From the
The rat type 1a (AT
The rat type 1a (AT
The Tyr
The type 1 Ang II receptors, like other G
protein-coupled receptors, undergo rapid endocytosis following agonist
binding
(22, 23, 24) . Although receptor
internalization and signaling both require the active conformation of
the receptor, the structural determinants of the internalization and
signaling processes are not identical
(6, 10, 25) . For example, we have recently
identified a sequence in the cytoplasmic tail of the rat AT
The data presented in this paper provide direct evidence
for the importance of the highly conserved tyrosine residue located in
the cytoplasmic aspect of the fifth transmembrane domain in the
activation process of a heptahelical receptor. These data are in
accordance with the predicted role of this residue based on its
location in modeling studies of G protein-coupled receptors. The
parallel impairment of the internalization, signaling, and G protein
coupling of the mutant AT
) angiotensin II (Ang II)
receptor contains a highly conserved tyrosine residue in the fifth
transmembrane region that is present in most G protein-coupled
receptors. The role of this amino acid in AT
receptor
activation was analyzed in a mutant receptor (Y215F) created by
replacing Tyr
with phenylalanine. The mutant receptor was
highly expressed in transfected COS-7 cells, and its binding affinity
for the peptide antagonist
[Sar
,Ile
]Ang II was similar to that
of the wild type receptor. Although the structural integrity of the
peptide ligand binding domain was preserved in the Y215F mutant
receptor, its affinity for the native agonist, Ang II, was
significantly reduced. Also, whereas guanosine
5`-3- O-(thio)triphosphate markedly reduced Ang II binding to
the wild type receptor, it had little effect on agonist binding to the
mutant receptor. Agonist-induced internalization of the mutant receptor
was also impaired, and its ability to mediate inositol phosphate
responses to Ang II stimulation was abolished. The concomitant
decreases in receptor internalization and G protein-mediated signaling
of the Y215F mutant receptor indicate that Tyr
has a
critical role in AT
receptor activation. In view of its
conservation among members of the seven transmembrane domain receptor
superfamily, this residue is likely to be of general importance in
signal transduction from G protein-coupled receptors.
)
(
)
Ang II
receptor is a heptahelical, G protein-coupled receptor and is widely
distributed in cardiovascular, neural, and endocrine cells
(1, 2) . Binding of Ang II to the AT
receptor is followed by phospholipase C activation and
Ca
signaling via the G
family of G
proteins
(2, 3, 4) . Although all G
protein-coupled receptors share the basic seven-transmembrane domain
structure, relatively few amino acids are highly conserved among the
numerous receptors of this type (Fig. 1). One of these conserved
amino acids is a tyrosine residue located in the fifth transmembrane
helix, adjacent to the amino-terminal end of the third cytoplasmic
loop. The location of this amino acid, which is Tyr
in
the rat AT
receptor, suggests that it might play an
important role in the receptor activation process. The amino-terminal
region of the third cytoplasmic loop adjacent to this residue has been
shown to be important in the signal generation and internalization of
several G protein-coupled receptors
(5, 6, 7) .
Furthermore, modeling of G protein-coupled receptors based on the low
resolution crystal structure of bovine rhodopsin molecule has suggested
that this residue is in molecular proximity to regions that are
important in receptor activation, including the conserved
acidic-arginine-aromatic (DRY) triplet of the second intracellular loop
(8, 9) . We have shown previously that AT
receptor internalization and signal transduction have different
structural requirements
(10) . However, a 6-amino acid deletion
of the amino-terminal end of the third cytoplasmic loop, which included
Tyr
, prevented both AT
receptor
internalization and signaling responses. To evaluate the role of the
highly conserved Tyr
residue in the activation process of
G protein-coupled receptors, we created a mutant receptor by
substituting Tyr
with phenylalanine (Y215F). This mutant
receptor was expressed in COS-7 cells and its binding, internalization,
and signaling properties were compared with those of the wild type
AT
receptor.
Figure 1:
Conserved amino acids in the rat
AT receptor. The helices were positioned and the most
conserved residues were selected based on a comparative study and
modeling of more than 200 G protein-coupled receptors (9,
27).
Materials
The cDNA clone (pCa18b) of the rat
smooth muscle ATreceptor subcloned into the mammalian
expression vector pCDM8 (Invitrogen, San Diego, CA) was kindly provided
by Dr. Kenneth E. Bernstein
(1) . 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. Lipofectamine was from Life Technologies, Inc.
I-Ang II and
I-[Sar
,Ile
]Ang II were
obtained from Hazleton Laboratories (Vienna, VA), and
[
H]inositol was from Amersham Corp.
Mutagenesis and Expression of the Rat Smooth Muscle
AT
A HindIII- NotI
fragment of the receptor cDNA was subcloned into pcDNAI/Amp
(Invitrogen, San Diego, CA) as described earlier
(10) . The
Y215F mutant rat ATReceptor cDNA
receptor was created using the
Mutagene kit (Bio-Rad), which is based on the method of Kunkel et
al. (11) . The sequence of the mutant colony was identified
by dideoxy sequencing using Sequenase II (Amersham-U. S. Biochemical
Corp.). The wild type and mutant AT
receptor cDNA were
transiently expressed in COS-7 using Lipofectamine as described
previously
(12) .
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
(10) . 24 h later the cells were washed twice, incubated in
inositol-free modified Medium 199 in the presence of 10 mM
LiCl for 30 min at 37 °C, and stimulated with 30 nM Ang II
for 20 min. Inositol phosphates were extracted and analyzed by high
pressure liquid chromatography as described previously
(13) .
Ang II Binding to COS-7 Cells and Membranes
48 h
after transfection,
I-[Sar
,Ile
]Ang II
binding was performed with intact COS-7 cells at 4 °C as described
previously
(10) , and the binding parameters were determined for
a one site model using the computer program LIGAND
(14) . To
measure binding to COS-7 cell membranes, the transfected cells were
washed with ice-cold phosphate-buffered saline and scraped into 3 ml of
ice-cold 10 mM Tris/HCl (pH 7.4), 1 mM EDTA, then
lysed by freezing on dry ice. Crude membranes were prepared by
centrifuging the samples at 16,000
g. The pellet was
washed in the same medium, and the protein content was determined.
Binding assays were performed in 0.2 ml of binding buffer (containing
100 mM NaCl, 5 mM MgCl
, 2 g/liter bovine
serum albumin, and 20 mM Tris/HCl (pH 7.4)) at 25 °C. Each
sample contained approximately 0.05 µCi
I-Ang II or
I-[Sar
,Ile
]Ang II,
15-25 µg of crude membranes, selected concentrations of
unlabeled Ang II or [Sar
,Ile
]Ang II,
and the indicated concentrations of GTP
S. After 90-min incubation
at 25 °C the unbound tracer was removed by rapid filtration and the
bound radioactivity was measured by
-spectrometry. The IC
values of the displacement curves were estimated by nonlinear
least squares curve fitting using the computer program ALLFIT
(15) .
Receptor Internalization in Transiently Transfected COS-7
Cells
The experimental conditions for these studies were as
described previously
(10) . Briefly, the internalization
kinetics of the mutant and wild type rat ATreceptors were
measured 48 h after transfection using
I-Ang II
(0.05-0.1 µCi, 0.2-0.5 nM). The percent
internalization at each point was calculated from the ratio of the
acid-resistant (internalized) binding to the total (acid-resistant
+ acid-released) binding. The internalization rate was calculated
by determining the slope of the initial linear phase of the
internalization kinetic plot. Internalization rates of the wild type
AT
receptor were measured over the first three minutes,
and those of the Y215F mutant receptor were measured over the first 5
min.
Expression and Signal Transduction of AT
COS-7 cells were transfected with cDNA encoding
either the wild type or the Y215F mutant rat ATReceptors
receptor
to analyze the signaling and internalization properties of the two
receptors. To determine the surface expression level and structural
integrity of the Y215F receptor, binding of the Ang II antagonist
analog, [Sar
,Ile
]Ang II, to the
mutant and wild type receptors transiently expressed in intact COS-7
cells was measured at 4 °C. Scatchard analysis of the binding data
showed that the expression of Y215F receptors was 69.1 ± 7.1% of
that of the wild type receptor ( n = 3, Fig. 2,
upper left panel). The K
of
[Sar
,Ile
]Ang II under these
conditions was 1.6 ± 0.1 nM for the wild type receptor
and 1.7 ± 0.1 nM for Y215F mutant receptor ( n = 3). Despite the unimpaired affinity for
[Sar
,Ile
]Ang II and its relatively
high expression level, cells transfected with the Y215F mutant receptor
showed no detectable inositol phosphate responses after stimulation
with a maximally effective concentration of Ang II (Fig. 2).
Figure 2:
Expression and inositol phosphate
responses of Y215F mutant and wild type AT receptors in
COS-7 cells. The number of binding sites for the expressed receptors
was determined by Scatchard analysis of
[Sar
,Ile
]Ang II binding data as
described under ``Experimental Procedures'' ( n = 3, upper left panel). Levels of inositol
phosphate ( InsP, upper right panel), inositol bisphosphate
( InsP
, lower left panel), and inositol
trisphosphate ( InsP
, lower right panel)
labeling after 30-min LiCl (10 mM) pretreatment were measured
in control ( C) and Ang II ( AII, 20 min, 1
µM)-treated COS-7 cells expressing wild type and Y215F
mutant rat AT
receptors. Data are shown as means ±
S.E. for three experiments each performed in
duplicate.
Ang II Binding to AT
To estimate the efficiency of G protein coupling of
the Y215F receptor, the binding of the native agonist,
Receptors in COS-7 Cell
Membranes
I-Ang II, was studied in membrane preparations from COS-7
cells expressing mutant and wild type AT
receptors. In
contrast to its unaltered affinity for
[Sar
,Ile
]Ang II, Y215F showed
significantly reduced binding affinity for Ang II. The IC
values for inhibition of radioligand binding by Ang II in
membranes of COS-7 cells transfected with wild type and Y215F mutant
AT
receptor were 0.35 ± 0.02 nM and 2.7
± 0.1 nM, respectively ( n = 3). In
addition, GTP
S markedly reduced Ang II binding to the wild type
receptor but had only a minor effect on agonist binding to Y215F
(Fig. 3). The impairment of Ang II binding to the wild type
receptor in the presence of GTP
S reflected a decrease in the
affinity of the receptor (data not shown), secondary to its uncoupling
from G protein(s). These data, in good agreement with the inositol
phosphate results, suggest that the Y215F mutant receptor has impaired
ability to couple to G proteins.
Figure 3:
Inhibitory effects of GTPS on Ang II
binding to Y215F mutant and wild type receptors AT
. Crude
membranes were prepared from COS-7 cells expressing wild type (
)
or Y215F mutant (
) AT
receptors.
I-Ang
II binding was measured in the presence of the indicated concentrations
of GTP
S as detailed under ``Experimental Procedures.''
The B
levels (100%) for the wild type and Y215F
mutant were 7152 ± 1616 cpm and 1016 ± 186 cpm,
respectively. Data are shown as means ± S.E. for three
experiments each performed in duplicate.
Internalization of the Y215F Mutant AT
As reported earlier, the rat ATReceptor
receptors expressed in COS-7 cells undergo rapid internalization
during agonist activation. The role of Tyr
in the
internalization process was studied by comparing the kinetics of
I-Ang II sequestration in Y215F mutant and wild type rat
AT
receptors. As shown in Fig. 4, the
internalization of
I-Ang II was significantly slower in
cells expressing the mutant receptor than in those expressing the wild
type AT
receptor (Fig. 4, left panel). This
difference was particularly evident when the internalization rates were
compared by estimating the slope of the initial linear phase of the
time course of receptor internalization (Fig. 4, right
panel).
Figure 4:
Internalization of wild type () and
Y215F mutant (
) AT
receptors. The internalization
kinetics ( left panel) and the initial internalization rates
( right panel) were calculated as described under
``Experimental Procedures.'' Data are shown as means ±
S.E. for four experiments each performed in
duplicate.
residue of the type 1 Ang II receptor
is a highly conserved amino acid in the fifth transmembrane domain of
numerous G protein-coupled receptors
(8, 9) . Recent
modeling data have suggested that this tyrosine residue clusters with
the highly conserved DRY sequence located at the end of helix 3 and
that this region is a likely site for interaction with G proteins
(8) . While the importance of the DRY sequence at the bottom of
helix 3 has been established for the AT
receptor
(16) as well as for other G protein-coupled receptors
(5, 6) , the present study provides experimental
evidence for a role of the conserved Tyr
residue in
receptor activation and signal transduction. Although further studies
are required to clarify its exact function, our data, together with the
above-mentioned modeling studies, indicate the importance of this
conserved residue in the activation process of G protein-coupled
receptors. Earlier studies have identified other residues in the
transmembrane domains as well as in the second and third intracellular
loops that are critical for receptor activation
(5, 6, 7, 17, 18, 19, 20, 21) .
Although the intramembrane residues are probably important for the
active conformation of the receptor, the residues located in the
intracellular domains are more likely to participate in G protein
coupling. In the m3 muscarinic receptor, which also couples to the
G
family of G proteins, a tyrosine residue located 4
residues downstream of the conserved tyrosine residue in the fifth
transmembrane domain (corresponding to Tyr
) was found to
be important for inositol phosphate signaling
(19) . Since this
distance is approximately one turn in an
-helical structure, it is
possible that Tyr
participates in propagation of the
agonist-induced conformational change from the binding site to the
intracellular loops.
receptor that is a critical determinant of receptor
internalization, but has no significant role in Ang II-induced signal
transduction
(12) . Conversely, receptors with impaired
signaling ability have been shown to undergo rapid endocytosis
(10, 26) . In this context it is important to note that
the internalization rate of the Y215F receptor was substantially
reduced, but unlike the inositol phosphate response, was readily
demonstrable. Despite the parallel impairment of the internalization
and signaling properties of the Y215F mutant receptor, replacement of
this single amino acid did not cause a major perturbation of receptor
structure as indicated by its retention of high affinity for the
peptide antagonist, [Sar
,Ile
]Ang II.
The concomitant loss of agonist binding affinity was consistent with
the impaired ability of the mutant receptor to interact with G
proteins.
receptor suggest that
Tyr
is a crucial determinant of multiple aspects of its
activation mechanism.
receptor, type 1 angiotensin II receptor; Ang II, angiotensin II;
GTP
S, guanosine 5`-3- O-(thio)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.