Institut National de la Santé et de la Recherche Médicale Unité 36, Collège de France, 75005 Paris, France
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ABSTRACT |
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The structural
determinants of the rat angiotensin (ANG) II
AT1A receptor involved in receptor
internalization, desensitization, and activation are investigated by
producing six mutants that had progessively larger deletions of the
cytoplasmic tail (13,
19,
24,
31,
46, and
56 residues, respectively). After stable transfection of the cDNAs into Chinese hamster ovary cells, all mutants, except the most truncated, exhibit normal
[Sar1]ANG II
affinities [dissociation constant
(Kd) = 0.19-0.70 nM] compared with the wild-type (WT) receptor
(Kd = 0.62 nM)
and are able to activate a Gq/11
protein and a phospholipase C as measured by the ANG II-induced
inositol phosphate (IP) turnover in the different clones. However, one
of these mutants,
329 (deletion of 31 residues), exhibits a peculiar
phenotype. This mutant shows a reduced ligand-induced internalization
as measured by the acid-washing procedure (only 32% of receptors are
internalized vs. 83% for WT). Moreover, the
329 mutant is less
desensitized by a pretreatment with either ANG II (15% desensitization
of ANG II-stimulated IP turnover vs. 60% for WT receptor) or the
phorbol ester phorbol 12-myristate 13-acetate (no desensitization vs.
29% for WT receptor). These functional modifications of the
329
mutant are associated with the transduction of an amplified signal as
demonstrated on both IP turnover and an integrated physiological effect
of ANG II. Taken together, these data indicate that the sequence
329SLSTKMS335
of the rat AT1A receptor is
involved in both receptor internalization and desensitization. This is
the first demonstration that a desensitization- and
internalization-defective AT1A
receptor mutant is also hyperreactive and mediates augmented cellular
responses.
angiotensin II receptor; mutagenesis; endocytosis; Chinese hamster ovary cells
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INTRODUCTION |
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ANGIOTENSIN II (ANG II) is a vasoactive peptide that
acts on its target tissues through the interaction with cell surface receptors. These receptors, members of the seven transmembrane domain
receptor family, have been divided into several types
(AT1 and
AT2) and subtypes
(AT1A and
AT1B) based on their
pharmacological properties, amino acid sequence, and/or tissue
distribution (for review, see Ref. 7).
AT1 receptors are G
protein-coupled receptors, which are responsible for most of the ANG II
physiological actions. The signal transduction via these receptors
follows a classical pathway, which involves
Gq/11 protein(s) and
phospholipase(s) C, which produce second messengers and result in
Ca2+ mobilization and protein
kinase (PK) C activation (7). More recently, other signaling pathways,
such as the JAK-STAT pathway or the Shc-p21ras pathway, were
demonstrated to be activated by the
AT1 receptor, but the exact
physiological significance of this observation is not known.
In parallel to the activation of this signal transduction pathway, the AT1 receptor, as for many other receptors of the same family, undergoes internalization of the ligand-receptor complexes (1) and phosphorylation (25). These two modifications are generally considered to participate in the process of receptor desensitization, defined as the attenuation of the signal due to serial agonist applications.
On one hand, the morphological aspects of the
internalization-sequestration process, which involves specific
clustering of the receptors in clathrin-coated pits (32), are well
known, whereas the molecular mechanisms and the functional consequences of this internalization are still a matter of debate. For the AT1 receptors, this process is
induced by peptidic but not by nonpeptidic ligands and is independent
of G protein coupling (9). The AT1
sequence involved in the internalization process is not the consensus
sequence
[NPX(1-2)Y]
located at the junction of the seventh transmembrane domain and the
COOH-terminal tail (17), as described for the
2-adrenergic receptors (3) or the low-density lipoprotein (LDL) and tyrosine kinase receptors (27),
but more probably a sequence in the COOH-terminal tail. Indeed, several
authors (16, 33) have shown that deletions or mutations in different
segments of the proximal part of the COOH-terminal tail impaired the
AT1A receptor internalization.
On the other hand, receptor phosphorylation on serine and threonine
residues is a major event, which follows receptor activation and
largely participates to receptor desensitization. G protein-coupled receptors can be phosphorylated by two different types of kinases: 1) second-messenger-activated
kinases, such as PKA or PKC, which produce a negative feedback and a
nonspecific mechanism of desensitization; and
2) specific kinases that form the
growing family of G protein-coupled receptor kinases (GRK) (26). One
example of such an homologous desensitization is the phosphorylation by
GRK2 of the -adrenergic receptor, which subsequently displays a high
affinity for a cytosolic protein,
-arrestin. This interaction
between the
-arrestin and the phosphorylated receptor
suppresses G protein interaction and uncouples the receptor. The sites
and pattern of phosphorylation vary from one receptor to another but
are located mainly in the COOH-terminal tail or the third intracellular
loop; this also varies for the same receptor from one cellular model to
another (29).
Despite a better understanding of the molecular mechanisms involved in receptor internalization, phosphorylation, and desensitization, the precise relationships between these three phenomena are still a matter of debate and vary from one receptor to another.
To establish precisely the sequences of the COOH-terminal tail of the AT1A receptor involved in activation, internalization, and desensitization, six mutants of this receptor with progressive deletions of the COOH-terminal were characterized. We studied the signaling, physiological actions, internalization, and desensitization of these mutants. This enabled the identification of a deletion mutant that was neither internalized nor desensitized. Interestingly, this mutant receptor transduces an amplified signal through the membrane.
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MATERIALS AND METHODS |
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Construction of truncated AT1A mutants.
The expression plasmid used for the construction of the truncated
mutants was pEAT1A and has been
described previously (8). This plasmid consists of a synthetic
AT1A cDNA containing multiple unique endonuclease restriction sites inserted into the
Hind III and
Xba I sites of the eukaryotic
expression vector pECE (11). The mutants
347,
341,
336, and
329 were generated by double digestions of
pEAT1A
with
Sma I (site located after the stop codon of AT1A cDNA) and
Sac I,
Stu I,
Sal I, and
Xho I, respectively. The cohesive ends
generated by each of these enzymes was blunted with either T4 DNA
polymerase or DNA polymerase I (Klenow fragment), and the linear
construction was subsequently recircularized using T4 DNA ligase. The
presence of stop codons in each of the three frames located immediately
after the Xba I site in pECE allowed this direct ligation but resulted in the presence of two to three additional amino acids at the COOH-terminal of all the mutants (Fig.
1).
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Cell culture: stable transfection. Chinese hamster ovary (CHO) K1 cells were cotransfected with 2 µg of pSVNeo (Pharmacia) and 10 µg of the expression plasmids by the calcium phosphate coprecipitation method (31). Transfected cells were selected by their resistance to neomycin (GIBCO-BRL). The cell populations giving high levels of binding were subcloned by limiting dilution to obtain pure cell lines with high expression levels of the mutant receptors. The CHO AT1A/V3 cell line was obtained by transfection of the already characterized CHO AT1A cell line with the expression plasmid pECE containing the vasopressin V3 receptor cDNA. Because these cells were already neomycin resistant, the selection was achieved by cotransfection with a plasmid that confers resistance to hygromycin.
Cells were then maintained at 37°C in a 5% CO2 atmosphere in Ham's F-12 medium supplemented with 10% fetal calf serum plus 0.5 mM glutamine, 100 U/ml penicillin, and 100 µg/ml strepromycin (all from Boehringer Mannheim).Binding studies. [Sar1]ANG II and ANG II (Sigma) were labeled by the chloramine-T method and the monoiodinated product was purified by high-performance liquid chromatography.
Cells were subcultured into 24-well culture trays and incubated for 45 min at 22°C with 0.5 nM 125I-[Sar1]ANG II or 125I-ANG II in the presence of increasing amounts of nonlabeled [Sar1]ANG II or ANG II, respectively, in 50 mM tris(hydroxymethyl)aminomethane HCl, 6.5 mM MgCl2, 125 mM NaCl, 1 mM EDTA, and 1 mg/ml bovine serum albumin (BSA, pH 7.6). Nonspecific binding was determined in the presence of 1 µM [Sar1]ANG II or ANG II. Each experiment was carried out in duplicate. Binding data were analyzed with a nonlinear least squares curve-fitting procedure (Ebda-Ligand, Elsevier-Biosoft, Cambridge, UK). Analysis of the guanosine 5'-O-(3-thiotriphosphate) (GTPInternalization assay. Internalization of wild-type and mutant AT1A was measured as a percentage of 125I-[Sar1]ANG II resistant to acid wash, as described previously (9). Transfected cells were incubated with 0.4 nM 125I-[Sar1]ANG II in binding buffer with or without 1 µM [Sar1]ANG II for 180 min at 4°C, washed twice, and placed in binding buffer alone at 37°C for various times. Finally, cells were placed at 4°C, and for half of the replicate wells, total and nonspecific binding was measured after cell lysis with 1 M NaOH. In the other wells, surface-bound 125I-[Sar1]ANG II (total and nonspecific) was determined after incubating the cells in 50 mM glycine and 125 mM NaCl (pH 3) for 5 min. Internalized radioactivity (total and nonspecific) was determined after lysis of the cells in 1 M NaOH.
Inositol phosphate production. ANG II stimulation of inositol phosphate (IP) production was performed as described previously (35). Cells were subcultured in 12-well culture trays, labeled with 2 µCi/ml myo-[3H]inositol for 24 h, preincubated for 10 min with 10 mM LiCl, and then incubated with increasing concentrations of ANG II for 30 min at 37°C in the presence of 10 mM LiCl. After purification on a Dowex 1×8 anion-exchange resin (Bio-Rad), the total IP fraction was measured. For desensitization experiments, labeled cells were preincubated for 15 min with 100 nM ANG II or arginine vasopressin (AVP) and rinsed three times, and after 15 min without agonist, they were incubated for a further 15 min with 100 nM ANG II or AVP. When used, phorbol 12-myristate 13-acetate (PMA) was added at the indicated concentration during the preincubation (30 min) and incubation (15 min) periods.
ANG II inhibition of insulin-induced [14C]glucose incorporation into glycogen. Confluent cells grown in 12-well plates were incubated for 1 h with the indicated concentrations of ANG II in phosphate-buffered saline (PBS), 0.1% BSA, 0.7 mM CaCl2, 0.5 mM MgCl2, and 100 nM insulin. Cells were then exposed for 3 h to [14C]glucose (5 mM, 2 µCi). After three washes with ice-cold PBS, the cells were lysed with 30% KOH and transferred to glass tubes. Total glycogen was precipitated with ethanol as described previously (20), and the amount of radioactivity incorporated was determined using a scintillation counter.
Statistics. Results are expressed as means ± SE. Statistical significance was assessed by analysis of variance.
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RESULTS |
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The COOH-terminal cytoplasmic tail of
AT1 receptors consists of ~60
amino acids. To test the role of this region in the biological functions (binding, activation, internalization, and desensitization) of the AT1A receptor, six mutants
were constructed in which progressively larger portions of the
cytoplasmic tail were removed. Thus, as shown in Fig. 1, receptor
mutants 347,
341,
336,
329,
314, and
304 lack the
last 13, 19, 24, 31, 46, and 56 residues of the cytoplasmic tail,
respectively.
CHO cells were stably transfected with the cDNAs encoding these
truncated receptors and were tested for their
125I-[Sar1]ANG
II binding activity. No
125I-[Sar1]ANG
II binding could be detected in cells transfected with 304 cDNA.
Therefore this form of the AT1A
receptor in which 56 COOH-terminal cytoplasmic residues are lacking is
either unable to bind the ligand or cannot be properly transported to
the cytoplasmic membrane.
The binding characteristics of
125I-[Sar1]ANG
II to CHO pure cell lines expressing the full-length receptor and the
five other truncated mutants are shown in Table
1. All these receptors display comparable
affinities for this peptidic agonist. Moreover, the wild-type
AT1A receptor [dissociation
constant (Kd) = 0.645 ± 0.027 nM] and the 329 mutant
(Kd = 0.343 ± 0.051 nM) present similar affinities for the natural peptide ANG II.
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The internalization of the mutant receptors from the cell surface after
exposure to
125I-[Sar1]ANG
II was measured using the acid-wash method. As shown in Fig. 2, treatment of cells expressing the
wild-type AT1A with this agonist
resulted in a rapid loss of the receptors from the cell surface, with a
maximal internalization of 83% of the total specific binding within 20 min at 37°C. The time necessary to internalize 50% of the
ligand-receptor complexes
(t1/2) was 4.22 min. Time courses of internalization of 347,
341, and
336 were
comparable to the time course of the full-length receptor, with an
acid-resistant 125I-[Sar1]ANG
II fraction reaching a maximum of 76, 86, and 83%, respectively, after
20 min at 37°C and similar
t1/2 (4.05, 3.63, and 4.19 min, respectively). The truncation of >31 COOH-terminal
residues of AT1A resulted in a
drastic decrease of its ligand-mediated internalization (32% for
329 and 18% for
314).
329-reduced internalization had a
time-course pattern comparable to
AT1A, since the maximal effect
occurred after 20 min at 37°C, and the t1/2 was 5.18 min, whereas the
314 internalization pattern appeared to be
different: the maximum was reached only after 90 min at 37°C. For
this mutant, the
t1/2 was ~14
min. These results are summarized in Fig. 1.
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The ability of cells expressing the full-length receptor and the
truncated mutants to stimulate IP production in response to increasing
concentrations of ANG II was analyzed. LiCl was added to allow an
accumulation of total IP. A correlation between the receptor density
and the level of second-messenger response has been demonstrated (13).
To normalize the amplitudes of the stimulation observed for the
different mutants according to their respective maximal binding
capacities (Bmax), the results
shown in Fig. 3 were expressed as a ratio
of the percentage of IP production over basal to the
Bmax. The ANG II concentration
required to elicit 50% of the maximal response
(EC50) in CHO
AT1A was 0.75 nM, and the maximal
IP production
(Emax/Bmax)
reached 479% over basal. The EC50
and
Emax/Bmax
obtained with cells expressing the different truncated mutants, except
329, were comparable. For
329, the maximal stimulation of 1,014%
was more than twofold higher, and the
EC50 observed (0.09 nM) was
significantly lower compared with the values obtained for
AT1A.
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The ability of the 329 mutant to interact with G proteins was
further analyzed by measuring the effect of GTP
S on the binding of
the native agonist ANG II to membranes from transfected cells. As shown
in Fig. 4, GTP
S displayed a slightly
better effect on the
329 mutant compared with the wild-type
receptor, but this difference was not significant. However, GTP
S
remained almost without effect on the binding of ANG II to the
314
mutant, suggesting a major alteration of the interaction between this
mutant and G proteins.
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This hyperreactivity of the 329 mutant was also investigated on an
integrated biochemical effect of ANG II in CHO cells. The inhibitory
action of ANG II on insulin-dependent incorporation of glucose into
glycogen was analyzed for wild-type and
329 receptors. This
physiological effect of ANG II was first described in hepatocytes (19)
and could be reproduced in CHO cells transfected with the AT1 receptors (B. Teutsch et al.,
unpublished observation). As shown in Fig.
5, in the CHO
AT1A cells, ANG II induced a 73%
inhibition of the glucose incorporation triggered by 100 nM insulin,
with an EC50 of 1.40 nM. In CHO
329 cells, the inhibitory effect of ANG II reached a maximum of
91%, and the EC50 of 0.15 nM was
statistically different from the wild type
(P < 0.05).
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For some G protein-coupled receptors, internalization and the
desensitization-resensitization processes have been shown to be closely
related. We therefore studied whether the enhanced response observed
with 329 reflected a modification of the desensitization pattern of
this mutant. The stimulation of IP turnover in response to serial ANG
II applications was measured for the wild-type and truncated receptors.
Because our interest was to measure not a time-dependent IP
accumulation but the effect of two consecutive agonist applications,
the experiment was performed without LiCl. Therefore the following
results cannot be compared with those described above. In addition, the
time course of this IP production measured by the present assay is
longer than the duration of the desensitization process. This explains
why the second application of ANG II was done before the return of the
IP values to the basal level.
Under these conditions, basal cellular IP content was 318 ± 25 counts/min (cpm) (see Fig. 6A, bar 1). After a single treatment with ANG II, IP production reached 675 ± 31 cpm (bar 2). However, two sequential treatments with ANG II gave rise to a significantly lower level of IP production (bar 4: 592 ± 29 cpm; P < 0.01) compared with treatment with a single application of ANG II. Moreover, because the pretreatment with ANG II raised the basal level of IP production somehow (to 448 ± 39 cpm, bar 3), the actual stimulation induced by the second application of ANG II was that much smaller at 144 cpm compared with 357 cpm after a single treatment with ANG II. This would suggest a 60% desensitization of the IP response to ANG II via the AT1A receptor (see also Fig. 7).
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For the 329 mutant, the basal IP cellular content was comparable to
that of the CHO AT1A cells (305 ± 37 cpm). The IP production reached 1,275 ± 126 cpm after a
single ANG II stimulation and 1,499 ± 120 cpm after two sequential
treatments with ANG II. Fifteen minutes after the ANG II pretreatment,
the IP level remained above the basal initial value (673 ± 55 cpm).
Taking this into account, a second ANG II application induced an IP
production of 826 cpm. Thus the IP production after two successive ANG
II treatments was only 15% lower than after a single agonist exposure
(823 cpm vs. 970 cpm; see also Fig. 7).
Therefore the IP response induced by ANG II via the
329 mutant
appeared only slightly desensitized.
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The same procedure was applied to the 347 and
341 mutants, which
behave as the wild-type receptor (data not shown). Unfortunately, the
336 and
314 mutants could not be tested because the low density
of sites did not allow any measurable IP production in the absence of
LiCl.
In the mutant 329, which had a modified internalization pattern and
was less desensitized with exposure to ANG II, the three potential PKC
phosphorylation sites present in the wild-type receptor were deleted.
Thus we investigated whether a short-term application of the phorbol
ester PMA (30 min), by activating PKC, would differentially modify the
amplitude of the ANG II-induced IP production via wild-type and mutated
receptors (Fig. 6B). In CHO
AT1A cells, PMA produced a
dose-dependent inhibition of the ANG II-induced IP production, which
reached, for 10
6 M PMA,
29% of the stimulation observed without PMA. Because, under the same
conditions, PMA had no effect on the ANG II-induced IP production in
CHO cells expressing the
329 mutant, the implication of PKC-mediated
phosphorylation in the desensitization process of the
AT1A receptor could be postulated.
To study homologous vs. heterologous desensitization, a CHO cell line stably expressing both AT1A and vasopressin V3 receptors (10), which are coupled to the same signaling pathways, was produced. When these cells were submitted to ANG II stimulation, with or without an ANG II pretreatment, the responses were similar to those described for CHO AT1A cells (data not shown). To verify that the procedure of serial ANG II applications described above measures the desensitization of the receptor and not of an element further downstream in this signaling pathway, the effect of an ANG II pretreatment on the AVP-evoked IP production in these cells was analyzed (Fig. 7). When cells were preincubated with 100 nM ANG II before the 100 nM AVP application, the amplitude of the response reached 114% of the maximal stimulation obtained without pretreatment. This experiment demonstrated that an ANG II pretreatment did not promote the desensitization of any other element of this signaling pathway. Therefore the results described above represented exclusively the desensitization of the receptor.
However, the amplitudes of the ANG II-induced IP production with or without an AVP pretreatment were comparable. Therefore the activation of phospholipase C and subsequently of PKC via the V3 receptor was not able to induce a desensitization of the AT1A receptor and vice versa.
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DISCUSSION |
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In the present study, the functional consequences of successive
deletions of the COOH-terminal tail on signaling, internalization, and
desensitization of the rat AT1A
receptor were investigated. The first result of this study is that
these successive deletions up to amino acid 314 do not drastically
modify the expression at the cell surface or the binding affinity of
the receptor for agonist ligands (Table 1). This is in accordance with
the report of Ohyama et al. (22), which demonstrated that the deletion of the COOH-terminal sequence upstream of amino acid 309 does not
drastically alter the expression and pharmacological properties of the
AT1A receptor. However, the 304
mutant of the present study does not present any detectable binding for
ANG II, which indicates that the sequence between residues 304 and 309 is involved in the binding site of ANG II or more probably plays a role
in the folding, processing, and/or transport process at the
cell surface of the receptor.
The most interesting mutant characterized in this study is the 329
mutant, which displays reduction of both internalization and
desensitization associated with the transduction of an amplified signal. Several classes of functional alterations of G protein-coupled receptors resulting from natural or site-directed mutations have been
described. These include loss-of-function mutations as well as
gain-of-function mutations, such as those resulting in the constitutive
activation of the receptor and its corresponding signaling pathways
(21). However, the
329 mutant of the
AT1A receptor belongs to a new
class of functional mutants, presenting an amplification of the
physiological response, probably due to an alteration of
densensitization and/or internalization. Approaching phenotypes
have been described for the
2-adrenergic receptors (14),
and it would be interesting to investigate whether mutations of other
receptors could determine such a "phenotype" and whether natural
mutations of receptors could correspond to this phenotype in vivo.
In our series of mutants, the 329 is the largest deletion mutant,
which presents a major impairment of its internalization. Indeed, the
deletion mutants
347,
341, and
336 exhibit a ligand-induced internalization similar to the wild-type receptor, whereas
314 and
329 mutants are poorly internalized despite their ability to
transduce the signal. Several sequences of the receptors have been
identified as important for internalization of membrane-bound receptors. An example is the
NPX(1-2)Y sequence, which is essential for the internalization of the LDL, tyrosine kinase (27), and
2-adrenergic receptors (3).
However, this sequence corresponding to the sequence
300NPLFY304
of the AT1A receptor is not
involved in the internalization of this receptor (17). In the present
paper, a
329SLSTKMS335
sequence seems to be essential for
AT1A receptor internalization, and
an additional 15-amino acid deletion further reduces this internalization (17.9 vs. 31.8%; Figs. 1 and 2). This result is in
agreement with a previous report, indicating that two regions of the
AT1A receptor COOH-terminal tail
are important for internalization (33). This result should also be
compared with that reported by Hunyady et al. (16), who identify a
332TKMSTLS338
sequence as essential for internalization. This sequence only partially
overlaps the sequence identified in this paper. This apparent
discrepancy could be explained by the present strategy of tail
deletions, which uses a synthetic
AT1A cDNA with multiple unique
restriction sites leading to the addition of two or three amino acids
at the COOH-terminal of each deletion mutant. Therefore the
336
mutant presents a COOH-terminal sequence (TKMSRV), which may represent
a fairly well-conserved substitution of the sequence described by
Hunyady et al. The internalization sequence described in this paper
could be classified in those serine- and threonine-rich sequences
located within the COOH-terminal tail or the third intracellular loop
and identified as important for internalization of the G protein-coupled receptors. The precise role of this
AT1A receptor sequence is not
known. Given the fact that this sequence is serine and threonine rich,
it has been proposed that this sequence is phosphorylated after
receptor activation (16). Indeed, there are two PKC phosphorylation
sites (Ser331 and
Ser338) in or close to this
sequence and several other serines or threonines. This could suggest a
link between AT1A receptor
phosphorylation and internalization. However, it is unlikely for this
receptor, since several reports including ours show that ANG II
peptidic antagonists are able to internalize the receptor without
activating its G protein coupling and signal transduction (9, 15); in addition, an AT1A receptor mutant,
which binds ANG II normally but is not coupled to G protein, is still
internalized (9, 15). This relationship between
phosphorylation and internalization has been investigated extensively
for other G protein-coupled receptors. For some receptors, such as the
gastrin-releasing peptide receptor, internalization is at least
partially dependent on PKC phosphorylation of the receptor (5).
Phosphorylation of the
-adrenergic receptor was not considered as
necessary for receptor internalization; however, recent data suggest
that GRK2 and
-arrestin are implicated in
-adrenergic receptor
internalization (12). Similarly, the involvement of GRK2 in muscarinic
M2 receptor internalization is
also controversial (24, 36). These data suggest that the relationship
between receptor phosphorylation and internalization considerably
varies from one receptor to another, and it is difficult today to
delineate a common paradigm to define these relationships among this
family of receptors.
Another explanation for the role of this internalization sequence would be that it interacts with a specific intracellular protein implicated in the internalization process. This protein could belong to, for example, the adaptin family, which is one of the main components of the plasma membrane coat pits, a major structural and morphological element of internalization (30).
In addition to its impaired internalization, the 329 mutant exhibits
a reduced ANG II-induced desensitization. Because the deleted sequence
contains several serines and threonines, one possible explanation is
that this altered desensitization is due to a modification of the
AT1A receptor phosphorylation
pattern. This hypothesis is indirectly supported by the observation
that PMA, via PKC activation, is able to desensitize the wild-type AT1A receptor but not the
329
mutant. This absence of PMA-induced desensitization of the
329
mutant is similar to the observations of Balmforth et al. (2) for a
319 mutant. Such a role of PKC in the
AT1A receptor desensitization was
previously reported using PKC inhibitors (4). However, the use of
pharmacological concentrations of PKC inhibitors or activators in these
studies and their limited effect on desensitization both suggest that
this mechanism is only subsidiary in the physiological state. Indeed,
further experiments using a cell line expressing both the ANG II
AT1A and the vasopressin V3 receptors, which are both
coupled to phospholipase C and PKC (6), do not indicate that the
desensitization of the AT1A
receptor proceeds from a heterologous mechanism involving PKC but
instead from a homologous or another mechanism.
Therefore the reduction of homologous desensitization of the 329
mutant receptor could be due to either a reduction of ligand-induced internalization or an impairment of specific phosphorylation by an
unidentified GRK. The possibility that the desensitization defect of
the
329 mutant could be the consequence of its impaired internalization is unlikely. Indeed, in CHO
AT1A and CHO
329 cells,
concanavalin A drastically reduces receptor internalization but has no
effect on ANG II-induced IP production (data not shown). Moreover,
there is accumulating evidence from the literature that the inhibition
of ANG II receptor internalization in different cell types using KCl,
sucrose, or other inhibitors either has no effect or leads to a
reduction of the signal transduction (18, 28). Therefore the most
likely hypothesis is that the loss of homologous desensitization of the
329 mutant is due to the deletion of a sequence phosphorylated by a
specific GRK or to an interaction with a specific protein involved in
this desensitization process. Taken together, these data support the
hypothesis that desensitization of the
AT1A receptor is closely related
to receptor phosphorylation either by a nonspecific PKC-associated
mechanism or more probably by a GRK. A recent paper (23) analyzing the
phosphorylation mechanisms of the
AT1A receptor confirms these
hypotheses, showing that a tagged recombinant
AT1A receptor expressed in HEK 293 cells is desensitized and that this desensitization correlates
temporally with receptor phosphorylation by both PKC and GRK.
Finally, probably as the result of its impaired desensitization, the
329 mutant is unique in its ability to transduce an amplified signal
compared with the other mutants and the wild-type receptor. The
329
mutant amplifies stimulation of the IP turnover induced by ANG II
compared with the wild-type receptor. Despite similar values of basal
IP turnover and a similar number of binding sites in both cell lines,
the EC50 is eightfold lower (0.09 nM for
329 mutant vs. 0.75 nM for wild-type receptor), and the
maximal stimulation of IP turnover is twofold higher for the
329
mutant. This result is confirmed by another effect of ANG II in these cell lines: the ANG II-mediated inhibition of insulin-induced stimulation of glycogen synthesis is amplified in the CHO cell line
expressing the
329 mutant compared with that expressing the
wild-type receptor. These data should be compared with those of the
literature. On one hand, the
319 mutant expressed in HEK 293 cells
(2) induces a similar maximal stimulation of the IP turnover compared
with the wild-type receptor, despite a 2.5-fold reduction of the number
of sites, a fact not pointed out by the authors. If the correlation
between maximal IP stimulation and Bmax is taken into account, the
above result can be interpreted as a signal amplification for the
319 mutant. On the other hand, the
314 mutant presented in our
study does not transduce an amplified signal (IP production similar to
wild-type receptor) and presents a major alteration of G protein
interaction as demonstrated by the action of GTP
S on the ANG II
binding (Fig. 4). In addition, a closely related mutant induces a
normal Ca2+ mobilization in
response to ANG II (34). Taken together, these results suggest that the
deletion of the amino acid sequence 329-336 is responsible for the
transduction of an amplified signal, whereas further deletion of the
amino acid sequence 314-319 reduces the signal transduction
ability of the AT1A receptor.
Therefore the most probable explanation of the signal amplification is
the impairment of receptor desensitization via phosphorylation. In
addition, the reduction of the coupling efficiency by further deletion
is likely due to an alteration of the G protein coupling.
In conclusion, the phenotype described for 329 could reflect major
impairments of two mechanisms dependent on ANG II binding: receptor
internalization and homologous desensitization, which could result from
receptor phosphorylation or other mechanisms. In the future, it will be
very interesting to analyze in detail the amino acids of this sequence
involved in the desensitization process by performing single or
multiple amino acid mutations or deletions. The parallel
characterization of the receptor phosphorylation will be possible using
functional epitope-tagged receptors. Finally, the analysis of the
phenotype produced by the expression of this internalization- and
desensitization- defective AT1A
mutant in transgenic animals could be of physiological and pathological significance.
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ACKNOWLEDGEMENTS |
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We acknowledge Catherine Monnot and Tracy Williams for many helpful discussions. We are grateful to Nicole Braure for secretarial assistance.
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FOOTNOTES |
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This work was supported by Institut National de la Santé et de la Recherche Médicale and by a grant from Roussel Uclaf. S. Conchon is the recipient of a Studentship Award from Association pour la Recherche contre le Cancer.
Address for reprint requests: E. Clauser, INSERM U36, Collège de France, 3 rue d'Ulm, 75005 Paris, France.
Received 24 January 1997; accepted in final form 17 October 1997.
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