(Received for publication, July 20, 1994; and in revised form, October 24, 1994)
From the
Phosphorylation of serine and threonine residues in the
carboxyl-terminal region of many G-protein-coupled receptors directs
the rapid uncoupling from signal transduction pathways. In Chinese
hamster ovary cells, we have stably expressed a truncated mutant of the
angiotensin II (AT) receptor devoid of the
carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine
residues. One clone, designated TL
to indicate truncation
after leucine 314, expressed a single class of angiotensin II receptors
with a dissociation constant of 1.08 nM and a receptor density
of 560 fmol/mg of protein (
75,000 receptors/cell). A
nonhydrolyzable analog of GTP accelerated the angiotensin II-induced
dissociation of [
I]angiotensin II from
TL
plasma membranes 3.6-fold, indicating G-protein
coupling. In TL
cells, angiotensin II stimulated the
release of intracellular calcium and the induction of mitogen-activated
protein kinase activity, the levels of which were comparable with the
full-length AT
receptor. The AII-stimulated calcium
response was rapidly desensitized in both full-length and truncated
AT
receptors. Interestingly, angiotensin II-induced
endocytosis of the truncated receptor was almost completely inhibited,
suggesting that a recognition motif within the carboxyl-terminal 45
amino acids of the AT
receptor promotes sequestration.
Thus, truncation of the AT receptor after leucine 314
inhibits agonist-induced internalization without affecting the capacity
of the expressed protein to adopt the correct conformation necessary
for high affinity binding of angiotensin II, coupling to G-proteins,
and activation of signal transduction pathways. The rapid
desensitization and refractoriness of the angiotensin II-induced
calcium transient in the TL
cell line, in which putative
carboxyl-terminal phosphorylation sites are absent, suggests that the
mechanism of AT
receptor desensitization differs from that
of other prototypical G-protein-coupled receptors.
Angiotensin II (AII) ()is a peptide hormone with
multiple actions(1) . The role of AII as a potent
vasoconstrictor and regulator of fluid and salt homeostasis is well
established, but the putative functions of this peptide as a
neuromodulator, growth factor, reproductive hormone, and cytokine
remain to be clarified. These diverse actions are mediated through a
number of AII receptor subtypes present in a variety of target tissues.
Molecular cloning studies have identified two major types of mammalian
AII receptors, designated AT
and AT
, with
multiple subtypes of AT
(e.g. AT
and
AT
)(2, 3, 4, 5, 6, 7, 8) .
Hydropathy analysis of the deduced amino acid sequences predicts that
the topology of both AT
and AT
receptors is
typical of seven-transmembrane guanyl nucleotide-binding protein
(G-protein) coupled receptors; however, only AT
receptors
appear to efficiently couple G-proteins(7, 8) .
Moreover, the AT
receptor shows a widespread tissue
distribution and appears to be the subtype that mediates most AII
actions. Binding of AII to the AT
receptor stimulates a
number of signal transduction pathways(9) , including the
activation of phospholipase C, to generate inositol triphosphate
(IP
) and diacylglycerol, resulting in the release of
calcium from intracellular stores and activation of protein kinase
C-dependent processes, respectively(10) . The AT
receptor has also been shown to modulate cAMP production, to
activate the mitogen-activated protein kinase (MAP kinase) cascade, and
promote tyrosine phosphorylation of cytoplasmic
proteins(10, 11) .
For G-protein-coupled receptors,
the seven transmembrane spanning helices presumably form a core that
positions amino acid residues in a conformation that specifically
recognizes the ligand. Binding of the ligand triggers conformational
changes within the intracellular regions of the receptor that activate
heterotrimeric G-proteins and initiate signaling. These signals are
rapidly terminated by desensitization mechanisms at the level of the
receptor(12, 13) . Studies on many G-protein
receptors, in particular the -adrenergic
receptor(12, 13) , have demonstrated two common
mechanisms for rapid receptor desensitization as follows: 1)
interference with G-protein coupling through phosphorylation of the
receptor at serine or threonine residues, particularly within the
carboxyl-terminal region, and 2) sequestration of the receptor away
from the plasma membrane so that it is inaccessible to extracellular
ligand.
Many responses to AII are rapidly attenuated (desensitized)
following the initial response to the peptide, although the
mechanism(s) has yet to be identified. The carboxyl-terminal region of
the AT receptor contains multiple serine and threonine
residues (13 out of the last 33 amino acids), and there are three
protein kinase C consensus sites(14) . Thus, it is tempting to
speculate that phosphorylation of the carboxyl terminus by protein
kinase C or a specific receptor kinase (13) modifies receptor
function and signaling. Bernstein and colleagues (15) recently
reported that in rat vascular smooth muscle cells the carboxyl-terminal
region of the AT
receptor is phosphorylated on serine and
tyrosine residues. This phosphorylation was constitutively present and
not temporally modulated by AII. Although this preliminary observation
needs to be confirmed, it implies that phosphorylation of the AT
receptor may not be involved in its dynamic regulation.
Internalization of AII receptors occurs rapidly upon ligand
binding(16, 17, 18) , and this process may
provide an efficient mechanism for the cell to prevent further agonist
stimulation.
We aimed to determine the role of the carboxyl-terminal
phosphorylation sites of the AT receptor in
agonist-induced desensitization and to identify the contribution of
receptor internalization to this process. Herein we describe the stable
expression in Chinese hamster ovary cells (CHO-K1) of a mutant
AT
receptor, truncated after leucine 314 to remove 45
amino acids from the carboxyl terminus region. This mutant receptor
displayed high binding affinity for AII, efficient G-protein coupling,
and activation of signal transduction pathways. Although deficient in
its ability to internalize, and despite the deletion of the thirteen
putative serine/threonine phosphate acceptor sites, this truncated
receptor rapidly desensitized in response to AII.
Figure 1:
Schematic representation of the rat
AT receptor. The site of truncation for the deletion
mutant, TL
, is indicated by a solid bar with the
deleted portion represented by unfilled circles. Shown underneath is the peptide sequence of the deleted region
(Gln
to Glu
). Multiple serine and threonine
residues (underlined), including three protein kinase C
consensus sites (asterisked), are shown. The palmitoylation
and membrane anchorage of Cys
is
presumed.
The effect of
GMP-PNP, a nonhydrolyzable analog of GTP, on the dissociation of bound
[I]AII was determined in plasma membranes
prepared from cultured cells. Confluent cultures were washed twice with
HBSS (4 °C) and scraped into ice-cold 50 mM Tris (pH 7.6)
containing 1 mM EDTA and 1 mM phenylmethylsulfonyl
fluoride, homogenized and centrifuged at 4000
g for 10
min. The supernatant was centrifuged at 30,000
g for
20 min, and the membrane pellet was washed once and resuspended in AII
receptor binding buffer(21) . Membranes (200 µg of
protein/ml) were allowed to bind 0.3 nM [
I]AII to equilibrium (60 min, 22 °C),
and unlabeled AII (100 nM) with or without GMP-PNP (100
µM) was added to the mixture and incubated for time
periods up to 40 min. Dissociation curves were analyzed using GraphPad
Prism, and dissociation rates were calculated.
The rate of
agonist-induced endocytosis for cells expressing either full-length or
truncated AT receptors was determined as follows: Cultures
were grown to confluence in 6-well 35-mm culture plates, washed three
times with ice-cold HBSS, and covered with 0.9 ml of AII receptor
binding buffer at 4 °C. Plates were placed on ice for 10 min to
ensure adequate cooling to 4 °C, and 100 µl of
[
I]AII was added to a final concentration of 1
nM. Equilibrium binding was reached in 3 h at 4 °C, a
temperature that prevents internalization. Wells were washed
extensively (5 times with 1.0 ml of binding buffer at 4 °C) to
remove unbound [
I]AII. To initiate
internalization, 1.0 ml of binding buffer (37 °C) was added to each
well, and the plates were immediately placed in a 37 °C incubator
for 0, 10, or 30 min. Plates were chilled on ice to terminate
endocytosis, and bound [
I]AII associated with
noninternalized plasma membrane receptors was removed by two 40-s
washes in 5 mM acetic acid in saline, pH 2.5. Internalized
radioactivity was collected by adding 1.0 ml of 0.2 M NaOH,
0.5% SDS to each well for 10 min and washing with an additional 0.5 ml
of the same solution. Radioactivity was measured with an LKB CompuGamma
-counter.
The pRC/CMV vector containing the cDNA insert coding for the
truncated AT receptor was transfected into CHO-K1 cells
using electroporation, and neomycin (G418) resistant colonies were
selected. Individual clones were isolated and screened for the
expression of functional receptors on the basis of
[
I]AII binding. Most clones displayed only low
level [
I]AII binding (<100 fmol/mg of
protein), and screening of more than 120 clones was necessary to obtain
several clones expressing
500 fmol/mg of protein. For comparison,
transfections run in parallel with the full-length AT
receptor DNA in the same vector consistently produced clones
expressing high levels of [
I]AII binding
(>500 fmol/mg of protein). One clone expressing the highest level of
binding for the truncated receptor, designated TL
, was
used for subsequent functional characterization. Confirmation that
TL
produced a truncated AT
receptor mRNA was
demonstrated by Northern blot analysis. Fig. 2shows a
comparison of total RNA from untransfected CHO-K1 cells, CHO-K1 cells
stably transfected with the full-length AT
receptor
(T
), and the TL
clone, probed with
Plabeled oligonucleotides either common to both
full-length and truncated mRNA or specific for the full-length
receptor. Whereas the untransfected CHO-K1 cell line expressed
undetectable levels of AT
receptor mRNA, the full-length
receptor clone (T
) showed a positive signal at about 1.2
kilobases with both probes. The truncated clone TL
was
only detected with the common probe at a reduced size, confirming the
truncation. Although no quantitation was attempted, both T
and TL
appeared to express equivalent amounts of
AT
receptor mRNA, which contrasts with a receptor density
6 times higher in the T
cell line(19) .
Figure 2:
Northern blot analysis of CHO-K1,
T, and TL
cell lines. Total RNA (10 µg)
extracted from untransfected CHO-K1 cells (lanes 1), CHO-K1
cells expressing full-length rat AT
receptor
(T
) (lanes 2), and CHO-K1 cells expressing
truncated AT
receptor (TL
) (lanes
3) was electrophoresed in a 1.5% agarose, formaldehyde gel and
transferred to nylon membrane. Blots were probed with
P-labeled oligonucleotides complementary to regions common (A) to both full-length and truncated mRNA species or
selective (B) for the full-length mRNA. This blot is
representative of three experiments. The positions of 28 and 18 S
ribosomal RNA are indicated.
A
competition binding curve for TL is shown in Fig. 3. The EC
(K
) of the
TL
AII receptor was 1.08 nM, which compares with
a K
of 1.9 nM for the full-length T
clone (19) and 1.0 nM for cardiac fibroblasts in
primary culture, which express predominantly high affinity AT
receptors(21) . The receptor density was 560 fmol/mg of
protein or
75,000 receptors/cell. High affinity binding requires
association of the receptor and the heterotrimeric G-protein with the
-subunit in the GDP bound form. Exchange of the GDP with GTP
results in a dissociation of the
-subunit and a functional
uncoupling of the
-subunit from the receptor with a subsequent
lowering of the receptor binding affinity. This GTP/GDP exchange effect
on receptor binding was used as an indicator of G-protein coupling (Fig. 4). Plasma membrane preparations from TL
cells, containing the truncated receptor and associated
G-proteins, bound [
I]AII to equilibrium, and
agonist was then displaced by unlabeled AII in the absence or presence
of the nonhydrolyzable GTP analog, GMP-PNP. Unlabeled AII caused a
rapid dissociation of [
I]AII from the membranes
with a half-life of 10.2 min. In the presence of GMP-PNP, this rate of
dissociation was increased with a half-life of 2.8 min. This 3.6-fold
increase in dissociation of [
I]AII indicates
that the receptor associates with a G-protein(s) and that the
-subunit was capable of exchanging GDP for GTP (GMP-PNP).
Figure 3:
Competition binding of
[I]AII to TL
cells expressing the
truncated AT
receptor. Cultures of confluent TL
cells were incubated with 36 pM [
I]AII for 60 min at 22 °C in the
presence of indicated concentrations of unlabeled AII. Each point
represents the mean ± S.D. of triplicate determinations.
Nonlinear least squares regression analysis gave an IC
(K
) of 1.08 nM and B
of 6646 cpm. B
(560
fmol/mg of protein) was calculated by the equation B
= B
/l
IC
(22) , where l represents the total
amount of radioactivity added, and corrected for protein content. This
dissociation curve is representative of three separate
experiments.
Figure 4:
Effect of the GTP analog (GMP-PNP) on
AII-induced dissociation of [I]AII from
TL
membranes. Plasma membrane preparations (
200
µg of protein) of TL
cells were incubated in the
presence of 0.3 nM [
I]AII for varying
times, and specific binding was determined. At equilibrium (60 min),
excess unlabeled AII (100 nM) in the presence (open
triangles) or absence (open circles) of GMP-PNP (100
µM) was added, and specific binding was determined over a
40-min period. Points are the means ± S.D. for
triplicate determinations. Similar results were obtained in two
additional experiments.
To
confirm that the truncated, high affinity, G-proteincoupled AT receptor stimulated known AII signal transduction pathways, the
ability of AII to induce a rise in intracellular calcium and stimulate
MAP kinase activity was determined ( Fig. 5and Fig. 6).
In Fura-2/AM-loaded cells expressing either full-length or truncated
receptors, exposure to 1 µM AII resulted in a rapid rise
in intracellular calcium, which immediately abated and returned to
preexposure levels within 50 s (Fig. 5). The peak level of the
calcium transient was approximately proportional to the receptor
density (T
, 3400 fmol/mg protein (19) versus TL
, 560 fmol/mg protein). In addition, another clone
(T
) expressing the full-length AT
receptor (K
= 0.82 nM; receptor density,
667 fmol/mg of protein) at levels comparable to TL
showed
a calcium transient of
200 nM in response to 1 µM AII. This observation suggests that truncated AT
receptors couple with similar efficiency to full-length
receptors. When extracellular calcium was chelated by addition of EGTA,
AII-induced transients were observed in TL
cells (data
not shown), indicating that the observed calcium transients were from
intracellular stores, similar to that described for T
cells(19) . MAP kinase activity is rapidly and
transiently induced following activation of AT
receptors by
AII(10, 11) , presumably via G-protein dependent
mechanisms(11) . Fig. 6shows the
4-fold
enhancement of MAP kinase activity in cytosolic extracts from both the
TL
and T
cells in response to a 2-min
exposure of AII.
Figure 5:
AII-stimulated calcium transients in T3
and TL cells. Confluent cultures of T
(Full-length receptor) (A) and TL
(Truncated receptor) (B) were serum-starved for
24 h, loaded for 1 h (37 °C) with the fluorescent dye Fura-2/AM,
and stimulated with 1 µM AII as indicated by the arrows. Intracellular calcium levels were determined as
described previously(19) .
Figure 6:
AII-induced MAP kinase activity in T and TL
cells. Confluent cultures were serum-starved
for 24 h, stimulated with 1 µM AII, and rapidly processed
for MAP kinase activity as described under ``Experimental
Procedures.'' Shown are means of triplicate determinations (S.D.
< 10% of mean). A 3-4-fold stimulation of MAP kinase activity
in TL
cells was observed in six separate
experiments.
Fig. 7compares the endocytosis at 37 °C
of full-length and truncated receptors. Equilibrium binding of
[I]AII to T
and TL
cells was performed at 4 °C to prevent internalization. After
transfer to 37 °C, radioactivity associated with acid-washed cells
was determined at 0, 10, and 30 min. It was assumed that radioactivity
associated with acid-washed cells reflected internalized receptors. At
equilibrium (3 h, 4 °C), when expressed as a percentage of the
total specific binding, 7.3% of full-length receptors and 8.9% of
truncated receptors were not susceptible to acid washing, representing
the small pool of internalized receptors at any given time. Full-length
AT
receptors (T
cells) rapidly internalized at
37 °C so that at 10 min 44% and by 30 min greater than 69% of the
surface receptors had been sequestered. In contrast, the
carboxyl-truncated mutant receptor showed a markedly inhibited capacity
to internalize with only 14 and 17% of receptors acid-resistant at 10
and 30 min, respectively (Fig. 7). This difference in
endocytosis rate is not the result of differential levels of receptor
expression because the T
clone also displayed rapid
receptor internalization (59% at 10 min).
Figure 7:
Endocytosis of full-length and Truncated
AT receptors. Confluent cultures of T
and
TL
cells were incubated with 1 nM [
I]AII for 3 h at 4 °C to allow
equilibrium binding with minimal internalization. Cultures were washed
to remove unbound ligand and switched to 37 °C for the times
indicated. At each time point, cells were acid-washed to dissociate
[
I]AII bound to cell surface receptors,
harvested, and counted. Acid-wash-resistant counts associated with the
cells are expressed as a percentage of the total specific binding. The
proportion of total receptors internalized at zero time varied between
6 and 9% for both cell lines.
Desensitization can be
termed as a process that occurs at the level of the receptor that
results in the termination of responses to a given ligand and an
unresponsiveness to additional challenges(12, 13) .
Previous studies have used the phospholipase C/IP/calcium
pathway to study AII receptor desensitization. AII-mediated
desensitization has been described in primary cell
cultures(26, 27) , and we have investigated this
process in T
cells stably expressing the AT
receptor(19) . Exposure of T
cells to 1
nM AII causes rapid desensitization and insensitivity to a
second dose of 100 nM AII(19) . As shown in Fig. 8, 100 nM AII gives a 70-80 nM calcium transient in the TL
cell line. This response
was maximal at a dose of 1-10 µM AII (150-200
nM), and the threshold of detection was at
1 nM AII. One hundred seconds after the initial 100 nM dose,
the cells were refractory to a second dose of 1 µM (Fig. 8A). As shown in Fig. 8B, an
initial challenge with 1 nM, a subsaturating concentration,
resulted in insensitivity to a 100-fold higher dose of 100 nM 100 s later, indicating rapid desensitization. ATPinduced calcium
transients were unaffected by preexposure to AII (Fig. 8B).
Figure 8:
Desensitization of the calcium transient
in TL cells. Representative trace (of five
experiments) showing intracellular calcium concentration in response to
sequential additions of AII. A, 100 nM AII, followed
by 1 µM AII. B, a 1 nM initial dose of
AII abolished the response to 100 nM AII but not
10
M ATP, which also releases calcium from
intracellular stores.
Angiotensin II receptors play a pivotal role in the
coordinated actions of AII, occupying a central position between the
generation of this peptide from its precursor angiotensinogen and
intracellular signaling pathways, which ultimately determine the fate
of cellular responses. Strict control of cellular responsiveness to AII
is important given the diversity and consequences of these actions. One
way to establish this control is for cells and tissues to use distinct
receptor subtypes to control different functions, whereas another is to
rapidly terminate the intracellular response following initial exposure
and response to AII. Receptor phosphorylation has become a hallmark of
this latter phenomenon for many G-protein
receptors(12, 13) . This acute (seconds to minutes)
process may be supplemented by internalization (minutes to hours) and
by down-regulation (hours to days) of the receptor from the plasma
membrane. In this study, we have detailed experiments with a mutant
AT receptor, truncated to delete potential
carboxyl-terminal phosphate acceptor sites, in which normal binding of
AII, coupling to G-proteins, and signaling pathways are intact. Our
primary observation is that this truncated receptor, devoid of
carboxyl-terminal serine and threonine residues, undergoes rapid
agonist-induced desensitization. In addition, we demonstrate that
desensitization occurs in the absence of receptor internalization and
hence the mechanism of AT
receptor desensitization is
obscure.
Three criteria were used to demonstrate that the truncated
receptor represents a functional, G-protein-coupled AT receptor. First, competition binding studies revealed a high
affinity binding site for AII (K
in the nM range), in agreement with previous determinations on native
receptors(28) . Second, experiments with a nonhydrolyzable GTP
analog confirmed G-protein coupling of plasma membrane truncated
receptors. Finally, the AII receptor coupled to two well established
signal transduction pathways: stimulation of intracellular calcium and
an increase in MAP kinase activity. We experienced, however, difficulty
in obtaining clones expressing high levels of truncated receptor.
Northern blot analysis, which we used to confirm our truncated
construct, showed that the TL
and T
cells
produced approximately the same amount of receptor mRNA. In contrast,
the level of functional receptor at the plasma membrane was
approximately 6-fold higher in T
cells. This suggests that
both expression constructs transcribe with equivalent efficiency but
that the truncated receptor is not efficiently transported and/or
inserted into the membrane. This may reflect a problem with folding and
obtaining correct conformation for the truncated receptor.
Alternatively, the removal of a putative palmitoylation site (cysteine
355) may prevent anchoring of the receptor in the membrane; or perhaps
the same cellular machinery that is responsible for receptor
internalization, for which the truncated receptor is deficient, is
involved in initial membrane insertion. Nevertheless, the proportion of
truncated receptors that are inserted into the membrane appear to
function with high binding, coupling, and signaling efficiency.
Our
results demonstrate that the last 45 amino acids of the AT receptor are not crucial for efficient coupling to G-protein.
Inagami and colleagues (29) reported that transient
transfection of an AT
receptor, truncated to remove the
last 50 amino acids (after phenylalanine 309) in COS cells, produced a
receptor with enigmatic properties. The mutated receptor showed high
affinity binding for AII, but no GTP effects on binding were observed,
and IP
production was markedly inhibited, suggesting
uncoupling from G-protein(s). This observation contrasts with the
current dogma that high affinity binding requires G-protein
interaction. We showed that truncation to leucine 314 results in a
functional mutant (at least with respect to G-protein coupling),
revealing that the proximal fifth of the carboxyl tail up to leucine
314 provides a site necessary for appropriate G-protein interaction.
Shortening to phenylalanine 309 (29) appears to abolish the
ability of the G-protein to exchange GDP/GTP and promote signaling.
Perhaps this region provides something required by the G-protein
complex for GDP/GTP exchange and IP
triggering, but points
of contact in other regions of the receptor (aspartic acid 74 (30) and the second intracellular loop(29) ) provide
stability for high affinity binding of AII.
Truncation of the
AT receptor to remove putative phosphorylation sites did
not affect the capacity of cells expressing this mutant receptor to
become refractory or desensitize to AII, at least with respect to
calcium signaling. We hypothesized that truncation of the AT
receptor would result in one of two observations: 1) after the
initial rise, intracellular calcium would remain elevated in a manner
analogous to a recent study(31) , where cAMP levels stayed
elevated when desensitization was prevented by inhibition of a specific
receptor kinase, or 2) following a nonsaturating dose of AII, a second
higher application of AII would initiate a second calcium transient.
Surprisingly, AII induced a calcium transient and desensitized this
response in a manner indistinguishable from that of the full-length
receptor, suggesting that the carboxyl-terminal region of the receptor
is not required for this process. The possibility exists, however, that
mechanisms downstream of the receptor are responsible for the observed
results (e.g. at the level of IP
production by
phospholipase C, at the IP
receptor, or at the calcium
channel itself), but indications are that desensitization of AII (19, 26, 27) and other (12, 13, 32) receptors occurs at the level of
the receptor. Concerns regarding delineation of desensitization at the
level of the receptor from desensitization of downstream pathways are
also applicable to studies with other G-protein receptors, where
phosphorylation of the receptor and desensitization have been only
temporally associated.
Our observation of desensitization in the
absence of the carboxyl-terminal region contrasts with a large body of
literature for other G-protein-coupled
receptors(12, 13, 32) . Most recently, a
gonadotropin-releasing hormone (GnRH) receptor, which naturally lacks a
carboxyl-terminal cytoplasmic tail, showed repetitive IP accumulation in response to multiple GnRH challenges and was
therefore incapable of short term desensitization(33) .
However, there is also recent evidence to support our results and the
idea that some G-protein-coupled receptors do not require
phosphorylation or the presence of a carboxyl-terminal region for
desensitization. First, Paxton et al.(15) reported
that phosphorylation of the carboxyl terminus of the AT
receptor was not modulated by AII and therefore would appear
incapable of dynamically regulating desensitization. Second, truncation
of the human Endothelin A receptor to remove the last 36 amino acids of
the carboxyl-terminal region, including 6 serine and 3 threonine
residues with two putative protein kinase C phosphorylation sites, had
no apparent effect on receptor signaling or
desensitization(34) . Third, a naturally occurring
carboxyl-terminal truncated dopamine D1 receptor, in which 80 amino
acids (including 9 serines and 3 threonines) are absent as compared
with prototypical mammalian D1 receptors, showed high affinity for
D1-specific ligands, dopamine-stimulated cAMP and calcium accumulation,
and desensitization in response to dopamine(35) . Thus, an
increasing literature suggests that the presence and phosphorylation of
the carboxyl-terminal region for some G-protein receptors is not a
prerequisite for desensitization. The mechanism(s) by which this
subgroup of G-protein-coupled receptors rapidly terminate intracellular
responses and remain refractory to additional challenges remains to be
determined. For the AT
receptor, desensitization may be
controlled by a phosphorylation event at other sites in the receptor.
In particular, the carboxyl end of the second intracellular loop of the
AT
receptor contains a serine and threonine residue, which
may be phosphorylated. These residues may be relevant given that
mutations in this region were very efficient at inhibiting G-protein
coupling(29) . Perhaps phosphorylation at these sites
interferes with G-protein coupling, a possibility that we are currently
investigating.
Truncation of the AT receptor markedly
reduced endocytosis of the receptor from the plasma membrane. This is a
key observation for a number of reasons, as follows: 1) it shows that
internalization is not required for AII signaling as has been suggested
previously(36, 37) ; 2) it demonstrates that
internalization is not a prerequisite for termination of receptor
signaling and desensitization, in agreement with our previous
observation that acute desensitization of the full-length AT
receptor cannot be explained on the basis of
internalization(19) ; and 3) it suggests that a site in the
last 45 amino acids of the AT
receptor promotes or
provides a recognition motif for receptor internalization. Whether the
phosphorylation sites are involved in internalization remains to be
determined, but the use of phosphorylation sites for internalization of
receptors has not been a common theme for plasma membrane receptors,
and internalization recognition motifs are disparate. Receptors like
those for transferrin, low density lipoprotein, and growth factors use
a NPXY or YXX-hydro motif (where X is any
amino acid and hydro is a large bulky hydrophobic amino acid) (38) or hydrophobic stretches of amino acids in the cytoplasmic
tails to control endocytosis(39) . The G-protein-coupled
thyrotropin-releasing hormone receptor uses two dissimilar domains for
internalization: two closely spaced cysteine residues in the proximal
region of the cytoplasmic tail and the sequence SDRFSTEL more
distally(40) . For the prototypical
-adrenergic G-protein-coupled receptor, the site
crucial for internalization resides in the N-terminal segment of the
third intracellular loop(41) . Comparison with the deleted
region of our truncated AT
receptor reveals that these
sequences and motifs are not present. Recently, Barak et al.(42) identified a role for a tyrosine residue, highly
conserved in G-protein-coupled receptors and in the motif
NPXXY, which is involved in sequestration of the
-adrenergic receptor. This tyrosine (tyrosine 302 in
AT
) is maintained, as is the NPXXY motif, in our
truncation at leucine 314, and therefore it does not appear to play a
critical role in endocytosis of the AT
receptor. For a
yeast pheromone G-protein-coupled receptor, the recognition motif is
DAKSS with an absolute requirement for the central lysine(43) .
A similar AKS site is present in the carboxyl-terminal region of the
AT
receptor, which was deleted by truncation to leucine
314. Whether this constitutes the recognition motif for AT
receptor internalization remains to be established.
In
summary, we have expressed in CHO-K1 cells a truncated AT receptor, which displays most characteristics of a functional
receptor, apart from a significantly reduced capacity for
agonist-induced endocytosis. Remarkably, this mutant receptor,
truncated to remove potential carboxyl-terminal phosphorylation sites,
appears to maintain an ability to rapidly terminate G-protein signaling
and undergo desensitization in response to AII. It is intriguing that
both AT
and endothelin A receptors, for which this
phenomenon has now been described(34) , are receptors for small
peptide ligands, the major function of which is potent
vasoconstriction. Whereas the reason(s) for this is unclear, these
results are important because they support the concept that different
subclasses of G-protein-coupled receptors have evolved divergent
mechanisms for controlling desensitization. Future experiments will
focus on identifying the mechanism(s) of AT
receptor
desensitization and the site(s) of the receptor that are responsible
for desensitization and internalization.