![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 54, Issue 2, 427-434, August 1998
Department of Physiology, Semmelweis University of Medicine, H-1088 Budapest, Hungary (L.H., Z.G., B.M.), Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510 (H.J., M.Z., K.J.C.), and Division of Cardio-Renal Drug Products, Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20857 (G.J.)
 |
Summary |
|---|
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1 angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1,Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.
| |
Introduction |
|---|
|
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The
AT1 angiotensin receptor is a GPCR that mediates
the physiological actions of the octapeptide pressor hormone Ang II
(Spät et al., 1991
; Ganguly and Davis, 1994;
Griendling et al., 1996). GPCRs share a common basic
structure of seven transmembrane helices connected by alternating
intracellular and extracellular loops. All members of this receptor
family couple to specific G proteins that mediate the activation of
several plasma membrane effector systems (Probst et al.,
1992; Strader et al., 1994). AT1
receptors, which occur as highly homologous and functionally similar
AT1a and AT1b subtypes in
rodents, are primarily coupled through the Gq/11
group of G proteins to stimulation of phospholipase C activity and
Ins(1, 4, 5)P3-induced Ca2+
signal generation. This is usually accompanied by activation of protein
kinase C, mediated by increased diacylglycerol production in
concert with the agonist-evoked intracellular
Ca2+ signal (Spät et al., 1991;
Ganguly and Davis, 1994; Griendling et al., 1996). The
molecular mechanisms by which activated GPCRs are coupled to signal
generation are currently the focus of intensive studies investigating
their structure-function relationships.
In many peptide hormone receptors, the cognate physiological ligands
bind to a site formed by the extracellular loops and the outermost
regions of the transmembrane helices (Strader et al.,
1995; Hunyady et al., 1996). The conformational changes
evoked by agonist binding are transmitted by the transmembrane helices to the intracellular loops, which are believed to comprise the regions
that couple the receptor to its intracellular signaling systems. The
current structural information about GPCRs is based on low-resolution
maps of bovine and frog rhodopsins (Schertler et al., 1993;
Schertler and Hargrave, 1995). These studies, together with recent data
obtained by electron cryomicroscopy of crystals of frog rhodopsin
(Unger et al., 1997), have confirmed the seven-transmembrane helix nature of GPCRs and have provided information about the positioning of the helices but have not elucidated the structural changes that are involved in the mechanism of activation of these receptors. For this reason, several theoretical models of the three-dimensional conformation of the AT1
receptor and other GPCRs have been constructed (Baldwin, 1993; Donnelly
et al., 1994; Joseph et al., 1995; Underwood
et al., 1995; Yamano et al., 1995; Inoue et
al., 1997). Although predictions based on such studies have been
helpful in defining the binding sites of several GPCRs, the manner in
which agonist binding changes the conformation of the receptor molecule
has not been clarified.
Pharmacological studies have identified two major classes of nonpeptide
compounds that bind selectively to angiotensin
AT1 and AT2 receptors
(Griendling et al., 1996). Structure-function studies have
shown that the binding site of such nonpeptide antagonists is located
within the intramembrane binding pocket, with contributions from
several residues located in the transmembrane helices (Ji et
al., 1994; Hunyady et al., 1996; Inoue et
al., 1997; Karnik et al., 1997). More recent studies on
nonpeptide analogs have led to the identification of compounds that act
as partial agonists of the AT1 receptor, causing
increased blood pressure in vivo and stimulation of signal
transduction in vitro (Perlman et al., 1995;
Kivlighn et al., 1996). Mutational studies focusing
primarily on the extracellular regions have failed to identify residues that contribute to the binding of nonpeptide agonists (Perlman et
al., 1995). However, the nonpeptide agonists and antagonists are
closely related chemically, and it is likely that some of the residues
that participate in the binding of nonpeptide antagonists also
contribute to the binding of nonpeptide agonists.
The seventh transmembrane helix has been suggested to be an important
area for both ligand binding and activation of the
AT1 receptor and several other GPCRs (Luo
et al., 1994; Marie et al., 1994; Hunyady
et al., 1995, 1996; Strader et al., 1995; Laporte et al., 1996; Inoue et al., 1997). Mutations of
amino acid residues in the seventh helix have been reported to
interfere with the binding of nonpeptide Ang II antagonists. These
residues include Tyr292 (the amino acid located in the position at
which the retinal chromophore binds covalently to the rhodopsin
molecule), Leu300 and Phe301 (two apolar amino acids located in the
center of the conserved NPXXY sequence), and Asn294 and Asn295 (two
polar residues located between Tyr292 and the NPXXY sequence) (Marie
et al., 1994; Schambye et al., 1994b; Ji et
al., 1995; Hunyady et al., 1996; Inoue et
al., 1997; Karnik et al., 1997). In addition, mutations in the seventh transmembrane helix have been found to interfere with
the binding of peptide ligands and with signal transduction from the
receptor (Marie et al., 1994; Hunyady et al.,
1995; Ji et al., 1995; Laporte et al., 1996).
Recent studies have suggested that an interaction between Asn295 and
Asn111 in the third helix stabilizes the inactive conformation of the
receptor, based on the finding that mutation of Asn295 or Asn111 caused
constitutive activation of the AT1 receptor (Noda et al., 1996; Balmforth et al., 1997; Groblewski
et al., 1997). The Asn111 residue has also been reported to
interact with the side chain of Tyr4 of Ang II, and this has been
suggested to initiate the process of receptor activation (Noda et
al., 1996). Because the available structure-function data suggest
that the seventh transmembrane helix of the AT1
angiotensin receptor is a region in which the binding sites of peptide
and nonpeptide ligands overlap (Hunyady et al., 1996), the
role of this region in the binding of nonpeptide agonists was analyzed.
Receptors bearing single amino acid substitutions of two polar residues
(Asn294 and Asn295) and an adjacent apolar residue (Phe293) were
created and expressed in COS-7 cells to study the role of this region
in the ligand binding and activation of the AT1
receptor.
| |
Materials and Methods |
|---|
|
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Mutagenesis of rat smooth muscle AT1a receptor
cDNA.
The rat AT1a receptor cDNA was
subcloned into the mammalian expression vector pcDNAI/Amp (Invitrogen,
San Diego, CA), as described earlier (Hunyady et al., 1994).
The AT1a receptors used in this study also
contained an octapeptide tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) inserted
after the starting methionine of mutant and wild-type rat
AT1a receptors, to permit the immunodetection of
binding-deficient mutant receptors. In accordance with a recent report
(Hein et al., 1997), the octapeptide tag had no major effect
on the binding and inositol phosphate signaling properties of the
wild-type rat AT1a receptor. The amino acid
numbers used in this study refer to the positions in the published rat
AT1a receptor sequence (Murphy et al.,
1991); the presence of the octapeptide tag does not affect the
numbering. Mutant rat AT1a receptors were created
using the Mutagene kit (Bio-Rad, Hercules, CA). Each mutant contained a silent restriction site to facilitate the screening of colonies. Oligonucletides were from Midland Certified Reagent Co. (Midland, TX).
All mutations were verified by dideoxy sequencing using Sequenase II
(Amersham-USB, Arlington Heights, IL).
Transient expression of AT1a receptor mutants. COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. For measurements of inositol phosphate responses or [Sar1,Ile8]Ang II binding to intact cells, the cells were seeded in 24-well plates (50,000 cells/well) 3 days before transfection. Transient transfection was performed by replacing the culture medium with 0.4-ml aliquots of Opti-MEM I containing 8 µg of Lipofectamine and 1 µg of plasmid DNA for each sample. The cells were incubated for 5-6 hr in this solution, which was then replaced with culture medium. All experiments were performed 48 hr after the initiation of the transfection procedure.
[Sar1,Ile8]Ang II binding to
intact cells.
To determine the surface expression level and
structural integrity of the mutant receptors, the number of Ang II
binding sites was determined by incubating the transfected cells with
125I-[Sar1,Ile8]Ang
II (0.05-0.1 µCi/sample) and increasing concentrations of unlabeled
[Sar1,Ile8]Ang II in
medium 199 (with HEPES) for 6 hr at 4°. The cells were washed twice
with ice-cold Dulbecco's phosphate-buffered saline, and the
radioactivity associated with the cells was measured by 
-counting
after solubilization with 0.5 M NaOH/0.05% SDS. The displacement curves were analyzed with the Ligand computer program, using a one-site model, as described earlier (Hunyady et
al., 1995).
Binding to COS-7 cell membranes.
COS-7 cells were seeded in
15-cm tissue culture dishes 72 hr before the transfection, which was
performed by the calcium phosphate precipitation method (Life
Technologies, Gaithersburg, MD), using 50 µg of DNA. After 48 hr, the
cells were washed, scraped into 1.5 ml of ice-cold 10 mM
Tris·HCl, pH 7.4, 1 mM EDTA, and then lysed by freezing.
Crude membranes were prepared by centrifuging the samples at
16,000 × g. The pellet was resuspended in binding buffer (containing 100 mM NaCl, 5 mM
MgCl2, and 20 mM Tris·HCl, pH 7.4),
and the protein content was determined. Binding assays were performed
at 25° in 0.2 ml of binding buffer supplemented with 2 g/liter BSA,
except when nonpeptide agonists were tested. In these experiments, BSA
was replaced with 0.1% lysozyme. Saturation binding experiments with
125I-Ang II and
125I-[Sar1,Ile8]Ang
II were performed by adding increasing concentrations of the respective
radioligand (up to 2 nM) to 15-30 µg of crude membranes. In displacement studies, each sample contained 0.05-0.1 µCi of 125I-[Sar1,Ile8]Ang
II, 15-30 µg of crude membranes, and the indicated concentrations of
unlabeled ligand. G protein coupling was evaluated by measuring the
binding of 125I-Ang II (0.05-0.1 µCi) under
similar conditions in the presence of the indicated concentrations of
GTPS. After 90-min incubations at 25°, the unbound tracer was
removed by rapid filtration, and the bound radioactivity was measured
by -counting.
Inositol phosphate measurements.
In these experiments, the
culture medium was replaced, 24 hr after transfection, with 0.5 ml of
inositol-free Dulbecco's modified Eagle's medium containing 1 g/liter
BSA, 20 µCi/ml [3H]inositol, 2.5% fetal
bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin, as
described earlier (Hunyady et al., 1994). After a 24-hr
labeling period, the cells were washed twice and incubated in
inositol-free modified medium 199 (with HEPES), in the presence of 10 mM LiCl, for 30 min at 37° and were then stimulated with
the indicated concentration of Ang II for 20 min. Incubations were
stopped by the addition of perchloric acid (5%, v/v, final), and the
inositol phosphates were extracted as described earlier (Hunyady
et al., 1995). After neutralization, the samples were
applied to a Bio-Rad AG1X8 column. The columns were washed four times
with 3 ml of water and twice with 3 ml of 0.2 M ammonium formate in 0.1 M formic acid, to remove inositol and
inositol monophosphates. After these washing steps, the combined
InsP2/InsP3 fractions were
eluted with two 3-ml aliquots of 1 M ammonium formate in
0.1 M formic acid, and radioactivities were determined by
liquid scintillation counting.
Materials.
The cDNA clone (pCa18b) of the rat smooth muscle
AT1a receptor (Murphy et al., 1991)
subcloned into the mammalian expression vector pCDM8 (Invitrogen) was
kindly provided by Dr. Kenneth E. Bernstein (Department of
Pathology, Emory University, Atlanta, GA). Restriction enzymes were
obtained from Boehringer Mannheim (Indianapolis, IN) or New England
Biolabs (Beverly, MA). Culture media were from Biofluids (Rockville,
MD). The medium 199 used in these experiments was modified to contain
3.6 mM K+, 1.2 mM
Ca2+, 1 g/liter BSA, and 20 mM HEPES.
Lipofectamine and Opti-MEM I were from Life Technologies. Losartan was
a gift from Dr. P. C. Wong (DuPont, Wilmington, DE).
L-162,313 and L-163,491 were kindly provided by
Dr. W. J. Greenlee (Merck Research Laboratories, Rahway, NJ).
125I-Ang II and
125I-[Sar1,Ile8]Ang
II were obtained from Hazleton Laboratories (Vienna, VA) or DuPont New
England Nuclear Research (Boston, MA), and
[3H]inositol was from Amersham (Arlington
Heights, IL).
Statistical analysis. The significance of changes in the binding affinities and inositol phosphate responses of the mutant AT1 receptors were determined by analysis of variance combined with Scheffe's range test.
| |
Results |
|---|
|
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Binding properties of mutant and wild-type AT1a receptors. The wild-type and F293A, N294A, and N295A mutant AT1a receptors were expressed in COS-7 cells, to study their radioligand binding and signaling characteristics. The positions of the mutated amino acids in the seventh transmembrane helix of the rat AT1a receptor are shown in Fig. 1. Binding affinities were determined by measuring the inhibition of binding of the radioiodinated peptide antagonist 125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of the unlabeled nonpeptide antagonist (losartan), the peptide antagonist (Fig. 2), or two nonpeptide agonists (L-162,313 and L-163,491) (Fig. 3). The Kd values are shown in Table 1. The binding affinities of the F293A receptor for [Sar1,Ile8]Ang II and losartan were not significantly different from those of the wild-type receptor. The N294A receptor had normal affinity for [Sar1,Ile8]Ang II, indicating that the structure of this mutant receptor was intact. However, its affinity for losartan was markedly reduced, with a 62-fold increase in Kd. The N295A mutant receptor exhibited diminished binding affinity for both [Sar1,Ile8]Ang II and losartan, with Kd values 16 ± 2 and 104 ± 6 times those of the wild-type receptor, respectively.
|
|
|
|
) differed from those for losartan in several respects; the conserved substitution with
glutamine in the N295Q receptor did not affect agonist binding, in
contrast to its major effect on losartan binding. The binding of
L-163,491 was substantially reduced by substitution with
serine (9.2-fold reduction) and was less affected by replacement with threonine, aspartic acid, lysine, leucine, or valine (2-4-fold) (Table
1).
Saturation binding studies. The affinities of the alanine mutant AT1a receptors for the physiological agonist Ang II and the peptide antagonist [Sar1,Ile8]Ang II were also evaluated in saturation binding experiments with 125I-labeled radioligands (Table 2). Under these conditions, Scatchard analysis of 125I-Ang II binding to the wild-type receptor detected only the high-affinity agonist binding site, with a Kd of 0.12 nM. For this reason, the number of Ang II binding sites measured in COS-7 cells expressing the wild-type receptor is approximately 16% of the sites calculated from binding analysis with [Sar1,Ile8]Ang II, which binds with a single affinity (Kd = 0.23 nM) to the entire AT1 receptor population (Table 2).
|
Inositol phosphate responses and G protein coupling of alanine
mutant AT1a receptors.
The signal transduction
properties of the alanine mutant receptors were studied by measuring
the inositol phosphate (InsP2 plus
InsP3) responses of the expressed mutant
receptors in [3H]inositol-prelabeled COS-7
cells in the presence of lithium. The data were normalized to the
measured receptor levels, because under these conditions the combined
InsP2 plus InsP3 response is directly proportional to the receptor number (Hunyady et
al., 1995). Representative curves for the inositol phosphate
responses of alanine mutant AT1a receptors are
shown in Fig. 4. The amplitudes of the
inositol phosphate responses for the F293A, N294A, and N295A mutant
receptors were 107 ± 27%, 16.3 ± 0.7%
(p < 0.01), and 73.0 ± 11.3%,
respectively, compared with those of the wild-type AT1a receptor (three experiments). The
EC50 values for the wild-type and F293A, N294A,
and N295A mutant AT1a receptors for inositol phosphate responses were 0.91 ± 0.36 nM, 1.1 ± 0.3 nM, 7.3 ± 1.6 nM
(p < 0.01), and 5.0 ± 1.1 nM
(p < 0.05), respectively (three experiments).
These data demonstrate that the reduced efficacy of the N294A mutant
receptor for inositol phosphate signal generation was accompanied by
decreased potency of Ang II to activate this receptor. The capacity of
the F293A mutant receptor for inositol phosphate generation was similar
to that of the wild-type receptor, whereas the decreased potency of the
N295A mutant receptor is in accordance with its reduced affinity for
Ang II. The maximal inositol phosphate response of the N295A mutant
receptor did not differ significantly from that of the wild-type
receptor. Thus, although the affinity of this mutant receptor was
reduced, the peptide agonist could induce receptor activation when
present at sufficiently high concentrations.
|
S on the binding of the physiological
agonist Ang II. As reported earlier (Hunyady et al., 1994),
GTPS substantially reduced the binding of
125I-Ang II to the wild-type
AT1a receptor in membranes of transiently transfected COS-7 cells (Fig. 5).
However, the inhibitory effect of GTPS on Ang II binding was
significantly decreased with the N294A mutant
AT1a receptor (four experiments)
(p < 0.001), in accordance with its impaired
coupling to inositol phosphate generation.
|
| |
Discussion |
|---|
|
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Alanine substitution of Asn294 affects agonist binding and signal
transduction.
In this study, mutational analysis has defined the
functional properties of three adjacent amino acids located in the
seventh transmembrane helix of the AT1 receptor.
According to predictions based on modeling studies of GPCRs, the two
consecutive polar residues (Asn294 and Asn295) are likely to face the
interior of the AT1 angiotensin receptor, whereas
Phe293 is expected to face the lipid environment of the plasma membrane
(Baldwin, 1993; Donnelly et al., 1994). In accordance with
these proposals, we found that alanine replacement of Phe293 had no
major effect on angiotensin receptor function, whereas similar
mutations of Asn294 and Asn295 significantly affected the binding and
signaling functions of the receptor.
, 1997). The most prominent feature
of the N294A receptor was its markedly attenuated capacity to engender
inositol phosphate responses, even in the presence of high
concentrations of Ang II. These data suggest that Asn294 is an
important determinant of agonist-induced receptor activation and signal
transduction. The moderately impaired agonist binding of the N294A
receptor may reflect its inability to achieve the optimal conformation
required for agonist-induced activation (Karnik et al.,
1997). This conclusion is supported by the finding that the effect of
GTPS on Ang II binding was reduced with the N294A mutant
AT1a receptor.
Replacements of Asn295 affect ligand binding of the AT1a receptor without interfering with its activation. Alanine replacement of Asn295 caused a substantial decrease in receptor binding affinity for all AT1 receptor ligands tested, without significant impairment of the maximal inositol phosphate response to Ang II stimulation. The role of Asn295 in the binding function of the AT1a receptor was evaluated with a series of mutant AT1a receptors bearing other replacements of this residue. In general, substitution with amino acids with polar, apolar, or charged side chains had less effect on the binding affinities for the tested ligands than did alanine substitution. In the case of [Sar1,Ile8]Ang II, substitution with amino acids with smaller side chains (e.g., serine and threonine) caused more marked impairment of binding than did substitution with residues with larger side chains (e.g., aspartic acid, glutamine, leucine, or lysine). Among the substituents with apolar side chains, leucine, with the largest side chain, caused the least impairment of [Sar1,Ile8]Ang II binding. Conversely, substitution with alanine, which has the smallest side chain, caused the most marked reduction of binding affinity. There was no clear requirement regarding the nature of the amino acid substitution in position 295. Substitution with polar (glutamine), charged (aspartic acid or lysine), or apolar (leucine) residues caused moderate impairment of binding. These data suggest that the Asn295 residue has a structural effect on [Sar1,Ile8]Ang II binding, and they argue against a direct contact between the bound ligand and this residue.
Losartan binding to the AT1a receptor was reduced by all tested substitutions of the Asn295 residue. These findings are in accordance with previous studies on the affinity of the N295S mutant AT1 receptor for losartan (Schambye et al., 1994a,b; Balmforth et al., 1997). The unique
requirement for asparagine in position 295 for normal losartan binding
may reflect a direct interaction between this ligand and Asn295 of the
receptor. The intramembrane location of Asn295 and the effects of its
replacement on the binding of peptide and nonpeptide antagonists
suggest that this residue is essential for the integrity of the
intramembrane binding pocket of the AT1 receptor.
Another interesting feature of the N295A mutation was its deleterious
effect on the binding of the nonpeptide agonists L-162,313 and L-163,491. This finding differs from those of previous
studies, in which replacement of Asn295 by aspartic acid had no effect on Ang II and nonpeptide agonist binding (Perlman et al.,
1995, 1997). Although aspartic acid was found to substitute
for Asn295 without detectable loss of binding affinity for
L-162,313, the efficacy with which L-162,313
and other nonpeptide agonists stimulated inositol phosphate responses
was greatly reduced with the N295D mutant (Perlman et al.,
1997). In our study, the affinity of the N295D mutant receptor for the
AT1-selective nonpeptide agonist L-163,491 was moderately impaired. All tested substitutions
of Asn295 caused less impairment of L-163,491 binding,
compared with that of losartan. In contrast to its marked effect on
losartan binding, the conserved substitution of glutamine for Asn295
had no effect on the binding affinity for L-163,491. These
data suggest that the structural requirements in position 295 for the
binding of L-163,491 differ from those for losartan.
Although these data do not provide evidence for a role of Asn295 in
nonpeptide agonist binding, the marked inhibition of binding to the
N295A and N295S mutant receptors suggests that the intact intramembrane
binding pocket is an important requirement for the binding of
nonpeptide agonists.
Our data also showed that alanine substitution of Asn295 reduced the
high-affinity binding of Ang II to the receptor. In accordance with
this finding, the potency with which Ang II stimulated inositol phosphate responses in cells expressing the N295A receptor was also
reduced. Although earlier studies suggested that mutation of Asn295 to
serine causes a relatively minor change in the Ang II binding affinity
of the receptor (Schambye et al., 1994a,b; Balmforth
et al., 1997), the present data, in accordance with previous
studies on the rat AT1b receptor (Ji et
al., 1995), demonstrate that the N295A mutation also affects the
binding of peptide ligands.
In a recent report, the finding that mutation of Asn295 to serine
caused constitutive activation of the AT1
receptor led to the proposal that an interaction between Asn295 and
Asn111 (in the third intramembrane helix) stabilizes the inactive
conformation of the AT1 receptor (Balmforth
et al., 1997). However, constitutive activation was not
observed when Asn295 was replaced by aspartic acid (Perlman et
al., 1997), and it was not detectable with the N295A mutant
AT1a receptor in this study, despite its
reasonably high levels of expression.
Mutations in the seventh transmembrane helix affect the binding of
nonpeptide agonists.
Initial studies on agonist binding to the
AT1 receptor demonstrated that several
extracellular residues are essential determinants of Ang II binding
(Hjorth et al., 1994). Based on these data, it has been
proposed that the peptide binding site of the AT1 receptor is located in the extracellular region (Hjorth et
al., 1994). However, more recent studies have emphasized the role
of the intramembrane helices in the agonist-induced conformational change (Noda et al., 1995, 1996; Hunyady et al.,
1996; Inoue et al., 1997; Karnik et al., 1997).
His256, in the sixth transmembrane helix, has been reported to interact
with the side chain of the carboxyl terminal Phe8 of Ang II (Noda
et al., 1995), and Tyr111 of the AT1
receptor has been proposed to interact with the side chain of Tyr4 of
Ang II (Noda et al., 1996). These data, as well as recent
modeling studies (Underwood et al., 1995; Inoue et
al., 1997), point to the importance of the intramembrane binding
pocket in the agonist-induced conformational change of the
AT1 receptor.
Asn294 has a role in the agonist-induced conformational
change.
The seventh transmembrane helix has been implicated
in several recent studies as the site of initiation of agonist-induced conformational changes in a variety of GPCRs. The retinal chromophore of rhodopsin binds covalently to the seventh transmembrane helix, which
has also been implicated in agonist binding to several different GPCRs
(Probst et al., 1992; Baldwin, 1993; Strader et
al., 1995; Hunyady et al., 1996). More recently,
conserved aromatic residues in the seventh transmembrane domain of the
same receptor were found to be essential for agonist binding and
stimulation of inositol phosphate production (Roth et al.,
1997). Rotation of transmembrane helices, which results in a new
hydrogen bond between Asp74 of the second transmembrane helix and
Tyr292 of the seventh transmembrane helix, has also been proposed as
the initial event of AT1 receptor activation
(Inoue et al., 1997).
;
Baldwin, 1993). In a published alignment of GPCR sequences, >60% of
the listed sequences contain asparagine in the position corresponding
to Asn294 of the AT1 receptor, and >90% of the
GPCRs have a polar amino acid that can participate in hydrogen bond formation (e.g., serine, threonine, histidine, or arginine) (Probst et al., 1992). In view of the location of Asn294 in the
seventh intramembrane helix (Baldwin, 1993; Liu et al.,
1995; Mizobe et al., 1996), this residue is unlikely to
directly mediate receptor coupling to Gq or other
G proteins. It is more probable that this residue is required for an
intramolecular interaction that in turn stabilizes the active
conformation of the receptor. In recent reports, an interaction between
Asn111 and Tyr292 (Groblewski et al., 1997) and/or Asn295
(Balmforth et al., 1997) was proposed to be a major
determinant of the inactive conformation of the AT1 receptor. It is possible that an interaction
between Asn294 and other amino acids is an additional structural
requirement for AT1 receptor activation, similar
to the proposed role for the interaction between Tyr292 of the seventh
transmembrane helix and Asp74 of the second transmembrane helix
(Bihoreau et al., 1993; Marie et al., 1994;
Joseph et al., 1995).
In summary, the present data provide evidence that the binding of
nonpeptide agonists to the AT1 receptor depends
on residues located in the intramembrane binding pocket. In addition,
Asn294 has a crucial role in receptor activation, and Asn295 is a major determinant of the binding of all tested Ang II ligands to the AT1 angiotensin receptor. The complementary
functions of these adjacent asparagine residues could provide a
molecular basis for the proposed role of the seventh transmembrane
helix in transforming agonist binding into the rearrangement of
intramembrane helices that is believed to result in receptor
activation.
| |
Acknowledgments |
|---|
We are grateful to Yue Zhang and Katinka Süpeki for expert technical assistance.
| |
Footnotes |
|---|
Received October 13, 1997; Accepted May 7, 1998
This work was supported in part by an International Research Scholar Award from the Howard Hughes Medical Institute (HHMI 75195-541702) and by grants from the Hungarian Ministry of Culture and Education (FKFP-0776/1997) and the Hungarian Ministry of Public Health (ETT 535/96). L.H. is an International Research Scholar of the Howard Hughes Medical Institute.
Send reprint requests to: Kevin J. Catt M.D., Ph.D., Endocrinology and Reproduction Research Branch, NICHD, NIH, Building 49, Room 6A-36, Bethesda, MD 20892-4510. E-mail: catt{at}helix.nih.gov
| |
Abbreviations |
|---|
GPCR, G protein-coupled receptor;
Ang II, angiotensin II;
InsP2, inositol bisphosphate;
InsP3, inositol trisphosphate;
GTPS, guanosine
5'-(-thio)triphosphate;
BSA, bovine serum albumin;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
| |
References |
|---|
|
Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
-helices.
Nature (Lond)
389:
203-206[Medline].This article has been cited by other articles:
|
|
 |
|
  L. Oliveira, C. M. Costa-Neto, C. R. Nakaie, S. Schreier, S. I. Shimuta, and A. C. M. Paiva The Angiotensin II AT1 Receptor Structure-Activity Correlations in the Light of Rhodopsin Structure Physiol Rev, April 1, 2007; 87(2): 565 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-H. Feng, L. Zhou, R. Qiu, and R. Zeng Single Mutations at Asn295 and Leu305 in the Cytoplasmic Half of Transmembrane {alpha}-Helix Domain 7 of the AT1 Receptor Induce Promiscuous Agonist Specificity for Angiotensin II Fragments: A Pseudo-Constitutive Activity Mol. Pharmacol., August 1, 2005; 68(2): 347 - 355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Clement, S. S. Martin, M.-E. Beaulieu, C. Chamberland, P. Lavigne, R. Leduc, G. Guillemette, and E. Escher Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay J. Biol. Chem., July 22, 2005; 280(29): 27121 - 27129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Reinhart, Q. Xie, X.-J. Liu, Y.-F. Zhu, J. Fan, C. Chen, and R. S. Struthers Species Selectivity of Nonpeptide Antagonists of the Gonadotropinreleasing Hormone Receptor Is Determined by Residues in Extracellular Loops II and III and the Amino Terminus J. Biol. Chem., August 13, 2004; 279(33): 34115 - 34122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Boucard, M. Roy, M.-E. Beaulieu, P. Lavigne, E. Escher, G. Guillemette, and R. Leduc Constitutive Activation of the Angiotensin II Type 1 Receptor Alters the Spatial Proximity of Transmembrane 7 to the Ligand-binding Pocket J. Biol. Chem., September 19, 2003; 278(38): 36628 - 36636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. C. Leclerc, M. Auger-Messier, P. M. Lanctot, E. Escher, R. Leduc, and G. Guillemette A Polyaromatic Caveolin-Binding-Like Motif in the Cytoplasmic Tail of the Type 1 Receptor for Angiotensin II Plays an Important Role in Receptor Trafficking and Signaling Endocrinology, December 1, 2002; 143(12): 4702 - 4710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Raiden, K. Nahmod, V. Nahmod, G. Semeniuk, Y. Pereira, C. Alvarez, M. Giordano, and J. R. Geffner Nonpeptide Antagonists of AT1 Receptor for Angiotensin II Delay the Onset of Acute Respiratory Distress Syndrome J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 45 - 51. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Le, P. M. L. Vanderheyden, M. Szaszak, L. Hunyady, and G. Vauquelin Angiotensin IV Is a Potent Agonist for Constitutive Active Human AT1 Receptors. DISTINCT ROLES OF THE N- AND C-TERMINAL RESIDUES OF ANGIOTENSIN II DURING AT1 RECEPTOR ACTIVATION J. Biol. Chem., June 21, 2002; 277(26): 23107 - 23110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hunyady, Z. Gaborik, G. Vauquelin, and K. J Catt Review: Structural requirements for signalling and regulation of AT1-receptors Journal of Renin-Angiotensin-Aldosterone System, March 1, 2001; 2(1_suppl): S16 - S23. [PDF]
|
|
|
|
 |
|
 M. de Gasparo, K. J. Catt, T. Inagami, J. W. Wright, and Th. Unger International Union of Pharmacology. XXIII. The Angiotensin II Receptors Pharmacol. Rev., September 1, 2000; 52(3): 415 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jayadev, R. D. Smith, G. Jagadeesh, A. J. Baukal, L. Hunyady, and K. J. Catt N-Linked Glycosylation Is Required for Optimal AT1a Angiotensin Receptor Expression in COS-7 Cells Endocrinology, May 1, 1999; 140(5): 2010 - 2017. [Abstract] [Full Text]
|
|
|
|
|
|
R. D. Smith, L. Hunyady, J. A. Olivares-Reyes, B. Mihalik, S. Jayadev, and K. J. Catt Agonist-Induced Phosphorylation of the Angiotensin AT1a Receptor Is Localized to a Serine/Threonine-Rich Region of Its Cytoplasmic Tail Mol. Pharmacol., December 1, 1998; 54(6): 935 - 941. [Abstract] [Full Text]
|
|
|
|
|
|
M. Zhang, X. Zhao, H.-C. Chen, K. J. Catt, and L. Hunyady Activation of the AT1 Angiotensin Receptor Is Dependent on Adjacent Apolar Residues in the Carboxyl Terminus of the Third Cytoplasmic Loop J. Biol. Chem., May 19, 2000; 275(21): 15782 - 15788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Marie, E. Richard, D. Pruneau, J.-L. Paquet, C. Siatka, R. Larguier, C. Ponce, P. Vassault, T. Groblewski, B. Maigret, et al. Control of Conformational Equilibria in the Human B2 Bradykinin Receptor. MODELING OF NONPEPTIDIC LIGAND ACTION AND COMPARISON TO THE RHODOPSIN STRUCTURE J. Biol. Chem., October 26, 2001; 276(44): 41100 - 41111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Olivares-Reyes, R. D. Smith, L. Hunyady, B. H. Shah, and K. J. Catt Agonist-induced Signaling, Desensitization, and Internalization of a Phosphorylation-deficient AT1A Angiotensin Receptor J. Biol. Chem., October 5, 2001; 276(41): 37761 - 37768. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) or F293A
(
), N294A (
), or N295A (
) mutant AT1a receptors
was measured in the presence or absence of the respective antagonists.
All values are expressed as percentages of the
B0 values determined in the absence of
unlabeled ligand. Data are shown as means of duplicate determinations
from a representative example of three to five experiments with similar
results. Kd values are shown in
Table 
