From the
A conserved NPX
The type 1 (AT
Another interesting feature of the NPX
To determine the internalization kinetics of prelabeled receptors,
the cells were incubated with
Since binding studies revealed significant
differences in the agonist affinities of the mutant AT
Detailed analysis of the NPLFY sequence in the seventh
transmembrane domain of the rat AT
As discussed above, recent modeling
studies of G protein-coupled receptors have suggested that the
NPX
The functional importance
of these residues has been demonstrated, and the existence of such a
receptor supports the suggested structural and functional relationship
of these residues(27) . More detailed studies will be necessary
to clarify the proposed hinge function of Pro
It has been reported that the tyrosine
residue of the NPX
The improved internalization kinetics of the triple
alanine mutant receptor strengthened our conclusion that Phe
Early structure-function studies on the
Ang II molecule revealed that the aromatic side groups of Tyr
The role of
clustered aromatic amino acids in stabilization of the tertiary
structure of proteins is well known(36) , and modeling studies
have suggested that such clusters provide an important frame for the
ligand binding site of many G protein-coupled
receptors(11, 14) . Since the conserved tryptophane and
phenylalanine residues in helices 4 and 6 that were suggested to
provide an aromatic floor for the binding sites of aminergic receptors
are also present in AT
The finding that the F301A receptor has much greater impairment
of binding affinity for Ang II and the nonpeptide antagonist losartan
than for the peptide antagonist is particularly interesting. The
Phe
In summary, this paper demonstrates
that Phe
The data are expressed as means ±
S.E. of three independent experiments each performed in duplicate.
The binding data are
expressed as means ± S.E. of three independent experiments each
performed in duplicate. The EC
Y sequence
that is located in the seventh transmembrane helix of many G
protein-coupled receptors has been predicted to participate in receptor
signaling and endocytosis. The role of this sequence (NPLFY) in
angiotensin II receptor function was studied in mutant and wild-type
rat type 1a angiotensin II receptors transiently expressed in COS-7
cells. The ability of the receptor to interact with G proteins and to
stimulate inositol phosphate responses was markedly impaired by alanine
replacement of Asn
and was reduced by replacement of
Pro
or Tyr
. The F301A mutant receptor
exhibited normal G protein coupling and inositol phosphate responses,
and the binding of the peptide antagonist,
[Sar
,Ile
]angiotensin II, was only
slightly affected. However, its affinity for angiotensin II and the
nonpeptide antagonist losartan was reduced by an order of a magnitude,
suggesting that angiotensin II and losartan share an intramembrane
binding site, possibly through their aromatic moieties. None of the
agonist-occupied mutant receptors, including Y302A and triple alanine
replacements of Phe
, Tyr
, and
Phe
, showed substantial changes in their internalization
kinetics. These findings demonstrate that the NPLFY sequence of the
type 1a angiotensin II receptor is not an important determinant of
agonist-induced internalization. However, the Phe
residue
contributes significantly to agonist binding, and Asn
is
required for normal receptor activation and signal transduction.
)
(
)receptor for
the vasoactive peptide Ang II is a member of the family of seven
transmembrane domain receptors(1, 2) . The AT
receptor has been reported to interact with several G proteins,
including G
, G
, and G
, but its
major physiological functions are expressed through
G
-mediated activation of phospholipase C followed by
phosphoinositide hydrolysis and Ca
signaling(1, 2, 3, 4, 5) .
During the last decade, hundreds of G protein-coupled receptors and
their subtypes have been cloned and sequenced(6, 7) .
Although these receptors all possess the basic seven-transmembrane
structure, the number of highly conserved amino acids shared by the
superfamily of G protein-coupled receptors is relatively
few(6, 8, 9) . One such conserved motif is the
characteristic NPX
Y sequence that is
located in the seventh transmembrane domain of most receptors, and in
the rat smooth muscle AT
receptor is
Asn
-Pro
-Leu
-Phe
-Tyr
(10) (Fig. 1).
Figure 1:
Amino acid sequence of the Ang II
molecule and localization of the conserved
NPXY sequence in the seventh transmembrane
domain of the rat AT
receptor. The position of the amino
acids is shown based on a recently published model of the human
-adrenergic receptor (9).
Several G protein-coupled receptor models
have been based on the structure of bacteriorhodopsin. This
heptahelical membrane protein shares no sequence homology with
mammalian G protein-coupled receptors, but it has an identical folding
pattern and functional resemblance to mammalian opsins(11) . The
recently reported low resolution structural image of bovine rhodopsin (12) has made it possible to further improve these
models(9, 13) . A recent model of aminergic G
protein-coupled receptors suggests that the
NPXY sequence is ideally placed to receive
a signal from agonist-induced conformational changes in the ligand
binding region(9, 14) . This sequence is in close
proximity to the functionally important asparagine-aspartic acid pair
located in transmembrane segments 1 and 2 and may participate in
important hydrogen bonding interactions. Most of the available models
also predict that the highly conserved intramembrane proline residues,
which disrupt the
-helical structure of the transmembrane domains,
serve as hinges that participate in the agonist-induced conformation
change of G protein-coupled receptors(9, 11) . One such
proline residue is located within the seventh transmembrane domain in
the conserved NPX
Y sequence, and this
residue (Pro
) of the m
muscarinic receptor
has been found to be important for signal transduction(15) .
Y
sequence is its similarity to the NPXY internalization
sequence that was first described in the cytoplasmic segment of the low
density lipoprotein receptor(16) . In recent studies, the
tyrosine residue in this sequence was found to be essential for
sequestration of the
-adrenergic receptor (17) but not for
the internalization of the gastrin-releasing peptide
receptor(18) . The gastrin-releasing peptide receptor and the
AT
receptors, unlike the
-adrenergic receptor, contain
an additional aromatic amino acid (Phe
and
Phe
, respectively) in their
NPX
Y sequences(1, 18) . The
presence of such residue might be important since phenylalanine can
substitute for the tyrosine residue in the NPXY
internalization sequence of nutrient receptors(16) . The present
study was performed to analyze the role of the
NPX
Y sequence in ligand binding,
internalization, and signaling of G protein-coupled receptors,
utilizing the rat AT
receptor as a model to evaluate its
participation in these critical aspects of receptor function.
Materials
The cDNA clone (pCa18b) of the rat
smooth muscle AT receptor subcloned into the mammalian
expression vector pCDM8 (Invitrogen, San Diego, CA) was kindly provided
by Dr. Kenneth E. Bernstein(10) . Restriction enzymes were
obtained from Boehringer Mannheim 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 Ca
, 1 g/liter
bovine serum albumin, and 20 mM HEPES. Lipofectin,
lipofectamine and Opti-MEM I were from Life Technologies, Inc. Losartan
was a gift from Dr. P. C. Wong (DuPont, Wilmington, DE).
I-Ang II and
[
I-Sar
,Ile
]Ang II were
obtained from Hazleton Laboratories (Vienna, VA) or DuPont NEN;
[
H]inositol was from Amersham Corp.
Mutagenesis of the Rat Smooth Muscle AT
The rat ATReceptor cDNA
receptor cDNA was
subcloned into the mammalian expression vector pcDNAI/Amp (Invitrogen)
as described earlier(19) . Mutant rat AT
receptors
were created using the Mutagene kit (Bio-Rad, Hercules, CA), which is
based on the method of Kunkel et al.(20) . Each mutant
contained a silent restriction site to facilitate the screening of
colonies. Oligonucleotides were obtained from Midland Certified Reagent
Co. (Midland, TX). All mutations were verified by dideoxy sequencing
using Sequenase II (U. S. Biochemical Corp.).
Transient Transfection of COS-7 Cells
COS-7 cells
were cultured in Dulbecco's modified Eagle's medium
containing 2 mML-glutamine, 10% heat-inactivated
fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml
streptomycin. To determine inositol phosphate responses,
internalization kinetics, or
[Sar,Ile
]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 0.5-ml aliquots of Opti-MEM I
containing 8 µg of lipofectamine and 1 µg of plasmid DNA/well,
or with increasing amounts of cDNA as indicated in the legend to Fig. 4. The cells were incubated for 5-6 h in this
solution, and the medium was replaced with culture medium. For membrane
binding studies, 10
cells were grown on 100-mm culture
dishes for 3 days. Transfections were performed for 5-6 h using 5
ml of Opti-MEM I containing 16 µg/ml lipofectamine and 10 µg of
plasmid DNA. After transfection, the medium was replaced with the
culture medium. All experiments were performed 48 h after the
initiation of the transfection procedure.
Figure 4:
Correlation between AT receptor expression level and the amplitude of inositol phosphate
responses. COS-7 cells were plated in 24-well plates and transfected
with increasing quantities of wild-type AT
receptor DNA
(0.03-2.5 µg) using lipofectamine (16 µg/ml) or
lipofectin (10 µg/ml). For inositol phosphate measurements, the
cells were prelabeled for 24 h with [
H]inositol
and stimulated with 1 µM Ang II in the presence of 10
mM LiCl. The extracellular Ang II receptor expression level
was measured by analyzing
[
I-Sar
,Ile
]Ang II
displacement curves. Methodological details are described under
``Experimental Procedures.'' The combined InsP
and InsP
responses are shown as means ± range
of duplicates from a representative experiment of three similar
observations.
[Sar
To determine the expression level
and structural integrity of the mutant receptor, the number of Ang II
binding sites was determined by incubating the transfected cells with
[,Ile
]Ang II
Binding to Intact Cells
I-Sar
,Ile
]Ang II
(0.05-0.1 µCi/sample) and increasing concentrations of
unlabeled [Sar
,Ile
]Ang II in Medium
199 (HEPES) for 6 h at 4 °C. The cells were washed twice with
ice-cold Dulbecco's phosphate-buffered saline, and the
radioactivity associated with the cells was measured by
-spectrometry 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(21) .
Binding to COS-7 Cell Membranes
48 h after
transfection, the cells were washed and scraped into 1.5 ml of ice-cold
10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 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 MgCl
, and
20 mM Tris-HCl (pH 7.4)), and the protein content was
determined. Binding assays were performed in 0.2 ml of binding buffer
supplemented with 2 g/liter bovine serum albumin at 25 °C. Each
sample contained 0.05-0.1 µCi of
I-Ang II or
[
I-Sar
,Ile
]Ang II,
15-30 µg of crude membranes, and the indicated concentrations
of unlabeled [Sar
,Ile
]Ang II,
losartan, or Ang II in the presence or absence of 5 µM GTP
S as indicated. After a 90-min incubation at 25 °C,
the unbound tracer was removed by rapid filtration, and the bound
radioactivity was measured by
-spectrometry.
Inositol Phosphate Measurements
In these
experiments, the culture medium was replaced 24 h after transfection
with 0.5 ml of inositol-free Dulbecco's modified Eagle's
medium containing 1 g/liter bovine serum albumin, 20 µCi/ml
[H]inositol, 2.5% fetal bovine serum, 100 IU/ml
penicillin, and 100 µg/ml streptomycin as described
earlier(19) . After 24 h of labeling, the cells were washed
twice and incubated in inositol-free modified Medium 199 in the
presence of 10 mM LiCl for 30 min at 37 °C, and then
stimulated with 30 nM Ang II for 20 min or, in the case of
F301A, with 1 µM Ang II for 20 min. Incubations were
stopped by adding perchloric acid (5% (v/v) final concentration).
Inositol phosphates were extracted and analyzed by high performance
liquid chromatography as described previously(22) .
Internalization of Wild-type and Mutant AT
Before each experiment the medium was
replaced by Hepes-buffered Medium 199. To determine the internalization
kinetics of the mutant and wild-type ATReceptors
receptors
I-Ang II (0.05-0.1 µCi) was added in the same
medium, and the cells were incubated at 37 °C for the indicated
times. Incubations were stopped by placing the cells on ice and rapidly
washing them twice with 1 ml of ice-cold Dulbecco's
phosphate-buffered saline. The cells were incubated for 10 min in 0.5
ml of acid wash solution (150 mM NaCl, 50 mM acetic
acid) to remove the surface-bound radioligand. The supernatant
containing the acid-released radioactivity was collected, and the cells
were treated with 0.5 M NaOH and 0.05% SDS to solubilize the
acid-resistant (internalized) radioactivity. The radioactivities were
measured by
spectrometry, and the percent of internalized ligand
at each time point was calculated from the ratio of the acid-resistant
binding to the total (acid-resistant + acid-released) binding.
I-Ang II (0.05-0.1
µCi) in 0.25 ml Medium 199 for 3-4 h at 4 °C to permit
binding in the absence of receptor internalization. The unbound tracer
was removed by washing the cells twice with 1-ml aliquots of ice-cold
Dulbecco's phosphate-buffered saline. After addition of 0.5 ml of
warm (37 °C) Medium 199, the cells were incubated for the indicated
times at 37 °C to allow internalization to proceed. Incubations
were stopped by placing the cells on ice, and the medium containing the
tracer released during the incubation was collected and replaced with
0.5 ml of ice-cold acid wash solution. The extracellular
(acid-sensitive) and internalized tracer were measured as described
above. The total binding was calculated as the sum of the released
(into the medium), extracellular (acid-sensitive), and internalized
(acid-resistant) radioactivities. The internalized or released
radioactivity was expressed as a percent of the total binding at each
time point.
Expression of Mutant AT
[SarReceptors in
COS-7 Cells
,Ile
]Ang
II binding was measured in intact COS-7 cells transfected with mutant
or wild-type AT
receptors to determine the expression
level and the functional integrity of these receptors at the plasma
membrane. As shown in , all mutant receptors analyzed in
this study bound the antagonist radioligand with high affinity. It is
interesting to note that all mutants in which Phe
was
replaced (F301A, L300A/F301A, F301A/Y302A, F301A/Y302A/F304A) showed
slightly but consistently lower binding affinity than the other mutants
or the wild-type receptor. The expression levels of the mutant
receptors showed more significant variations. While alanine replacement
for Asn
and Tyr
had no major effect on
receptor expression, mutation of the three inner residues
(Pro
, Leu
, and Phe
) reduced
expression to approximately one-third of that of the wild-type receptor (). The effect of amino acid replacement on receptor
expression appeared to be additive, since L300A/F301A showed further
reduction compared with L300A and F301A, and F301A/Y302A and
F301A/Y302A/F304A showed progressively reduced expression levels
compared with F301A or Y302A.
Ang II Binding to COS-7 Cell Membranes Expressing
Wild-type or Mutant AT
Previous
reports showed that the type 1 Ang II receptors can couple to multiple
G proteins including GReceptors
, G
, and G
(1). To evaluate the ability of the AT
receptor to
couple to G proteins, we measured the effect of GTP
S on Ang II
binding in COS-7 cell membranes. Addition of GTP
S (5
µM) caused a 3.45 ± 0.38-fold (n =
3) decrease in the affinity of the wild-type receptor for Ang II,
indicating that the expressed AT
receptor is coupled to G
proteins. In the presence of the GTP analogue, when the receptor is
uncoupled from G proteins, the binding affinities of the N298A, P299A,
L300A, and Y302A mutants for Ang II were similar to that of the
wild-type receptor (). In the absence of GTP
S, the
affinities of the mutants for Ang II were lower than that of the
wild-type receptor, suggesting that receptor-G protein interaction is
affected in these mutants. The GTP
S-induced change in affinity was
most impaired in the N298A and P299A mutant receptors, but it was also
evident in the Y302A mutant (Fig. 2). Interestingly, despite its
near normal affinity for [Sar
,Ile
]Ang
II (), the F301A mutant receptor had significantly reduced
affinity for the physiological agonist, Ang II ().
Figure 2:
Effect of GTPS (5 µM) on
the binding affinity of Ang II for the wild-type (w.t.), N298A (N), P299A (P), L300A (L), F301A (F), and Y302A (Y) mutant receptors. The
GTP
S-induced change in the receptor affinity is shown as percent
of the shift measured for the wild-type receptor. GTP
S caused a
3.4-fold (100%) increase in the IC
of the wild-type
receptor (n = 3). For the F301A mutant
[
I-Sar
,Ile
]Ang II was
used as tracer since the
I-Ang II labeling of this
receptor was low. Under these conditions GTP
S caused a 1.9
± 0.2-fold (100%) increase in the IC
of Ang II for
the wild-type receptor (n = 3). Data are shown as means
± S.E. for three experiments performed in
duplicate.
Displacement of
[
Since both the expression levels () and the agonist affinity () of the F301A
receptor were impaired, it was difficult to accurately characterize
this mutant using I-Sar
,Ile
]Ang
II in COS-7 Cell Membranes Expressing F301A or Wild-type AT
Receptors
I-Ang II as radioligand. For this
reason, [
I-Sar
,Ile
]Ang
II was utilized to measure the binding properties of F301A. Consistent
with the data obtained in intact cells, the F301A mutant receptor
showed only slightly decreased affinity relative to the wild-type
receptor when [Sar
,Ile
]Ang II was
used to displace the radioligand (IC
: wild-type, 0.53
± 0.09 nMversus F301A, 0.92 ± 0.05
nM; n = 3) (Fig. 3, upper
panel). In contrast, the ability of the agonist ligand (Ang II) to
displace [
I-Sar
,Ile
]Ang
II was reduced 9.7 ± 0.9-fold in the F301A mutant
(IC
: wild-type, 3.0 ± 0.1 nMversus F301A, 29.5 ± 2.6 nM; n = 3) (Fig. 3, middle panel). This selective reduction of Ang
II binding affinity was not related to impaired G protein coupling,
since the effect of GTP
S on binding was similar to that observed
in the wild-type receptor (Fig. 2). The nonpeptide AT
receptor antagonist losartan inhibited
[
I-Sar
,Ile
]Ang II
binding to the wild-type receptor with an IC
of 26.6
± 1.4 nM (n = 3). However, the affinity
of the F301A mutant receptor for losartan was reduced 9.9 ±
2.5-fold (IC
, 263 ± 66 nM; n = 3) (Fig. 3, lower panel), similar to its
loss of affinity for the native agonist, Ang II.
Figure 3:
[I-Sar
,Ile
]Ang
II binding of the F301A mutant (
) and wild-type (
) rat
AT
receptors. The tracer was displaced with the indicated
concentrations of [Sar
,Ile
]Ang II (upper panel), Ang II (middle panel), or
losartan (lower panel). Data are shown as means ± S.E.
for three experiments performed in duplicate.
Effects on Inositol Phosphate Signaling
To
determine the ability of the mutant receptors to couple to
phospholipase C via G and related proteins, we measured the
inositol phosphate response of transfected COS-7 cells to Ang II in the
presence of LiCl. As reported earlier, under these experimental
conditions the major accumulating products of phosphoinositide
hydrolysis are InsP
and InsP
in AT
receptor-transfected COS-7 cells(19) . Since the
expression levels of the mutant AT
receptors showed
significant variations (), the relationship between
receptor expression and the amplitude of the maximal inositol phosphate
response was determined after transfecting COS-7 cells with increasing
amounts of the wild-type AT
receptor cDNA. Despite the
wide range of receptor expression in such cells, there was a linear
relationship (r = 0.98) between the measured
extracellular receptor sites and the inositol phosphate responses to
agonist stimulation (Fig. 4). This finding indicates that valid
comparisons between cells expressing mutant AT
receptors
can be made by normalizing their inositol phosphate responses to the
number of plasma membrane binding sites (Fig. 5, lower
panel).
Figure 5:
Ang II-induced inositol phosphate
responses of wild-type (w.t.), N298A (N), P299A (P), L300A (L), F301A (F), and Y302A (Y) mutant AT receptors. Cells were prelabeled
for 24 h with [
H]inositol and preincubated in the
presence of 10 mM LiCl as described under ``Experimental
Procedures.'' The
H radioactivity of InsP
(upper panel) or InsP
(middle
panel) fractions after a 20-min treatment with (hatchedbars) or without (openbars) Ang II in
the presence of 10 mM LiCl is shown. Maximally active
concentration of 30 nM Ang II was used for each receptor
except for F301A where 1 µM Ang II was used. The lower
panel shows the combined InsP
+ InsP
responses normalized to the number of
I-[Sar
,Ile
]Ang II
binding sites. These data are expressed as percent of the wild-type (w.t.) response, which was 38,600 ± 4,600 cpm/pmol
binding sites (n = 3) and are shown as means ±
S.E. from three independent experiments each performed in
duplicate.
The inositol phosphate responses of cells expressing
mutant AT receptors were measured after maximal agonist
stimulation with 30 nM Ang II. However, 1 µM Ang
II was added in studies on the F301A receptor due to its reduced
binding affinity for the native agonist. Single alanine replacements in
the NPLFY sequence resulted in mutants showing various degrees of
reduction of the inositol phosphate responses. The most prominent
decrease was detected in the N298A, P299A, and F301A mutants during Ang
II stimulation (Fig. 5). After normalization of the data to the
receptor expression level, the impaired response of cells transfected
with the F301A receptor was attributable to its lower expression level,
while the L300A receptor appeared to activate phospholipase C more
effectively than the wild-type receptor (Fig. 5, lower
panel). The most significant impairment of inositol phosphate
signaling was observed in COS-7 cells expressing the N298A receptor,
which showed more than 60% reduction of inositol phosphate
accumulation. However, the P299A and Y302A receptors also mediated
consistently reduced inositol phosphate responses (Fig. 5, lower panel).
receptors, a detailed analysis of the dose-dependence of their
signaling responses was performed. The EC
values for
inositol phosphate responses of the wild-type and mutant AT
receptors showed a good correlation with the respective IC
values for inhibition of radioligand binding by native Ang II (). For example the dose-response curve for Ang II-induced
inositol phosphate production of the F301A mutant receptor was shifted
to the right compared with that of the wild-type receptor, consistent
with the reduced agonist affinity of this mutant (Fig. 6).
However, the maximum level of stimulation mediated by the F301A
receptor showed no reduction when the data were normalized for the
reduced number of binding sites (Fig. 5, lower panel).
On the other hand, the higher EC
values observed in cells
expressing the N298A receptor (Fig. 6) and the P299A and Y302A
receptors () were paralleled by impaired G
protein-coupling. This is indicated by the reduced maximal response (Fig. 5, lower panel) and decreased GTP
S effect on
Ang II binding (Fig. 2) to these mutant receptors.
Figure 6:
Dose-response curve of InsP (upper panel) and InsP
(lower
curve) responses of the wild-type (
), F301A (
), and
N298A (
) rat AT
receptors. Data are expressed as
percent of the maximum response obtained after stimulation with 1
µM Ang II, and are representative of three similar
observations. In the experiment shown, the basal and maximally
stimulated levels of inositol phosphates were as follows.
InsP
, 598 cpm versus 9,297 cpm for the wild-type
AT
receptor, 651 versus 2,433 cpm for the N298A
receptor, and 451 cpm versus 2,879 cpm for F301A receptor;
InsP
, 264 cpm versus 2,321 cpm for the wild-type
receptor, 300 versus 1,092 cpm for the N298A, and 268 cpm versus 929 cpm for the F301A
receptor.
Internalization of Wild-type and Mutant AT
The ATReceptors in COS-7 Cells
receptor
expressed in COS-7 cells undergoes rapid agonist-induced
internalization, similar to that of the native receptors of smooth
muscle, adrenal glomerulosa, and other cell
types(2, 19, 23, 24) . Single alanine
replacements in the NPLFY sequence caused relatively minor impairment
of the internalization kinetics of the hormone-receptor complex (Fig. 7). While the rates of internalization of the N298A, F301A,
and Y302A receptors were somewhat slower than that of the wild-type
receptor, all mutant receptors showed rapid agonist-induced
internalization, and the quantity of internalized radioligand exceeded
that of extracellular binding after a 30-min incubation at 37 °C (Fig. 7).
Figure 7:
Internalization kinetics of wild-type
(), N298A (
), P299A (
, upper panel), L300A
(
), F301A (
, middle panel), and Y302A (
, lower panel) AT
receptors in the continuous
presence of
I-Ang II. The dashedline represents the wild-type curve on the lower panels.
Results are expressed as percent of the total binding for each time
point. Data are shown as means ± S.E. for three experiments
performed in duplicate.
In the low density lipoprotein receptor, the
tyrosine residue of the NPXY motif can be replaced by phenylalanine
with no loss of function of the internalization signal(16) .
Since the AT receptor Tyr
is preceded by a
phenylalanine, the Y302A mutant contains an NPLF sequence that meets
the criteria of an internalization signal. To evaluate the possibility
that the neighboring phenylalanine residues could substitute for
Tyr
when the latter is replaced with alanine, double
(F301A/Y302A) and triple (F301A/Y302A/F304A) alanine replacement
mutants were analyzed. Like the F301A mutant receptor, these mutants
showed reduced expression levels and slightly reduced antagonist
binding () but exhibited markedly impaired agonist binding
(data not shown). However, these mutant receptors underwent rapid
agonist-induced endocytosis, indicating that the
NPX
Y sequence is not a major determinant
of the internalization of the AT
receptor (Fig. 8).
Figure 8:
Internalization kinetics of wild-type
(), F301A/Y302A (
) and F301A/Y302A/F304A (
) mutant
receptors in the continuous presence of
I-Ang II. Results
are expressed as percent of the total binding for each time point. Data
are shown as means ± S.E. for three experiments performed in
duplicate.
Internalization Kinetics in Prelabeled Cells
To
further analyze the role of Tyr in the endocytosis of
AT
receptors, internalization kinetics were measured in
cells prelabeled with
I-Ang II. Prelabeling was performed
at 4 °C to prevent internalization of the radioligand as described
under ``Experimental Procedures.'' After warming the cells to
37 °C, more than 60% of the tracer bound to the wild-type receptor
internalized within 5 min (Fig. 9, upper panel) similar
to the previously reported rapid kinetics of endogenous AT
receptors(23, 24) . At the same time (5 min), less
than 20% of the radioactivity was released into the medium (Fig. 9, lower panel), indicating that dissociation of
the agonist from the receptor is relatively slow in comparison with the
rapid kinetics of the internalization process. The release of bound and
internalized radioactivity into the incubation medium exhibited
biphasic kinetics. As shown for the wild-type receptor (Fig. 9, lower panel), the initial phase of release, which is largely
due to dissociation of the surface-bound ligand, began to plateau by 5
min, reflecting the concomitant decrease in surface-binding by 5 min.
After 5 min, the release showed a second increase that was associated
with a progressive decrease in intracellular labeling. This
radioactivity must be derived from recycling of the internalized
tracer, since by 5 min, the extracellular binding was reduced to 20% of
its initial value, and the internalized radioactivity is the only pool
that can account for the second phase of release into the medium (Fig. 9).
Figure 9:
Internalization and release kinetics of I-Ang II in prelabeled COS-7 cells. COS-7 cells were
transfected with wild-type (
), Y302A mutant (
), or combined
F301A/Y302A/F304A mutant (
) rat smooth muscle AT
receptor. Cells were prelabeled with
I-Ang II at 4
°C, and kinetics of the Ang II internalization (upper
panel) and release of the tracer into the media (lower
panel) were measured at 37 °C as described under
``Experimental Procedures.'' Data are expressed as percent of
the total binding, and shown as means ± S.E. for three
independent experiments.
The initial rate of internalization of the Y302A
mutant receptor was slightly reduced under these conditions (Fig. 9, upper panel) in accordance with the slower
initial kinetics detected in the continuous presence of the
radiolabeled agonist (Fig. 7). The Y302A mutant also displayed a
moderate decrease in the dissociation and recycling kinetics (Fig. 9), indicating that the turnover of this receptor is
modestly impaired. The F301/Y302A/F304A mutant AT receptor
showed more rapid dissociation, which is consistent with the lower
affinity of this receptor (Fig. 9, lower panel). The
initial rate of internalization of this combined mutant receptor showed
a similar minor impairment as the Y302A mutant (Fig. 9, upper
panel). Despite the modest impairment of the receptor
internalization kinetics of the Tyr
mutant receptors, the
above data are not consistent with the hypothesis that the NPLFY
sequence of the AT
receptor serves as an internalization
signal in the same manner as the NPXY sequence of the low
density lipoprotein receptor.
receptor has revealed
that, in addition to its structural role, this region is also an
important functional determinant of receptor expression, affinity, and
signal transduction. Alanine replacement of each amino acid,
particularly Pro
, Leu
, and
Phe
, caused a reduction in the number of extracellular
binding sites. While the affinity of the expressed mutant receptors for
the peptide antagonist
[
I-Sar
,Ile
]Ang II
showed only minor variations, the affinity of the F301A mutant
AT
receptor for the native agonist, Ang II, and the
nonpeptide antagonist, losartan, was markedly reduced. This finding is
of interest since Ang II contains aromatic residues that are critical
for its binding to the receptor.
Y sequence has an important role in the
agonist-induced conformation change that leads to receptor
signaling(9, 14) . In agreement with this hypothesis,
alanine replacement of Asn
in the rat AT
receptor markedly reduced both coupling to inositol phosphate
production and interaction with G proteins as measured by the effect of
GTP
S on Ang II binding. The model also predicts that Asn
is in close proximity to the conserved Asp
residue
in the second transmembrane helix, and may participate in important
hydrogen bonding interactions. The demonstrated importance of
Asp
in AT
receptor signaling (19, 25) is consistent with the role of the
corresponding residue in the activation of many other G protein-coupled
receptors(8) . Although a recent study suggested that Asp
interacts with Tyr
in the AT
receptor
(26), more recent models relying on the recently published low
resolution crystal structure of bovine rhodopsin have suggested that
Asn
is in an appropriate location to participate in such
an interaction(9, 13, 14) . The present data are
in good agreement with this prediction. It is interesting to note that
the gonadotropin-releasing hormone receptor contains an Asn in the
second transmembrane domain and an Asp in the seventh intramembrane
helix, an arrangement that may represent a natural reciprocal mutation
of these conserved residues(27) .
in the
receptor activation process. In the present work, replacement of
Pro
with alanine, which would stabilize the
-helical
structure of the seventh transmembrane domain, caused relatively minor
impairment of the inositol phosphate responses. However, the G protein
coupling of this mutant receptor was significantly impaired, suggesting
that Pro
might be important for interaction with G
proteins other than G
. Similar discrepancies between the
inositol phosphate response and G protein coupling of the AT
receptor have been previously observed (19) and could
likewise reflect the fact that the type 1 Ang II receptor interacts
with multiple G proteins.
Y sequence is required
for agonist-induced sequestration of the
-adrenergic
receptor(17) . Our data demonstrate that replacement of this
tyrosine residue (Tyr
) with alanine has only a minor
effect on the internalization kinetics of the AT
receptor,
and similar findings were reported for the gastrin-releasing peptide
receptor(18) , another Ca
-mobilizing hormone
receptor. However, we also examined the potential role of nearby
aromatic amino acids. This was important since replacement of
Tyr
with alanine changes the NPLFY sequence to NPLFA,
which might still serve as a fully functional internalization signal
since NPXF can substitute for the NPXY sequence in
the low density lipoprotein receptor(16) . In fact, the mutant
AT
receptor containing alanine replacements for the
Phe
and Tyr
residues (F301A/Y302A) was well
internalized, and an additional triple alanine mutant receptor, in
which Phe
was also replaced (F301A/Y302A/F304A) exhibited
internalization kinetics even closer to those of the wild-type
receptor.
and Tyr
in the NPLFY sequence of the AT
receptor do not participate in agonist-induced internalization of
the receptor. The minor differences detected in the internalization
kinetics of the mutant receptors are probably due to an overall
conformational effect, perhaps affecting the position of the
cytoplasmic tail of the receptor, which contains sequences essential
for the agonist-induced internalization process(28) . Most of
the identified sequences in G protein-coupled receptors that regulate
agonist-induced endocytosis have been found to be serine-threonine rich
(28-33). These observations, and the inability of the NPLFY
sequence to serve as an internalization signal in the AT
receptor, suggest that the endocytosis of G protein-coupled
receptors is regulated by an alternative mechanism than that utilized
for the tyrosine-containing signal-regulated endocytosis of nutrient
and growth factor receptors.
and His
, together with the guanidine group of
Arg
, are essential for the binding of the octapeptide
hormone, while the phenyl group of Phe
carries the
information needed for the biological response(34) . The most
frequently used peptide antagonists of Ang II contain sarcosine in
position 1 (which increases the binding affinity of the ligand) but do
not contain Phe
, which is essential for activation of the
receptor. The present data, in accordance with previous
structure-function studies on the ligand(34) , suggest that the
binding of the amino-terminal (charged) portion of the Ang II molecule,
and that of the more carboxyl-terminal aromatic residues, are
stabilized by different interactions and may occupy binding sites
located in different regions of the receptor. Thus, the amino-terminal
charged end of Ang II and its peptide antagonists is likely to interact
with the recently-defined extracellular ligand binding region of the
AT
receptor(35) , whereas the aromatic residues of
Ang II probably interact with amino acids that include residues as
deeply located in the membrane as Phe
.
receptors, it is conceivable that
aromatic interactions between the intramembrane helices and Ang II are
important determinants of agonist binding. The proposed intramembrane
location of the binding site that interacts with the carboxyl-terminal
residues of the Ang II molecule is also in agreement with site-directed
mutagenesis studies that suggested that Lys
in the fifth
intracellular helix of the bovine AT
receptor is important
for ligand binding and might interact with the carboxyl-terminal
carboxyl group of the ligand(37) . The selective effect of F301A
on agonist binding suggests that interactions between the aromatic
residues of the hormone molecule and those of the intramembrane segment
of the AT
receptor are important for binding of the native
agonist. The binding of [Sar
,Ile
]Ang
II to the F301A mutant receptor is less affected since
Sar
-substituted Ang II peptides have increased affinity for
the AT
receptor due to the increased basicity of their
amino-terminal region(38) , while the aromatic interaction has
less effect due to the absence of the phenylalanine residue in position
8.
residue of the AT
receptor is likewise
important for the binding of losartan but not the peptide antagonist
(39). The participation of this residue in both Ang II and losartan
binding suggests that the binding of the agonist and the nonpeptide
antagonist share an overlapping site that is likely to be stabilized by
aromatic interactions. This finding accounts for the competitive nature
of Ang II and losartan binding to the AT
receptor. Despite
its reduced affinity for Ang II, the F301A receptor showed normal G
protein coupling and full inositol phosphate responses to maximal
agonist stimulation. This suggests that an additional site of
interaction is required for agonist activation of the receptor, in
accordance with the role of Phe
in both agonist and
nonpeptide antagonist binding.
in the NPLFY motif of the rat AT
receptor is essential for normal agonist binding to the receptor,
as well as for binding of the nonpeptide antagonist, losartan. These
findings may explain the competitive kinetics of the binding of many
nonpeptide Ang II antagonists, despite their additional dependence on
other residues that are not required for peptide
binding(39, 40) . Our data also support the predicted
importance of Asn
in the activation of G protein-coupled
receptors, but are not consistent with the general importance of the
conserved NPX
Y sequence in agonist-induced
endocytosis of these receptors.
Table: Parameters of
[I-Sar
,Ile
]Ang
II binding for wild-type and mutant AT
receptors
expressed in COS-7 cells
Table: IC of
I-Ang II binding in the absence or
presence of 5 µM GTP
S and EC
of
the combined InsP
+ InsP
responses for wild-type and mutant AT
receptors expressed in COS-7 cells
values are shown as means
± S.E. from independent dose-response curves of two to three
experiments.
receptor, type 1 angiotensin II receptor; Ang II, angiotensin II;
InsP
, inositol bisphosphate; InsP
, inositol
trisphosphate; GTP
S, guanosine 5`-3-O-(thio)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.