
is a 1-kilobase pair band. Asterisk indicates absence of band
due to specific reverse transcription of cDNAs containing the
carboxyl-terminal insertion.
Two single
point mutants K102A and K199A are unable to bind the peptidic agonists I-[Sar
]AngII (Table 1) and
[
H]AngII, or the non-peptidic antagonist
[
H]DuP753 (data not shown). These data suggest
that these mutations result in a loss of the structural integrity
necessary for peptide and non-peptide binding or that they are not
expressed at the membrane. Latter experiments (see below) show that
co-expression of these two receptor mutants results in normal ligand
binding. It is therefore concluded that these mutants are expressed at
the cell surface and that Lys
and Lys
are
essential for the AT
binding site. Two previous studies
also describe the important role of Lys
and
Lys
, respectively(14, 15) . In the case
of Lys
, it was proposed that its substitution would
provoke an overall alteration in receptor structure in view of its
position at the neighboring disulfide bridge. Our co-expression study
indicates that substitution of this residue does not cause a global
alteration in receptor structure as protein complementation can occur
to produce chimeric receptors, functional in binding AngII. Thus, it
seems likely that Lys
represents an overlapping point in
binding site for peptidic (AngII and [Sar
]AngII)
and non-peptidic ligands (DuP753). A similar argument can be made for
Lys
. Since substitution of this residue with Gln causes a
major decrease in affinity for AngII (14) and substitution with
Ala provokes the complete abolition of AngII and DuP753 binding, we
propose that Lys
is required for ionic interaction with
carboxyl-terminal COOH of AngII as well as with the acidic group of the
tetrazole of DuP753.
Five single point mutants in the TM-III, S105A,
S107A, S109A, N111A, and S115A, recognized I-[Sar
]AngII with K
values similar to those of the wild-type receptor (Table 1). The pharmacology of these five mutants for different
peptidic or non-peptidic ligands was then analyzed (Table 2). All
of these mutants exhibited AngII and
[Sar
,Ala
]AngII binding affinities
that did not markedly differ from those of the wild-type receptor.
However, some differences are observed in the binding affinity of the
pseudopeptidic CGP42112A or non-peptidic DuP753 compounds, specific for
AT
and AT
receptors, respectively, for the
N111A and S115A mutants. N111A showed a significant increase (8-fold)
in affinity for CGP42112A and a significant decrease (45-fold) in
affinity for DuP753. S115A had an affinity for the DuP753 similar to
the wild-type but a significantly increased affinity for CGP42112A
(7-fold).
These results indicate that the polar residues
Ser, Ser
, and Ser
are not
essential for the binding of peptidic or non-peptidic ligand to the
AT
receptor. The absence of a major role for Ser
in the binding of
[Sar
,Ala
]AngII and DuP753 has been
demonstrated previously(17) . This Ser residue is conserved in
mammalian AT
receptors, but it is replaced by an alanine in
the amphibian AngII receptor. Our data suggest that it is not
responsible for the specific pharmacological profile observed for the
amphibian receptor, which recognizes peptidic but not non-peptidic
antagonists. Furthermore, our results demonstrate that this residue is
not a contributor to the binding of either peptidic agonists, or of the
specific AT
/AT
non-peptidic ligands. In
contrast, these studies stress the importance of the residues
Asn
and Ser
in binding the specific
AT
/AT
ligands. These results could be related
to two previous reports; the substitution of Asp
, present
in TM-II, by an asparagine (16) and the substitution of the
Tyr
, present in TM-VII, by a phenylalanine (18) provoke a lower affinity for DuP753 and a higher affinity
for CGP42112A. Despite their locations on different transmembrane
domains, these 4 residues are probably positioned within a small
distance of each other in the plasma membrane, suggesting that
Asp
, Asn
, Ser
, and Tyr
could belong to a specific AT
/AT
ligand
binding site. Molecular modeling studies suggest that these residues
lie in a plane that is three or four
-helical turns below the
membrane surface and therefore buried deep in the lipid bilayer.
The maximal stimulation of IP production (Fig. 2) is similar to the wild-type receptor for S105A and
S109A and corresponds to a 6-fold increase above the basal production
of total IP. These results show a functional coupling for these two
mutants. In contrast, the maximal stimulation of IP production by the
S107A and N111A mutants (Fig. 2) was lower (2-3-fold
increase above the basal production), but these receptors were
expressed at lower levels in COS-7 cells (1.1-1.3
10
sites/cell) than the wild-type or other mutants (4.3 to
6.0
10
sites/cell). Similarly, after stable
expression in CHO cells, the expression level was also reduced by 5- or
6-fold for the S107A and N111A mutants as compared to the wild-type
receptor (Table 1). However, the ratio E
/B
10
sites and the EC
value were similar for the two
mutants as compared to the wild-type receptor (Table 3). These
results indicate that the S107A and N111A mutations do not alter the
ability of the receptor to couple to G-protein, but probably interfere
with the biosynthesis and/or cell surface expression of
AT
.
Figure 2:
AngII-induced stimulation of total
inositol phosphate production in COS cells expressing wild-type or
mutant AT receptors. Dose-response curves were performed
for the wild-type (
), S105A (
), S107A (
), S109A
(
), N111A (
), and S115A (
) AT
receptors. The results are expressed as the ratio of the
[
H]IP fraction (counts/min) derived from cells
after exposure to agonist versus those obtained from cells
exposed to buffer alone. Data points represent the mean ± S.E.
of three independent experiments carried out in
duplicate.
Mutation of Ser results in a dramatic
reduction of the ability of the receptor to mediate AngII-induced IP
formation, despite the fact that this receptor is expressed at similar
levels to those of the wild-type receptor in CHO cells (Table 3).
Therefore, this polar residue in TM-III plays a crucial role in
agonist-induced activation of the AT
receptor, responsible
for G-protein coupling and signal transduction. Two other polar
residues (Asp
and Tyr
) deeply located in the
TM-II and -VII, respectively, have also been shown to be essential for
receptor activation and coupling(16, 18) . According
to a computer model for AT
receptor activation, Marie et al. have proposed that the carboxylate group of Asp
and the hydroxyl group of Tyr
are linked by an
hydrogen bond. Further analysis of the polar residues interaction
within the seven transmembrane segments by computer modeling permits
the development of an hypothetical model for the role of these 3
residues (Asp
, Ser
, and Tyr
)
in receptor activation. In the absence of ligand, Tyr
and
Asp
could be linked by an hydrogen bond. This interaction
could be displaced by binding of AngII or other agonist, resulting in
the formation of a new hydrogen bond between Tyr
and
Ser
, required for the receptor activation and therefore
G-protein coupling. This hypothetical model needs to be confirmed
experimentally.
As the
K102A and K199A mutants, two deletion mutants (3-25) and
(168-188) are unable to bind the peptidic agonists
I-[Sar
]AngII and
[
H]AngII, or the non-peptidic antagonist
[
H]DuP753 (data not shown). Co-expression of all
four AT
mutants in different paired combinations was
performed to determine whether intermolecular complementation could be
observed for this peptide hormone receptor. COS-7 cells co-transfected
with the two deletion mutants
(3-25)/
(168-188)
did not display any specific binding for
I-[Sar
]AngII (data not shown). In
contrast, co-transfection of COS-7 cells with K102A/K199A resulted in
the appearance of specific binding sites for the
I-[Sar
]AngII (Table 4).
Theses sites displayed ligand binding properties similar to those of
the wild-type receptor in terms of K
and
specificities for different ligands (Table 5). Furthermore, the
maximum number of binding sites detected with
I-[Sar
]AngII depends on the
K102A/K199A ratios (Fig. 3).
Figure 3: Effect of co-transfection of varying K102A/K199A DNA ratios on receptor density. Data represent the means ± S.E. obtained from at least two separate experiments in which each point is performed in duplicate. *, p < 0.05 versus 10/40 and 40/10 ratios of K102A/K199A DNA (µg).
These results demonstrate that
the two single-point mutants K102A and K199A are expressed at the cell
surface and are able to complement each other, whereas their individual
expression in COS-7 cells does not result in functional receptors. The
increased number of binding sites is correlated to the increase of the
quantity of the two plasmid DNA, indicating that the appearance of
these binding sites requires the presence of the two deficient mutants
in equimolar amount. This protein complementation results in the
restoration of a binding site similar to that of the wild-type, since
it interacts with peptidic agonists and antagonists as well as with
non-peptidic compounds. In contrast, there was no restoration of a
ligand binding site when the two deletion mutants (3-25) and
(168-188) were co-expressed together, or with either the
K102A or K199A mutants (data not shown).
Co-expression of the K102A/K199A mutants did not result in activation of IP production (data not shown). Similarly, no IP response was detected when co-expression experiments were performed with mutant receptors (D74N and S115A) that are unable to mediate stimulation of IP production, although both have been shown to bind AngII peptides with wild-type affinities ( (16) and the present report, respectively).
Because of the
unexpected and somewhat provocative nature of these results, it was
necessary to verify that no homologous recombination had occurred
between the transfected cDNAs. This specific point was analyzed using
three AT cDNA constructs containing additional
heterologous sequences inserted at either the 5` or 3` end or at both
ends of the coding sequence (Fig. 4A). Individually
expressed, these constructions produce functionally normal AT
receptors (data not shown). Expression and co-expression of these
three constructions in COS cells allowed the analysis of the RNA
populations by RT-PCR using pairs of oligonucleotides located either in
the 5` (primer 1) or 3` (primer 4) insertions or in the 5` or 3` part
of the AT
coding sequence (primers 2 and 3). RT-PCR
products with primers 1 and 4 are obtained from the RNA template
derived from COS cells transfected with the construction
Ins[Nter-Cter] but not obtained with RNA isolated from COS
cells co-transfected with the constructions Ins[Nter] and
Ins[Cter] (Fig. 4B)). Thus, no homologous
recombination events were detected during co-transfection of two
similar constructions. The fact that no functional receptor was
observed when the two deletion mutants (
(3-25) and
(168-188)) were co-expressed is further evidence that
homologous recombination does not occur between co-transfected cDNAs.
These results show that AT receptors may be able to
physically interact with each other to create binding sites similar to
those of the wild-type. These binding sites may account for up to 5% of
the total receptor molecules, assuming that the number of mutant
receptors that reach the cell surface is the same as the wild-type
AT
density in the same experiments (Table 4). The
inability of these ``reconstituted'' receptors to transduce a
signal suggests either that the number of functional molecules is
insufficient for the response to be detected or that the conformation
of these ``chimeric'' molecules is not optimal for G-protein
coupling. Taken together, these experiments strongly suggest that de novo production of an AngII binding receptor from two
functionally defective mutants is secondary to protein-protein
interactions amongst transmembrane domains at the cell surface.
In
conclusion, the present report identified a role for charged residues
located in the external part of TM-III and TM-V (Lys and
Lys
) in AngII binding. Moreover, these residues and
others buried deep in TM-III (Asn
and Ser
)
are involved in the binding site of non-peptidic analogs. In addition
to the Tyr
and Asp
residues, Ser
is required for receptor activation. Finally, co-expression of
these different AT
mutants has permitted the first
demonstration of intermolecular complementation amongst peptide hormone
receptors.