From the
The angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors
belong to the seven transmembrane receptor superfamily. Previous
studies have suggested that the AT1R couples to a G
The vasopressor hormone angiotensin II (Ang II)
The association between an agonist-bound seven transmembrane
receptor and a G protein is mediated by the formation of a ternary
complex which includes the agonist, the receptor, and the
heterotrimeric G protein. Recent studies using the chimeric
In contrast,
less is known about the involvement of the AT1R intracellular loops in
receptor signaling. Previous studies using site-directed and deletion
mutagenesis have identified several polar residues located in the
second and third intracellular loops, as well as two residues conserved
in most G protein-coupled receptors (Asp-74 in the second transmembrane
domain and Tyr-292 in the seventh transmembrane domain) that are
important for AT1R coupling to
G
The AT1R activates phospholipase C through a pertussis
toxin (PTX)-insensitive G protein (5) which belongs to the
G
The cDNA of the human AT1R and rat AT2R were cloned using the
polymerase chain reaction (PCR); fos-luciferase reporter
vector (p2FTL) was a gift from Dr. Gordon N. Gill (University of
California, San Diego);
Binding assays for intact cells were
performed as follows: transfected CHO cells were rinsed twice with PBS
and detached from the culture dish by incubating with 2 mM EDTA/PBS for 5 min at 37 °C. Cells were suspended in 2 mM EDTA, 0.1% BSA, PBS at 10
In the present study, we have utilized receptor chimeras
between AT1R and AT2R to demonstrate that the third intracellular loop
of AT1R plays a key role in receptor signaling. Substitution of either
the IC2 or the C-tail of the AT1R for the corresponding region of AT2R
did not affect AT1R function, while substitution of the IC3 caused a
loss of receptor function. Previous reports have shown that the AT2R
does not couple to G
After identifying the role that the IC3 plays in
AT1R signaling, we have attempted to identify which specific portions
of the IC3 determines its interaction with the G protein. Previous data
have shown that the IC3 of different receptors which couple to similar
functional pathways are heterogeneous in amino acid sequence and size
in the IC3 (4, 5, 17, 19), suggesting that secondary structure, rather
than the specific length of the domain, is important in G protein
coupling. Previous studies on the m3 muscarinic acetylcholine receptor
and
Previous studies from muscarinic cholinergic receptors and
adrenergic receptors have demonstrated that co-expression of the IC3
was able to specifically inhibit its homologous receptor-mediated
signaling(47, 48) . This evidence has suggested an
alternative strategy for developing receptor antagonists at the level
of the receptor and G protein interaction. In the case of AT1R, our
studies have demonstrated the critical role that the IC3 plays in AT1R
signaling. This loop therefore provides a feasible target for
antagonist drug design which would be highly specific in blocking
angiotensin II-induced signaling.
Recent reports on the function of
AT2R are
controversial(6, 7, 12, 13, 14, 49) .
In the cell line PC12W, some studies have suggested that the AT2R
mediates inhibition of tyrosine phosphatase via a pertussis
toxin-sensitive G protein(7, 14) , while others have
shown that it activates a tyrosine phosphatase(12, 13) .
Previous studies have demonstrated that the AT2R does not couple to
G
This table shows the amino
acid sequences of the AT1R, AT2R, and various chimeric AT1/AT2
receptors in the third intracellular loop. The numbers refer to amino
acid positions. The bold letters represent the amino acids from AT1R,
the roman letters represent the amino acids from AT2R.
We thank Drs. H. Arai and D. Pot for their helpful
comments and Dr. P. Garcia for his generous gift of G protein plasmids
and advice. We also thank Drs. B. Eide, Q. Hu, and T. Quinn for their
critical reading and helpful discussions of this manuscript and B.
Cheung for her help in the preparation of this manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
signaling pathway, whereas the AT2R does not associate with
G
. To identify the role that individual intracellular
domains play in AT1R function, AT1R/AT2R chimeric receptors were
prepared by substitution of intracellular loops. CHO cells expressing
these chimeras were used to test angiotensin II-induced c-fos expression and Ca
mobilization which are
involved in the AT1R signaling pathway through G
coupling.
Substitution of the second intracellular loop (IC2) and the cytoplasmic
tail between the two receptors did not affect AT1R function. However,
exchange of the third intracellular loop (IC3) resulted in the loss of
function in the AT1R and conferred to the AT2R the ability to
constitutively activate the fos promoter. These findings
suggest that the third intracellular loop of the AT1R is critical for
G
coupling. Substitution of discrete amino acid sequences
of the third intracellular loop indicate that its N-terminal and
C-terminal portions, especially the seven amino acids 219-225 in
the N-terminal portion, are important for AT1R function, and that the
intermediate portion of this loop is not required for G
coupling.
(
)plays an important role in the maintenance of
electrolyte homeostasis and cardiovascular function. Pharmacological
studies of Ang II nonpeptidic antagonists suggest that multiple Ang II
receptor subtypes exist(1, 2, 3) . Two major
subtypes of Ang II receptors, the Ang II type 1 receptor (AT1R) and the
Ang II type 2 receptor (AT2R), have been
cloned(4, 5, 6, 7) . Hydropathy analysis
reveals that both receptors belong to the seven transmembrane receptor
superfamily. Furthermore, the amino acid identity between AT1R and AT2R
is approximately 34%(4, 5, 6, 7) .
Recent studies have shown that the AT1R receptor, by coupling to
heterotrimeric G proteins, activates multiple signal transduction
pathways including: inositol phosphate production, intracellular
calcium mobilization, activation of protein kinase C, inhibition of
adenylate cyclase, activation of mitogen-activated protein kinase, and
induction of c-fos gene
expression(5, 8, 9, 10, 11) .
However, the physiologic functions and signaling pathway of the AT2R
have not been well
defined(6, 7, 12, 13, 14) .
2/
2,
1B/
2,
1/
2 adrenergic receptors and
chimeric m2/m3 muscarinic cholinergic receptors have suggested that the
third intracellular loop (IC3), determines G protein coupling
specificity. Exchange of either the entire IC3 or even certain portions
of this loop confers G protein selectivity in these
receptors(15, 16, 17, 18, 19, 20, 21) .
Conversely, studies using chimeras of two other seven transmembrane
receptors, the thyrotropin stimulating hormone receptor and
2-adrenergic receptor, have indicated that the second
intracellular loop (IC2) of the thyrotropin stimulating hormone
receptor is important for G protein association (22).
(23, 24, 25, 26) . However,
these studies have not revealed the role that the individual
intracellular loops themselves play in the coupling of the G protein to
the AT1R.
subfamily(27, 28) . Previous studies have
demonstrated that activation of phospholipase C by this receptor
induces a multisignal pathway which includes Ca
mobilization and c-fos expression. In the current study,
we have attempted to assess the contribution of the intracellular
domains of AT1R to its interaction with G
. Taking advantage
of the apparent inability of the AT2R to interact with
G
(6, 7) , we constructed several chimeric
AT1R/AT2R receptors in which the intracellular loops of the AT1R were
substituted by the corresponding loops from the AT2R. One of these
chimeras, a loss-of-function mutant, was used to construct
regain-of-function chimeric receptors in order to identify the discrete
amino acid sequences responsible for AT1R signaling. Our data have
demonstrated that the IC3 of AT1R is a determinate loop for G
coupling and that the N-terminal and C-terminal portions of the
IC3 to be important for AT1R signaling.
I-[Sar
,Ile
]angiotensin
II was purchased from Amersham; LipofectAMINE
reagent was
manufactured by Life Technologies, Inc.; a luciferase assay system was
obtained from Promega; an AutoRead Sequencing Kit was from Pharmacia;
restriction enzymes were purchased from Boehringer and New England
Biolabs; a PCR kit was from New England Biolabs; pertussis toxin was
from BIOMOL; and other reagents were from Sigma.
Construction of Chimeric AT1R/AT2R Receptors
Unique
restriction enzyme sites were introduced into the AT1R and AT2R
receptor genes without change in the encoded amino acid residues by PCR
(29, 30) in a modified pKS vector in which HindIII, BamHI, and KpnI sites had been
previously deleted. Sites for the following restriction enzymes: HindIII, StuI, BclI (native), BamHI, and NdeI were introduced into the putative
third, fourth, fifth, sixth, and seventh transmembrane regions of the
AT1R receptor gene, respectively. An NdeI site was introduced
into the sixth transmembrane region in the AT2R receptor gene. The cDNA
fragment encoding the IC2 of AT2R was amplified by using
oligonucleotide primers containing HindIII and StuI
restriction sites. The oligonucleotide primers for amplification of the
IC3 of AT2R and C-tail of AT2R also have the corresponding restriction
sites. In the construction of the IC3 partial region chimeras,
synthesized oligonucleotides encoding the chimeric IC3 were used as a
template for PCR amplification. These cDNA fragments were subcloned
into pKS
/AT1R vector using corresponding restriction
sites. The AT2R fragment containing the IC3 of AT1R between KpnI (native site located in IC2) and NdeI was
amplified by overlap extension PCR, and this fragment was subcloned
into the pKS
/AT2R vector. The DNA sequences of these
chimeras were confirmed by dideoxynucleotide sequencing. Finally, the
chimeric construct was subcloned into a eukaryotic expression vector
under a hybrid promoter (SV40/HTLV-1) designated pBJ-1 or under a
cytomegalovirus promoter, pCMV1.
Cell Culture and Transfections
COS-7 cells and CHO
cells were grown in Dulbecco's modified Eagle's medium and
Ham's F-12 medium, respectively, each supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin
at 37 °C in a CO incubator. COS-7 or CHO cells lacking
endogenous angiotensin II receptors were transiently transfected by
lipofection as described in the manufacturer's protocol provided
by Life Technologies, Inc.
Ligand Binding
Membranes from COS-7 cells
expressing different chimeric receptors were prepared as described
previously (25). Competition binding assays were carried out by
incubation of membrane proteins with 0.5 nMI-[Sar
,Ile
]Ang II and
various concentrations of nonradioactive
[Sar
,Ile
]Ang II
(10
-10
M) at room
temperature for 1 h. Cell-bound radioactivity was separated from free
ligand by filtration through Whatman GF/C filters presoaked in 1% BSA. K
values were calculated according to
Cheng and Prusoff(31) .
cells/ml. For the binding
assay, 2.5
10
cells were incubated with 0.1
nM
I-[Sar
,Ile
]Ang II in
the absence or presence of a 1,000-fold excess of Ang II for 1 h at
room temperature. To separate ligand-bound cells from free ligand,
cells were loaded on a 28.5% Ficoll cushion and centrifuged at 3,500
g for 5 min.
fos-luciferase Assay
1.5 10
CHO cells were seeded in 12-well plates and co-transfected with
wild type or chimera receptor DNA and fos-luciferase reporter
gene (p2FTL) using the method described above. The fos-luciferase reporter gene consists of two copies of the
c-fos 5`-regulated enhancer element (-357 to
-276), the herpes simplex virus thymidine kinase (TK) gene
promoter (-200 to +70), and luciferase
gene(32, 33) . The ratio of receptor and fos-luciferase DNA was 1:1. Forty-eight hours after
transfection, transfected cells were incubated with Ham's F-12
medium with 0.5% BSA for 24 h. Quiescent cells were treated with 100
nM or 1 mM Ang II for 3.5 h, washed with PBS, and
lysed for 15 min with 200 µl of cell lysis buffer at room
temperature. 10 µl of cell extract was mixed with 100 µl of
luciferase assay reagent, and the light produced was measured for 5 s
using a Monolight Luminometer (Analytical Luminescence Laboratory).
Measurement of Intracellular Free
Calcium
Intracellular Ca concentration was
determined using the calcium-sensitive dye Fura-2. Confluent CHO cells
transfected with receptor were detached from plates with 2 mM
EDTA/PBS, spun at 1,200 rpm for 5 min, and then resuspended in
Ham's F-12 medium with 25 nM HEPES (pH 7.4) and 1 mg/ml
BSA and incubated in the dark with 4 mM Fura-2/AM at 1
10
cells/ml for 20 min at 37 °C in a CO
incubator. The Fura-2-loaded cells were then washed and
resuspended in the same medium at 1
10
cells/ml.
The fluorescence was measured after 100 nM Ang II treatment
with a CAF-2000 Spectrofluorometer (Hitachi Corp.) with excitation at
340 nm and 380 nm and emission at 500 nm.
Identification of the IC3 As a Key Loop for AT1R
Receptor Signaling
Previous studies of seven transmembrane
receptors have shown that either the IC3 or IC2 is crucial in
determining G protein
coupling(15, 16, 17, 18, 19, 20, 21, 22) .
In order to identify which intracellular loop of the AT1R directs its
interaction with the G protein, we constructed chimeric receptors by
switching these loops between the AT1R and AT2R. As shown in Fig. 1, the C1 chimera was prepared by replacing the amino acid
residues 125-144 of AT1R for residues 141-159 of AT2R. In
chimera C2, the amino acid residues 219-236 of AT1R were replaced
by residues 235-252 of AT2R. Chimera C3 contains the cytoplasmic
tail, amino acid residues 315-363, of the AT2R. To assess the
effect of these loop and tail substitutions, the ability of these three
chimeric receptors to signal in response to Ang II treatment was
determined. Ang II-induced c-fos gene expression was measured
by transiently co-transfecting the chimeric receptor and fos-luciferase reporter gene into CHO cells. The fos-luciferase reporter construct contains the serum response
element of the c-fos promoter (32, 33) which
previous studies have demonstrated to be sufficient for Ang II-induced
activation of c-fos promoter(11) . The ability of the
receptor chimeras to induce c-fos gene expression was
determined by measuring the increase in fos-luciferase
activity in lysates of transfected cells following Ang II treatment
(luciferase assay). This method provided a sensitive and quantitative in vivo assay for comparing the ability of Ang II to activate
the chimeric receptors with that of the wild type AT1R. As shown in Fig. 2, Ang II stimulation of wild type AT1R resulted in an
8-9-fold expression of c-fos, whereas the wild type AT2R
did not induce c-fos expression. Chimeras C1 and C3, in which
the IC2 and C-tail have been replaced, respectively, were still able to
mediate Ang II-induced c-fos expression. In contrast, Ang
II-induced c-fos expression was almost completely absent in
CHO cells expressing chimera C2, an AT1R chimera with the IC3 of AT2R (Fig. 2). To identify the effect of these chimeric receptors on
Ca/inositol 1,4,5-trisphosphate signaling, Ang
II-induced Ca
mobilization in transfected CHO cells
was examined by measuring the change in intracellular free calcium
concentration using Fura-2 as a Ca
indicator (Fig. 3). 100 nM Ang II evoked a rapid, transient
increase in the Ca
concentration in the CHO cells
transfected with the wild type AT1R, chimeras C1 and C3, but not in
cells transfected with C2.
Figure 1:
AT1R and AT2R chimeras. The chimeric
receptors were prepared as described under ``Materials and
Methods.'' In chimera C1, amino acids 125-144 of AT1R were
substituted for amino acids 141-159 of the AT2R. The chimera C2
was prepared by replacing amino acids 219-236 from AT1R with
amino acids 235-252 from AT2R. In chimera C3, the C-tail (amino
acids 300-359) was replaced by the equivalent portion of AT2R
(amino acids 315-363). Finally, chimera C10 was constructed by
replacing amino acids 235-256 (IC3) of AT2R with amino acids
219-240 of AT1R.
Figure 2:
Ang II-induced c-fos expression.
CHO cells expressing the AT1R, AT2R, or AT1R chimeras C1, C2, and the
C-tail chimera C3, were assayed for their ability to activate the fos-luciferase reporter gene following Ang II treatment.
Results are expressed as percentage over basal levels of fos-luciferase activity. Vector refers to the expression
vector without the AT1R cDNA. AII indicates angiotensin II.
This result is representative of three independent experiments. Each
determination was done in triplicate.
Figure 3:
Ang II-induced changes in intracellular
[Ca] in CHO cells transfected with
different receptor chimeras. CHO cells transfected with the different
receptor chimeras were loaded with Fura-2 and exposed to 100 nM Ang II in the presence of EDTA. The changes in Ca
concentration were measured at different times following Ang II
stimulation. The arrows indicate the time of agonist addition. AII indicates angiotensin II.
To eliminate the possibility that the
observed changes in function of chimeric receptors were a consequence
of lower receptor expression levels or failure to bind ligand, we
performed binding assays on COS cell membranes and intact CHO cells
expressing chimeric receptors. As shown in , the three
chimeric receptors C1, C2, and C3 specifically bound to antagonist I-[Sar
,Ile
]Ang II with
similar affinity (K
) as the wild type
AT1R. Maximal ligand binding values in COS and CHO cells indicated that
the expression levels of different chimeric receptors were comparable
to that of the wild type, suggesting that the failure of the chimera C2
to respond to Ang II results from the inability of this receptor to
activate the G
protein.
Constitutive Activation of fos Promoter by a Chimeric
AT2R Containing the IC3 of AT1R
To determine whether the IC3 of
AT1R is by itself sufficient to confer G-mediated signaling
to AT2R, we constructed a chimeric AT2R receptor (C10) in which the
amino acids 235-256 of AT2R were replaced by the corresponding
region (amino acids 219-240) of AT1R (Fig. 1). As shown in Fig. 4A, the ability of chimera C10 to bind Ang II was
significantly lower than that of the wild type AT2R and AT1R. However,
co-transfection of this chimera with the fos-luciferase
reporter gene into CHO cells resulted in a high basal value of fos-luciferase activity, suggesting a constitutive activation
of the fos promoter in a ligand independent fashion (Fig. 4B). To confirm this result, we co-transfected
different amounts of wild type AT1R, AT2R, or C10 receptor DNA with the
same amount of p2FTL (0.2 µg/well) into CHO cells and then measured
both basal and Ang II-induced fos-luciferase activity in these
cells. Fig. 5shows that in cells expressing chimera C10, the
basal level of fos-luciferase activity increased directly with
the amount of C10 DNA used in the transfection. The levels of fos-luciferase activity induced by C10 was comparable to that
mediated by the wild type AT1R after Ang II stimulation. In contrast,
no increase in basal level fos-luciferase activity was
detected in wild type AT1R and AT2R transfectants. Since both previous
studies (11) and our data have demonstrated that the Ang
II-induced c-fos expression is mediated by a PTX-insensitive G
protein (G
), we attempted to determine if the c-fos expression mediated by chimera C10 was also through coupling to
G
. As shown in Fig. 6, treatment with PTX did not
inhibit C10-induced expression of c-fos, suggesting that the
c-fos expression mediated by this receptor chimera occurs
through G
.
Figure 4:
Ang II binding and c-fos expression in CHO cells expressing the AT2R chimera (C10) encoding
the IC3 from AT1R. A, Ang II binding competition assay using
whole cells expressing the different receptor constructs. B,
activation of the fos-luciferase reporter gene following
stimulation of the wild type and chimeric receptors with Ang II. AII indicates angiotensin II. These graphs represent the
results of three different experiments performed in
triplicate.
Figure 5:
Constitutive activation of fos promoter by the AT2R chimera C10. CHO cells transfected with
different amounts of the AT2R chimera encoding the IC3 of AT1R were
tested for their ability to induce fos expression using the fos-luciferase assay. Basal refers to the amount of
luciferase activity in the cells prior to addition of Ang II. Response corresponds to levels of luciferase activity
following addition of Ang II. This graph represents the results of two
independent experiments performed in
triplicate.
Figure 6:
Effect of treatment with PTX on AT2
chimera C10-induced c-fos expression. CHO cells transfected
with AT2 chimeric receptor and AT1R were serum-starved for 24 h in the
absence or presence of PTX (20 ng/ml); basal and Ang II-induced
c-fos expression was measured by luciferase assay. These data
present two different experiments performed in
triplicate.
The change in intracellular free
Ca mediated by chimera C10 was also measured, but Ang
II-induced transient increase in Ca
concentration was
not observed. This lack of calcium mobilization was also observed in
CHO cells transiently transfected with a constitutively active mutant
G
(data not shown). This mutant G
is
known to constitutively activate phospholipase C(34) . One
potential explanation for this result is that the constitutive
activation of phospholipase C leads to depletion of intracellular
calcium storage(35, 36) . Taken together, substitution
of the IC3 conferred on AT2R the ability constitutively to activate the fos promoter, suggesting that the IC3 of AT1R plays a
determinate role in G
association.
Partial Replacement of the IC3 Defines Essential Portions
Required for AT1R Signaling
Our study has identified the IC3 of
AT1R to be an important loop for G protein coupling. To
further explore which portions within the IC3 are essential for AT1R
function, we substituted discrete sequences of the IC3 from AT1R back
into the loss-of-function receptor mutant (chimera C2) to determine
which amino acid residues were required to regain function (). shows that all receptor chimeras bound to
I-[Sar
,Ile
]Ang II with
affinities similar to that of the wild type AT1R and with similar
maximal levels of binding in COS and CHO cells. The ability of these
chimeras to activate the fos promoter and mobilize
intracellular free calcium was tested as shown in , Fig. 3, and Fig. 7. The C4 chimera, which was constructed
by substituting amino acids 219-225 in the N-terminal portion of
the IC3 of AT1R into chimera C2, resulted in approximately a 57%
recovery of the ability to activate the fos promoter. C5, in
which the amino acids 226-236 in the C-terminal portion of the
IC3 from chimera C2 were replaced by the corresponding region of AT1R,
yielded approximately 30% functional recovery. However, replacement of
amino acids 226-231 in the intermediate portion of IC3 from AT1R
back into C2 (chimera C6) did not produce a gain of function. A limited
replacement of the residues 234-236 adjacent to the sixth
transmembrane region (chimera C7), resulted in a 24% gain in the
ability of Ang II to induce c-fos promoter activity. These
results suggest that residues adjacent to the transmembrane regions of
the IC3 of AT1R are important for G
signaling. To further
confirm these results, we constructed two additional chimeras: C8, in
which amino acids 219-225 and amino acids 226-231 of AT1R
were replaced into C2, and C9, in which amino acids 219-225 and
amino acids 234-236 from AT1R were replaced into C2. As shown in and Fig. 7, chimera C8 mediated a similar function as
the chimera C4 in which only amino acids 219-225 of IC3 from AT1R
were replaced back. Interestingly, chimera C9 resulted in a 92%
recovery of wild type receptor function. These data support the
conclusion that the N-terminal portion and C-terminal portion,
especially the seven amino acids 219-225 adjacent to the fifth
transmembrane region of the receptor, are critical for signaling, and
amino acids 226-231 in the intermediate portion of IC3 are not
essential for G
signaling by AT1R.
Figure 7:
Ang II-induced c-fos expression
in CHO cells expressing different IC3 domain chimeras. CHO cells
transfected with different AT1R chimeras were used to test the ability
of receptors to induce c-fos transcription by Ang II
stimulation using the fos-luciferase assay. AII represents angiotensin II. The results are expressed in percentage
over basal and are representative of three different experiments
performed in triplicate.
and fails to activate the
phospholipase C signaling pathway(6, 7) . Here, we have
found that replacement of the IC3 of AT2R with the corresponding region
of AT1R converts the AT2R into a constitutively active mutant which
activates the fos promoter by G
coupling. This
result is consistent with previous chimeric receptor studies of the
adrenergic and muscarinic cholinergic receptors(15, 16, 17, 18, 19, 20, 21) which show
that these receptors also specifically coupled to G proteins through
their IC3 domains.
1 adrenergic receptor have demonstrated that the N-terminal
segment of the IC3 plays a major role in determining the selectivity of
receptor coupling to a G protein(16, 18) . Secondary
structure analysis and insertion mutagenesis (37, 38) predict that a portion of this loop forms an
amphipathic
-helix. Studies of peptides, including GTPase
stimulation peptides and peptides corresponding to the sequences of the
IC3, also indicate that these peptides may form a charged surface of an
amphipathic
-helix which is very important for activation of G
proteins(39, 40, 41) . Permutation and amino
acid insertion of these peptides suggest that the relative orientation
of basic residues within the proposed
-helix is important for
GTPase stimulation(42, 43) . In the present studies, by
using a regain-of-function strategy, we substituted selected sequences
of the IC3 from AT1R back into a loss-of-function mutant (chimera C2)
and found that seven amino acids (219-225) in the N-terminal
portion are critical for AT1R function. Substitution of this domain
back into chimera C2 produces a 57% recovery of the AT1R function. The
amino acid identity of the seven amino acids in this portion between
the AT1R and AT2R is 43%. The
-helix model predicts that the two
receptors differ in their distribution of hydrophobic and hydrophilic
residues. This structural model shows that in the N-terminal portion of
the AT1R, all basic residues are located on one side, and uncharged
residues are clustered on the opposite side, forming an amphipathic
-helix (Fig. 8). A similar
-helix conformation is
predicted in the N-terminal portion of the IC3 from m3 muscarinic
cholinergic receptor which also couples to G
(38) .
Our data from alanine scanning mutagenesis of the IC3 indicates that
substitution of individual amino acids for alanine in this region is
not sufficient for altering receptor function (data not shown).
However, deletion of this region causes a loss of function(26) .
Altogether, our data support the
-helix hypothesis, suggesting
that the
-helix conformation in this portion may be important for
G protein recognition.
Figure 8:
Putative -helix model of the
N-terminal portion of the IC3 of the AT1R and AT2R. Charged amino acids
are shown with white letters on black background, hydrophilic
amino acids are shown in hollow letters, and hydrophobic amino
acids are shown in solid black
letters.
We also found that three amino acids in the
C-terminal portion of IC3 play a role in AT1R signaling. Previous
mutation studies of the adrenergic receptors have also shown that this
region of the IC3 is important for the agonist-activated conformation
switch which results in receptor activation, and mutation of several
single amino acids located in this portion of the 1B and
2
adrenergic receptor have resulted in constitutively active
receptors(44, 45) . Our results agree with previous
observations suggesting that the N-terminal portion and C-terminal
portions of the IC3 are important in
1B adrenergic receptor and m3
muscarinic receptor coupling to G
(17, 46) .
(6, 7) . In our present study, substitution
of the IC3 between the two angiotensin II receptors results in the
inability of AT1R to couple to G
and confers the ability of
AT2R to constitutively activate the fos promoter. These
results imply that there is a possibility that the IC3 of AT2R may be
involved in coupling a unique G protein. The peptides corresponding to
the IC3 of AT2R may be used to find the G
subunit which couples to
AT2R as previously done in the
2 adrenergic
receptor(50, 51) . Once the nature of the AT2R
interaction with its specific G protein has been characterized, further
studies identifying the function and signaling pathways of the AT2R can
be pursued.
Table: Amino acid sequence of chimeric angiotensin II
receptors in third intracellular loop
Table: Ligand binding parameters of chimeric
angiotensin II receptors
, q subfamily of G protein;
G
,
subunit of G
protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.