(Received for publication, August 19, 1996, and in revised form, October 4, 1996)
From the Institute for Cardiovascular Research and § Department of Pharmacology, University of Leeds, Leeds LS2 9JT, United Kingdom
A model of the angiotensin AT1 receptor and site-directed mutagenesis were used to identify key residues involved in ligand binding. Receptors were stably expressed in human embryonic kidney 293 cells, and their binding properties compared. Wild type receptors exhibited low and high affinity binding sites for peptides. Substitution of Asn111, situated in the third transmembrane helix, resulted in a significant alteration in ligand binding with only high affinity binding of the peptides, angiotensin II, angiotensin III, and [p-amino-Phe6]angiotensin II and a marked loss in the binding affinity of the AT1 receptor selective non-peptide antagonist losartan. From our model it was apparent that Asn111 was in close spatial proximity to Asn295 in the seventh transmembrane helix. Substitution of Asn295, produced identical changes in the receptor's pharmacological profile. Furthermore, the Ser111AT1A and Ser295AT1A mutants did not require the association of a G-protein for high affinity agonist binding. Finally, the Ser295AT1A mutant maintained higher basal generation of inositol trisphosphate than the wild type, indicating constitutive activation. We propose that substitution of these residues causes the loss of an interaction between transmembrane helices III and VII, which allows the AT1 receptor to "relax" into its active conformation.
The renin-angiotensin system plays a vital role in the regulation of cardiovascular function, and its activity may be abnormal in a number of disease states. The effector molecule of this system, the octapeptide angiotensin II (Ang II),1 has a number of actions in a variety of cell types (1). Two distinct Ang II receptor subtypes, AT1 and AT2 have been identified (2). This characterization is based on the differential selectivity for these two receptors of the non-peptide Ang II receptor antagonists, DuP 753 (losartan; AT1 selective) and PD123177 (AT2 selective). In addition, the modified peptides CGP42112A (3) and [p-amino-Phe6]angiotensin II (pNH2F6AII) (4), both display selectivity for the AT2 receptor over the AT1 receptor. Furthermore, several studies suggest that AT1 and AT2 receptors display subtle differences in their binding affinities for Ang II and angiotensin III (Ang III). AT2 receptors bind Ang II and Ang III with equal affinity (5-8), while AT1 receptors exhibit a slightly higher affinity for Ang II, between 3- and 20-fold greater than Ang III (9-11). Many of the recognized actions of Ang II appear to be mediated through AT1 receptors, predominantly through activation of the phospholipase C signal transduction pathway.
AT1 receptors have been cloned from a variety of species
and tissues (9-15). All these AT1 receptors consist of a
single polypeptide, 359 amino acids in length, arranged with a
topography comprising seven -helical transmembrane regions (TM
I-VII), typical of the G-protein-coupled receptor family. They display
a high degree of sequence identity at the amino acid level (over 94%
identical between all mammalian species). Two closely related isoforms
(AT1A and AT1B) encoded by different genes have
been identified in the rat and mouse (13, 16) Recently AT2
receptors have been cloned (17-19), which are also members of the
G-protein-coupled receptor family. They consist of 363 amino acids and
share only 34% sequence identity at the amino acid level to the
AT1 receptors.
Mutagenesis studies of the AT1 receptor have recently identified residues important in the binding of the non-peptide antagonist, losartan, and the natural ligand Ang II. These residues appear to be mutually exclusive, since amino acid substitutions, which affect losartan binding, do not alter Ang II binding, and vice versa. The major determinants of losartan binding appear to be residues located within the transmembrane helices III-VII (20-23). In contrast, the major determinants of Ang II binding appear to be residues present in the extracellular regions, particularly in the N-terminal extension adjacent to the first transmembrane helix and in the C-terminal part of the third extracellular loop (24, 25). However, a conserved residue, Lys199 in the fifth transmembrane helix (TM V) of the AT1 receptor, has recently been reported to be crucial for the binding of both peptide and non-peptide ligands (26). In the present study we constructed a three-dimensional model of the AT1A receptor using the method of Donnelly et al. (27) to guide site-directed mutagenesis aimed at identifying potential residues involved in ligand binding. The consequences of substituting either Asn111, in the third transmembrane helix (TM III), or Asn295, in the seventh transmembrane helices (TM VII), on ligand binding and receptor coupling are presented. These data suggest that a specific interaction between TM III and TM VII may be a major determinant of AT1 receptor isomerization from an inactive to active conformation.
Ang II, Ang III, bacitracin, bovine serum albumin
(protease-free), GTPS, and fura 2-AM were purchased from
Sigma.
125I-[Sar1,Ile8]angiotensin II
and 125I-angiotensin II from DuPont NEN.
pNH2F6AII and CGP42112A from Bachem. Losartan
(DuP 753) was generously donated by DuPont (Wilmington, DE). PD123319,
an analogue of PD123177, was generously donated by Parke-Davis. All
cell culture reagents were purchased from Life Technologies, Inc. Human
embryonic kidney (HEK293) cells were obtained from the European
Collection of Animal Cell Cultures (ECACC no. 85120602, Porton Down,
Salisbury, UK). Hygromycin B was from Calbiochem, Transfectam from
Integra Biosciences Ltd., and Mutagene Phagemid In Vitro Mutagenesis
Version 2 kit from Bio-Rad. pBluescript KS+ and M13K07
helper phage was from Stratagene Ltd. and pCEP4 and Escherichia
coli CJ236 cells from Invitrogen. The cDNA clone-encoding the
rat AT1A receptor was kindly provided by Glaxo Group
Research Limited.
Modeling was performed according
to the method of Donnelly et al. (27). Briefly, following
sequence alignment of the rat AT1A receptor to the human
2-adrenergic receptor, the seven identified
-helices
were constructed using the interactive graphics program InsightII
(Biosym Technologies). The projection map of bovine rhodopsin was used
to position these helices into a three-dimensional helical bundle, the
order of which was assumed to be analogous to the arrangement in
bacteriorhodopsin. The internal faces and relative depth of each helix
in the bilayer were deduced by Fourier transform analysis. The
connecting intracellular and extracellular loops are not included in
the model due to the lack of structural information.
The entire coding region of the rat AT1A receptor (a HindIII-NotI fragment of 2 kilobases) was subcloned into the polylinker sites of pBluescript KS+, and single-stranded DNA was rescued from CJ236 cells using M13K07 helper phage. Mutagenesis was performed with a Mutagene Phagemid In Vitro Mutagenesis Version 2 kit and confirmed by dideoxy sequencing using Sequenase II (U. S. Biochemical Corp.). The wild type and mutated AT1A cDNAs were subcloned into the mammalian expression vector pCEP4 using the restriction enzymes HindIII and NotI.
Permanent Expression of Receptors in HEK293 CellsHEK293 were cultured in modified Eagle's medium containing Earle's salts, supplemented with 10% (v/v) fetal calf serum, 1% nonessential amino acids, 50 µg/ml gentamicin, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin at 37 °C in a humidified atmosphere of air/CO2 (19:1). Cells were transfected with 12 µg of pCEP4 containing either the cDNA for the wild type or mutated AT1A receptors using Transfectam, according to the manufacturer's instructions. Stable expression of AT1A receptors in HEK293 cells was achieved by addition of 200 µg/ml hygromycin B to the medium 3 days after transfection and for all subsequent passages of the cells.
Binding AssayPreparation of transfected HEK293 cell
membranes and subsequent radioligand-receptor binding assays were
undertaken as described previously (28) with the following
modifications. All competitors were diluted in assay buffer which
consisted of 100 mM NaCl, 20 mM Hepes (pH 7.4),
1 mM EDTA, 10 mM MgCl2, bacitracin
(0.1% w/v), and bovine serum albumin (0.1% w/v). Nine to 11 concentrations of competitor were examined, each concentration in
duplicate. Membranes were incubated with
125I-[Sar1,Ile8]angiotensin II
and competitors for 60 min at room temperature. In the experiment to
determine the effect of GTPS on Ang II binding, the total reaction
volume was 300 µl, consisting of 200 µl of membranes (10-50 µg
of protein), 50 µl of 125I-angiotensin II (1.2 nM), and 50 µl of GTP
S (60 µM) or assay buffer.
Cells were subcultured on glass coverslips and used at subconfluence. The coverslips were incubated with the cell permeant fluorescent probe fura 2-AM (5 µM) in a Krebs-Ringer buffer (pH 7.4) consisting of 145 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 1.3 mM CaCl2, 10 mM glucose, 20 mM Hepes, at 20 °C for 1 h. Loaded cells were then washed free of extracellular dye, and the coverslip was mounted in an open culture chamber which was then placed on the stage of an inverted Nikon microscope and maintained at 37 °C in Krebs-Ringer buffer. Dynamic video imaging was carried out as described previously (29), using Magical hardware and TARDIS software (Applied Images, Sunderland, UK) and a low light level intensified charged coupled device camera (Photonic Science, Robertsbridge, UK).
Measurement of Inositol 1,4,5-Trisphosphate (IP3) Production in Transfected HEK293 CellsCells were subcultured on 30-mm Petri dishes and used at confluence. Each dish was washed with assay medium (inositol-free Dulbecco's modified Eagle's medium containing 20 mM HEPES, 0.25% (w/v) bovine serium albumin, 0.25% (w/v) bacitracin, pH 7.4; three times at 2 ml each). After washing, 1 ml of medium containing either no reagents or 100 nM angiotensin II was added, and incubations were performed for 15 s. The assay was stopped by the addition of ice-cold 20% trichloroacetic acid (0.5 ml). Subsequent extraction and measurement of IP3 production was undertaken using an inositol 1,4,5-trisphosphate 3H radioreceptor assay kit according to the manufacturer's instructions. Functional studies for both wild type and Ser295AT1A receptors were routinely performed in a single assay.
Data AnalysisRadioligand binding results are expressed as a percentage of the control (i.e. specific binding in the absence of any competitor). The binding data were analyzed and IC50 values determined by nonlinear regression analysis using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Competition curves were simultaneously fitted to one- and two-site models, and an F test was used to determine the most appropriate model. All IC50 values are quoted as the mean ± S.E.
The wild type AT1A receptor stably expressed
in HEK293 cells was characterized by examining the ability of ligands
to compete with 0.1 nM
125I-[Sar1,Ile8]angiotensin II
binding to membrane preparations (Fig. 1A and Table I) and the ability of Ang II to stimulate a rise
in intracellular calcium (Fig. 2A). GraphPad
Prism Analysis of the Ang II, Ang III, and
pNH2F6AII competition curves fitted best a
two-site model. These ligands competed for both high and low affinity
binding sites, with estimated IC50 values (nM)
of 0.8 ± 0.1, 3.5 ± 0.2, and 63 ± 30 for high, and
12 ± 2.8, 120 ± 12, and 51810 ± 4075 for low affinity
binding sites, respectively. The AT1 selective non-peptide
antagonist losartan competed for a single high affinity binding site
(IC50 value of 26.1 ± 1.5 nM), while the
AT2 selective ligands, CGP42112A, and PD123319 (an analogue
of PD123177) competed for single low affinity binding sites
(IC50 values of 5930 ± 1466 and 102650 ± 6582 nM, respectively). The functional coupling of the receptor was demonstrated by measuring intracellular calcium. Addition of Ang II
(100 nM) to transfected cells resulted in a rapid,
transient increase in intracellular calcium, which reached maximum
levels by 5-10 s and then declined toward resting levels by 60 s
(Fig. 2A). Neither this response nor specific binding of the
radioligand 125I-[Sar1,Ile8]angiotensin II
was observed in untransfected cells (data not shown).
|
Characterization of the Ser111 and Ala111 Mutant AT1A Receptors
The replacement of an asparagine residue (Asn111), located in the third transmembrane helix, with a serine, resulted in both subtle and large changes in the binding affinities of the AT1- and AT2-selective ligands studied here (Fig. 1B and Table I). In contrast to the findings with the wild type receptor, all of the ligands now competed for binding to a single site. Of particular interest was the observation that the resulting IC50 values for Ang II, Ang III, and pNH2F6AII (0.9 ± 0.1, 0.6 ± 0.1, and 68 ± 13.6 nM, respectively) were very similar to the IC50 values recorded for the binding of these ligands to their high affinity sites displayed by the wild type receptor. One consequence of this altered binding was that the mutated receptor now had equal affinity for Ang II and Ang III; an observation typical of an AT2 receptor. Furthermore, the AT2 selective peptide ligand, CGP42112A, also displayed an increased binding affinity (28 fold), in keeping with this trend toward AT2 receptor pharmacology. However, the AT2 selective non-peptide ligand PD123319 showed only a modest 3-fold increase in binding affinity for the mutated receptor.
In contrast to the above increases in binding affinity, the ability of the specific AT1 non-peptide antagonist losartan to bind to the mutated receptors was severely impaired (a 148-fold decrease in binding affinity). The rank order of potency was thus Ang III = Ang II > pNH2F6AII > CGP42112A > losartan > PD123319. The pharmacological profile of the Ala111AT1A receptor was identical to that of the Ser111AT1A receptor, with only minor differences in the IC50 values for all the ligands tested (Fig. 1C and Table I). Addition of Ang II (100 nM) to HEK293 cells transfected with the two Asn111 mutant AT1A receptors resulted in a rapid, transient increase in intracellular calcium, similar to that observed in HEK293 cells transfected with the wild type receptor (Fig. 2, B and C).
Characterization of the Ser295AT1A ReceptorAnalysis of the molecular model of the AT1A
receptor revealed that Asn111 located in the third
transmembrane helix was in spatial proximity to Asn295 in
the seventh transmembrane helix (Fig. 3, A
and B), raising the possibility of an interaction between
these two residues. Since this interaction may be disrupted by the
mutation of Asn111 to either an alanine or serine residue,
its loss may be responsible for the observed changes in ligand binding.
This hypothesis was tested by creating a
Ser295AT1A receptor. The
pharmacological profile of the Ser295AT1A
receptor was identical to that of the Asn111 mutants, with
only minor differences in the IC50 values for all the
ligands tested (Fig. 1D and Table I). Furthermore, addition of Ang II (100 nM) to HEK293 cells transfected
with the Ser295AT1A receptor resulted
in a rapid, transient increase in intracellular calcium, similar to
that observed in HEK293 cells transfected with the wild type receptor
(Fig. 2D).
Effect of GTP
The effects of
GTPS on 125I-angiotensin II binding were examined in
membranes prepared from HEK293 cells expressing wild type, Ser111AT1A, and
Ser295AT1A receptors (Fig. 4).
GTP
S reduced Ang II binding to the wild type receptor to 13.8 ± 0.4% of that in the absence of GTP
S (n = 3),
whereas it had no effect on Ang II binding to
Ser111AT1A (98.6 ± 2.1%,
n = 3) or Ser295AT1A
(102.7 ± 3%, n = 3) receptors. This suggests
that, in contrast to the wild type receptor, coupling to G-proteins is
not required for these mutated receptors to maintain their high
affinity binding.
Inositol 1,4,5-Trisphosphate Production in Transfected HEK293 Cells
IP3 production was examined in HEK293 cells
expressing either wild type AT1A or
Ser295AT1A receptors (Fig. 5).
Even though the level of expression of the
Ser295AT1A receptor (1385 ± 123 fmol/mg
total cell protein, n = 3) was less than that of the
wild type AT1A (1990 ± 178 fmol/mg total cell
protein, n = 3), the cells expressing the
Ser295AT1A receptor exhibited higher levels of
agonist-independent (basal) production of IP3 (2.2 ± 0.4-fold higher than wild type receptor basal value, n = 6; Fig. 5, left). Furthermore, although 100 nM Ang II stimulated both wild type AT1A and
Ser295AT1A receptors, the total IP3
production by cells expressing the Ser295AT1A
receptor was lower (64 ± 7% of the wild type AT1A,
n = 6) than that produced by cells expressing the wild
type AT1A receptor (Fig. 5, right).
Using our model of the rat angiotensin AT1A receptor, we selected to mutate the asparagine residue at position 111, located in TM III. Substitution with either a serine or alanine residue resulted in a marked alteration in the binding of several peptides and the AT1 selective non-peptide ligand losartan. Importantly, only high affinity binding was observed following Asn111 substitutions, in contrast to the wild type receptor, which displayed both high and low affinity binding for Ang II, Ang III, and pNH2F6AII. Furthermore, the binding affinity of the AT1 receptor-selective non-peptide antagonist losartan decreased (148-248-fold). Identical results were obtained when we substituted a serine residue for an asparagine residue located in TM VII (Asn295), identified by our three-dimensional model of the rat AT1A receptor as being in spatial proximity to Asn111. All these mutations retained Ang II mobilization of intracellular calcium, indicating that these substitutions did not perturb coupling of these receptors to the phospholipase C signal transduction pathway.
The binding of agonists, but not antagonists, to G-protein-coupled receptors in membrane preparations is characterized by high and low affinity binding states. The high affinity state is believed to be the active conformation of the receptor, consisting of a ternary complex involving the agonist, the receptor, and the G-protein, while the low affinity state is believed to be an inactive binary complex of agonist and receptor (30). In the presence of guanine nucleotides, which cause dissociation of G-proteins from their receptors, the high affinity state appears to be converted into the low affinity state (31).
Ang II receptors are typical of G-protein-coupled receptors, existing
in high and low affinity agonist binding states in various tissues, the
higher being converted to the lower in the presence of guanine
nucleotides (32-35). We have also observed that wild type recombinant
AT1A receptors display both high and low affinity agonist
binding states for Ang II. Furthermore, using a low concentration of
125I-angiotensin II, which would preferentially bind to the
high affinity state, we demonstrated that this high affinity state was
lost when binding was undertaken in the presence of the nonhydrolyzable guanine nucleotide GTPS. This suggested that the high affinity state
was also dependent on formation of a ternary complex involving Ang II,
the AT1A receptor and a G-protein.
Ang III and pNH2F6AII also bound to high and
low affinity states of the wild type AT1A receptor,
suggesting both are agonists. While Ang III is accepted as an agonist,
there are no direct reports of the agonist actions of
pNH2F6AII. We included
pNH2F6AII in our studies since it had been
reported that it preferentially bound to AT2 receptors and
might therefore be useful as a subtype selective ligand (4). However,
in keeping with our observations of its agonist properties, these
authors also reported that the potency of binding of
pNH2F6AII to AT1 receptors was
diminished in the presence of GTPS but did not attempt to estimate
the high and low affinity agonist binding state values (4).
There was a striking similarity between the estimated high affinity
agonist binding state values for Ang II, Ang III, and pNH2F6AII displayed by the wild type
AT1A receptor and their corresponding single site binding
affinities in the Asn111 and Asn295 mutant
AT1A receptors. This suggests that a consequence of these substitutions is to allow all of the receptors expressed to adopt the
high affinity agonist binding state conformation. However, the effects
of GTPS on Ang II binding indicate that, whereas the wild type
receptor requires the association of a G-protein to form the ternary
complex to adopt the conformation of a high affinity agonist binding
state, the Ser111AT1A and
Ser295AT1A mutants do not (Fig. 4). While, in
many instances, the lack of a GTP
S effect on agonist binding merely
indicates that a mutant receptor is no longer coupled, this was clearly
not the case here. All three mutants produced transient increases in
intracellular calcium similar to the wild type receptor. Thus
substitutions at Asn111 and Asn295 produce a
G-protein-independent, high affinity agonist binding state conformation
of the AT1A receptor which is still capable of coupling to
a G-protein. Such features are characteristic of constitutively active
receptors.
In a subsequent experiment (Fig. 5) we compared IP3
production in cells expressing either wild type AT1A or
Ser295AT1A receptors. In agreement with the
intracellular calcium data we confirmed that the
Ser295AT1A receptor was capable of coupling to
a G-protein. However, we also noted two discrepancies between the cells
expressing the wild type AT1A and
Ser295AT1A receptors. First, although the level
of expression of the Ser295AT1A receptor was
less than that of the wild type AT1A receptor, cells
expressing the Ser295AT1A receptor exhibited
higher levels of agonist-independent production of IP3
(approximately 2-fold higher basal), suggesting that this receptor is
constitutively active. Second, Ang II stimulated IP3 production by cells expressing the Ser295AT1A
receptor was lower than that produced by cells expressing the wild type
receptor. Since the Ser295AT1A receptor appears
constitutively active, the underlying mechanism may be constitutive
desensitization, as previously reported for a constitutively active
2-adrenergic receptor (36). Similar findings are
anticipated for the Asn111 mutants, but so far have not
been undertaken.
Constitutive activation of a G-protein coupled receptor was first
described in the 1B-adrenergic receptor (37), with
substitution of amino acids in the C terminus of the third
intracellular loop resulting in increases in both agonist binding
affinity and basal inositol phosphate production (suggesting receptor
activation in the absence of an agonist). Mutations that cause
constitutive activation appear to occur at widely distributed sites in
the sequences of G-protein-coupled receptors (38). However, relevant to
our work is the observation that substitution of Glu113 in
TM III or Lys296 in TM VII cause constitutive activation of
rhodopsin (39). According to our model, equivalent residues to these in
the AT1A receptor would be positioned two helical turns and
one helical turn above Asn111 and Asn295,
respectively. Of particular interest is that substitution of Cys128 in the
1B-adrenergic receptor leads
to constitutive activation (40). This residue is equivalent in position
to Asn111 in the AT1A receptor and both
receptors are coupled to phospholipase C.
Previous approaches to identifying residues important in the binding of ligands to AT1 receptors have, in general, been undertaken comparing the effects of substituting residues on the binding of non-peptide antagonist(s) and at most only one or two peptide ligands. Although such studies have established that peptides bind differently to non-peptide Ang II receptor antagonists (20-24), the limited range of ligands used may have resulted in inappropriate conclusions. For example, Schambye et al. (21) reported that substitution of Asn295 with a serine residue substantially reduced non-peptide antagonist binding without affecting the binding of Ang II or [Sar1,Leu8]angiotensin II. They concluded that this residue was directly or indirectly interacting with the non-peptide antagonists, but was not involved in the binding of peptides. Recently, similar results and conclusions were reported independently by two groups (22, 23) for Asn111. However, although the present study confirms some of the above observations, our alternative approach suggests a very different conclusion regarding the function of these two residues.
We determined which residues to mutate using our three-dimensional model and chose to explore a wide series of peptides and non-peptide antagonists to test predictions from the model. These included peptides that display differential binding to AT1 and AT2 receptors. Our findings indicate a pivotal role for the asparagines at positions 111 and 295 in the isomerization step described in the allosteric ternary complex model (41). In this model it is proposed that the receptor undergoes an isomerization from an inactive (R form) to an active (R* form) state which couples to G-proteins (R*G). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal `constraining' function of the receptor, allowing it to "relax" into the active conformation. The identical effects of mutating Asn111 in TM III and Asn295 in TM VII on the binding of a series of agonists and antagonists, suggests that these two Asn residues may have a complementary role in the receptor isomerization. Due to the potential spatial proximity of these two residues identified by our three dimensional model (Fig. 3, A and B), this role may be through direct hydrogen bonding. Substitution of a serine residue would be expected to reduce and alanine to eliminate, hydrogen bonding associated with the asparagine residues. Thus one of the major determinants of the isomerization of the AT1A receptor from the R to R* form may be the breaking of hydrogen bonding between Asn111 in TM III and Asn295 in TM VII.
Inverse agonists are ligands which are postulated to posses a higher affinity for the inactive R form of the receptor when compared to the active R* and R*G forms and are capable of shifting the equilibrium from R*G by stabilizing the R conformation (for review, see Milligan et al. (42)). In the present study, the marked reduction in the binding affinity of losartan observed following substitution of either Asn111 or Asn295, raises the possibility that it is not an antagonist, but an inverse agonist.
A further interesting but more speculative implication of the present
study is that the interaction between Asn111 and
Asn295 may be fundamental in determining the apparent
peptide binding selectivity of AT1 and AT2
receptors. The pharmacological profile of the mutant receptors,
particularly of the peptides, was closer to that of an AT2
receptor (5-8), than an AT1. Only the change in the
affinity of CGP42112A is not entirely consistent with the typical
AT2 binding of peptides. This discrepancy may be due to the
fact that CGP42112A also contains non-peptide modifications and thus is
quite unlike Ang II, Ang III, or pNH2F6AII and
therefore would be less likely to share similar "peptide" binding
epitopes. Furthermore, in addition to displaying similar peptide
binding affinities, the binding of Ang II to AT2 receptors is also unaffected by the presence of guanine nucleotides (4, 6, 35,
43) as observed here for the mutant receptors. Finally, the cloning of
the AT2 receptor has now revealed that it also has an
asparagine residue in TM III at an equivalent position to
Asn111 of the AT1 receptor. However, in TM VII
of the AT2 receptor, a serine residue corresponds to the
asparagine at the equivalent 295 position in the AT1
receptor (Fig. 6). This therefore raises the possibility
that the loss (AT2 receptor) or gain (AT1
receptor) of this interaction between TM III and TM VII is a major
determinant of peptide selectivity between Ang II receptor subtypes. It
also raises the possibility that in vivo AT2
receptors may be constitutively active.
In summary, we have established that substitution of either Asn111 in TM III or Asn295 in TM VII leads to an identical conformational change in the receptor. The conformation adopted appears to be the active (R* form) of the receptor. The spatial proximity of these two residues suggests that the mechanism responsible for this conformational change may be the breaking of a hydrogen bond interaction between these two residues. The proximity of these two helices has been postulated by us and others in three dimensional models based upon the projection map of bovine rhodopsin (27, 44, 45). Earlier models based upon the bacteriorhodopsin structure tend to result in TM III and TM VII being more distant (46). To our knowledge, this is the first evidence of a direct interaction between TM III and TM VII in a G-protein-coupled receptor.