From INSERM Unité 367, 17 rue du Fer à
Moulin 75005 Paris, France and the ¶ Institute of Physiological
Chemistry and Pathobiochemistry, Johannes Gutenberg University at
Mainz, Duesbergweg 6, D-55099 Mainz, Federal Republic of Germany
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ABSTRACT |
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The binding of bradykinin (BK) to
B2 receptor triggers the internalization of the
agonist-receptor complex. To investigate the mechanisms and the
receptor structures involved in this fundamental process of receptor
regulation, the human B2 receptor was mutated within its
cytoplasmic tail by complementary strategies of truncation, deletion,
and amino acid substitution. Ligand binding, signal transduction,
internalization as well as phosphorylation were studied for the mutated
receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail
corresponding to positions 321-364 did not alter the kinetics of BK
binding and the receptor coupling to phospholipase C and phospholipase
A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as
alanine substitution of serine and threonine residues within segment
335-351 diminished the internalization capacity of the mutant
receptors. Mutants with a markedly reduced internalization potential
failed to produce BK-induced receptor phosphorylation suggesting that
phosphorylation may be involved in receptor internalization. The
mutagenesis approaches converged at the conclusion that three serines
in positions 339, 346, and 348 and two threonines in positions 342 and
345, contained in a sequence segment that is highly conserved between
species, have a critical role in the ligand-dependent
internalization and phosphorylation of kinin receptors and can
intervene in these processes in an alternative manner. However, mutants
lacking these residues were still sensitive to dominant-negative forms
of The capacity of most G protein-coupled receptors
(GPCRs)1 to respond to
agonists becomes altered after previous exposure to their ligands, a
phenomenon referred to as desensitization. Desensitization can be a
consequence of receptor endocytosis (1) and may also result from
receptor phosphorylation that prevents further activation of G proteins
(2, 3). Negative cooperativity in bradykinin (BK) binding due to
receptor-receptor interaction in the cell membrane can also contribute
to the desensitization of some receptors including the BK
B2 receptor (4). For few GPCRs, internalization appears to
be a prerequisite for resensitization prior to receptor recycling to
the plasma membrane (5). Internalization is believed to involve
clathrin-coated vesicles and/or caveolin-rich vesicles, and to result
from an interaction of components of the endocytic machinery with
specific motifs located in the cytoplasmic domains of the receptor (6).
For many receptors, the carboxyl tail has been found to play a critical
role in receptor internalization (2, 3, 7) but other critical motifs
have been identified in the second or third intracellular loops (8, 9)
as well as in the seventh transmembrane domain (10).
The molecular mechanisms that trigger internalization of the
agonist-occupied receptor have been studied for only a few receptors. Phosphorylation has been implicated in the desensitization process uncoupling the receptor from G proteins, and in the initiation of
internalization sequestering the receptor from the cell surface (2, 3).
Indeed, mutations of potential phosphorylation sites of the GLP-1,
muscarinic m3, and the cholecystokinin receptors have been
shown to reduce their internalization (11-13). However, receptor
internalization and desensitization are not always causally linked,
e.g. mutations of certain GPCRs suppressed desensitization but not internalization (14, 15). Conversely mutations in some GPCRs
altered internalization but not desensitization (10, 15). Clearly, the
two processes can proceed independently although they may involve the
same type of post-translational modifications such as phosphorylation.
Prototypical GPCRs that differ by their ligand-induced
densensitization, phosphorylation, and internalization are the
receptors for the vasoactive kinin peptides. Two subtypes of mammalian
kinin receptors, B1 and B2, have been
recognized so far (16-18). The B2 receptor is responsible
for most of the physiological actions of BK including vasodilation (19)
through activation of G proteins (20) that stimulate the activity of
phospholipase C (PLC) and phospholipase A2
(PLA2) and increase the cytoplasmic calcium concentration, [Ca2+]i (21). More recently, other signaling
pathways such as the mitogen-activated protein (MAP) kinase pathway
were found to be triggered via the B2 receptor (22, 23).
BK-induced B2 receptor internalization has been reported in
several cell systems including cultured human fibroblasts HF-15 (24,
25) and CHO-K1 cells transfected with the human B2 receptor
cDNA (4), and we have recently reported that in HF-15 cells BK
induces the phosphorylation of serine and threonine residues located in
the COOH terminus of the B2 receptor (21). The time course
of BK-stimulated phosphorylation paralleled the kinetics of
desensitization/resensitization and of internalization/recycling of the
receptor, suggesting that these phenomena may be interrelated (21).
The present work was aimed at studying the role of the COOH-terminal
tail of the human renal B2 bradykinin receptor (referred as
wild-type, B2wt) in BK binding, coupling to signaling
pathways, internalization, and phosphorylation. For this purpose, we
generated a series of human B2 receptor mutants and
transfected them into COS-7 cells, CHO-K1, and HEK 293 cells. Three
complementary strategies were followed for mutagenesis, by generating
mutants with truncated COOH-terminal tail, mutants with deletion of
internal regions of the COOH-terminal tail, and mutants with serine
and/or threonine residue(s) replaced by alanine. This allowed us to
demonstrate that a cluster of three serine and two threonine residues
located in the center portion of the COOH-terminal tail region that
becomes phosphorylated in response to BK is involved in the
internalization of the B2 receptor. Co-transfection of the
B2wt and its mutants with Construction of the Mutant Receptor cDNAs--
Mutant
cDNAs were constructed by using the previously cloned
B2wt cDNA placed under the control of cytomegalovirus
promoter into the eucaryotic expression vector pcDNA3
(Invitrogen, Leek, Netherlands) (4) as a template in site-directed
mutagenesis using the TransformerTM Site-directed
Mutagenesis Kit (2nd version, CLONTECH,
Palo Alto, CA). All mutations were sequenced using an
AmplicycleTM sequencing kit (Perkin Elmer, Langen,
Germany). Three truncated B2 mutants, tR351, tI334, and
tY320 were created by single nucleotide substitutions (indicated by
bold face in the sequences given below) at the appropriate sites so as
to create stop codons (underlined) terminating translation at positions
351, 334, and 320, respectively, of the protein sequence (numbering
according to Hess et al. (17)). The nucleotide sequences of
the oligonucleotide primers used were: 5'-CGTGGAACGCTAGATTCACAAACTG-3' for the mutant
tR351, 5'-CAGAACCCATTTAGATGGAGAACTCC-3' for
tI334, and 5'-GGAGGTGTACTAGGGAGTGTG-3' for
tY320. Hence deletion mutants tR351, tI334, and tY320 lacked the
terminal 13, 30, and 44 amino acid residues, respectively, of the human
B2 receptor. Deletions of regions within the COOH-terminal
tail of the B2 receptor were obtained by mutagenesis with
the following oligonucleotide primers:
5'-CAGGTCAGAACCCATTCAGATTCACAAACTGC-3' for the mutant del[335-351],
5'-CAGGTCAGAACCCATTCTGCGGACCTCCATC-3' for del[335-342] and
5'-CTCCATGGGCACACAGATTCACAAACTGC-3' for del[343-351]. The resulting
mutants del[335-351], del[335-342], and del[343-351] lacked
17, 8, and 9 amino acid residues, respectively; their relative positions within the protein sequence are given in brackets. Point mutations were created by the same procedure to replace Ser and/or Thr
residues in the COOH-terminal tail by Ala using the following oligonucleotide primers:
5'-GGAGATCCAGGCCGAGAGGAGG-3' for the mutant
T237A, 5'-CGAAAGAAGGCTTGGGAGGTG-3' for
S316A, 5'-GGCTGCAGGGCAGAACCC-3' for S331A,
5'-GATGGAGAACGCCATGGGCAC-3' for S339A,
5'-CTCCATGGGCGCACTGCGGAC-3' for T342A,
5'-GCACACTGCGGGCCGCCATCTCCGTG-3' for
T345A/S346A, 5'-GACCTCCATCGCCGTGGAAC-3' for
S348A, 5'-GGAGAACGCCATGGGCGCACTGCGG-3'
for S339A/T342A and
5'-GAACGCCATGGGCGCACTGCGGGCCGCCATCGCCGTGG-3' for B2 Cell Culture and Receptor Expression--
COS-7 and HEK 293 cells (American Type Culture Collection, Manassas, VA) were grown in
Dulbecco's modified Eagle's medium (Biological Industries, Israel)
and CHO-K1 cells in Ham's F-12 medium (Seromed, Berlin, Germany) both
supplemented with 10% fetal calf serum, 2 mM glutamine,
and 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml
amphotericin B. For immunoprecipitation and phosphorylation studies,
transient receptor expression was achieved by transfecting COS-7 cells
grown in 6-well plates using the LipofectAMINETM method
(Life Technologies). Otherwise, transfections were performed with cells
grown in T75 flasks (COS-7 cells) by using the DEAE-dextran method (26)
and the cells were then subcultured into 24-well plates where binding,
internalization, PLC, and PLA2 activation experiments were
performed or with cells directly grown in 24-well plates (HEK 293 and
CHO-K1 cells) by using Superfect Transfection Reagent (Qiagen,
Courtaboeuf, France). CHO-K1 cell clones that were selected with
geneticin (0.75 µg/ml) for stable expression and with
[3H]BK binding and coupling assays for receptor
expression, were expanded to obtain the CHO-K1 cell lines used in some
experiments. All cell types were maintained at 37 °C in a humidified
water-jacketed incubator with 5% CO2. Functional studies
of the transfected cells were done at confluence. Control cells were
created by transfecting the pcDNA3 vector without insert.
Radioligand Binding and Internalization of
[3H]BK--
Cells were incubated as described previously
(4) in Hank's balanced salt solution (HBSS, 0.33 mM
Na2HPO4, 0.44 mM
KH2PO4, 127 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 20 mM HEPES, 1 mM MgCl2, 0.8 mM MgSO4, 1.5 mM CaCl2,
5 mM glucose, 10 mM sodium acetate, pH 7.4)
containing protease inhibitors (10 Inositol Phosphate Production--
Cells were loaded for 18 h at 37 °C with 3 µCi/ml
myo-[2-3H]inositol (10-20 Ci/mmol, Amersham
International) added to the culture medium. The cells were washed twice
with HBSS, preincubated for 10 min at 37 °C with 10 mM
LiCl in HBSS containing 0.1% BSA and the protease inhibitors used for
binding assay, and stimulated for 15 min with BK at varying
concentrations (10 Measurement of Phospholipase A2 activity--
Cells
were labeled to equilibrium with 1 µCi/ml
[3H]arachidonic acid (150-230 Ci/mmol; Amersham
International) for 18 h. After washing steps to eliminate unbound
radiolabel (4), phospholipase A2 activation experiments
were performed at 37 °C for 10 min in HBSS containing 0.1% BSA,
protease inhibitors, and the test compounds or vehicles. The medium
radioactivity containing the released [3H]arachidonic
acid plus derived 3H-labeled metabolites was counted and
expressed in percent of the total radioactivity, i.e. the
medium plus cell associated radioactivity.
35S Labeling and Immunoprecipitation--
Cells were
washed twice with sulfur-free HEPES-buffered Dulbecco's modified
Eagle's medium, incubated for 30 min at 37 °C in the same medium,
and labeled with 0.1 mCi/ml 35S-labeled amino acids
(Prox-mixTM, Amersham International) for 8 h (21).
After three washes with 50 mM Tris, pH 7.5, 150 mM NaCl (Tris-buffered saline), cells were scraped into
ice-cold lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (w/v) Nonidet P-40,
0.5% deoxycholate, 0.1% SDS, 0.1 mM Pefabloc
SCTM, and 10 µg/ml each of 1,10-phenantroline, aprotinin,
leupeptin, and pepstatin A, and incubated for 45 min at 4 °C under
gentle rocking. The resulting lysates were centrifuged for 10 min at 13,000 rpm and the supernatants were precleared with 50 µl of Staphylococcus aureus cell suspension
(PansorbinTM, Calbiochem).
To immunoprecipitate the receptor, 2.5 µl of antiserum AS346 (21)
diluted in 0.1 ml of 5% BSA in lysis buffer was added. After 15 min at
room temperature, 50 µl of PansorbinTM was added, and the
suspension incubated for 10 min at room temperature. The precipitate
was recovered by a centrifugation for 2 min at 6,000 rpm and washed
three times with lysis buffer and once with water. SDS sample buffer
(25 µl) was added to the immunoprecipitate followed by a 15-min
incubation at 45 °C. Proteins were resolved by 10%
SDS-polyacrylamide gel electrophoresis (PAGE) in the presence of 5 M urea. After fixation with 20% (w/v) trichloroacetic acid for 20 min, the gels were washed several times with water and subjected
to fluorography using 15% (w/v) sodium salicylate as the fluorophor.
For control, the antiserum was preadsorbed for 90 min at room
temperature with the peptide antigen coupled to Affi-Gel 10 (Bio-Rad).
Receptor Phosphorylation--
Cells were washed twice with
phosphate-free HEPES-buffered Dulbecco's modified Eagle's medium,
incubated for 1 h at 37 °C, and labeled with 0.25 mCi/ml
[32P]orthophosphate (ICN) for 8 h in the same
medium. After a 5-min exposure to 1 µM BK or vehicle at
37 °C, cells were scraped into 1 ml of ice-cold lysis buffer
containing protease inhibitors (see above) and phosphatase inhibitors
(50 mM sodium fluoride, 25 mM sodium
pyrophosphate, 1 mM sodium orthovanadate). To study the role of PKC and the effect of phosphatase inhibitor, cells were exposed
to 0.1 µM PMA and 0.1 µM okadaic acid,
respectively, in the absence or presence of 1 µM BK
before the cell lysis. Solubilization, immunoprecipitation, and gel
electrophoresis were carried out as above. Proteins labeled by
[32P]orthophosphate were revealed by autoradiography.
Our strategy to analyze the role of the COOH-terminal domain of
the B2 receptor in ligand binding, signal transduction,
sequestration, and phosphorylation involved the production of mutants
with progressive truncations of the COOH-terminal tail of the receptor,
deletions of defined portions of the tail region, or exchange of single or multiple residues of serine and threonine against alanine at defined
positions of the COOH-terminal domain of the receptor.
Endogenous Receptor of Cells Used for Transfection--
Initially
we tested for the presence of endogenous B2 receptor in the
COS cells using mock-transfected cells and comparing them with
transfected cells expressing the wild-type human B2 receptor (COSB2wt). The receptor density measured by
[3H]BK binding and the BK-sensitive PLC activity
determined by IPs production in mock-transfected COS cells represented
<5% of the values measured in COSB2wt, whereas the
ligand-induced [Ca2+]i transients of
mock-transfected cells were similar to those obtained with
COSB2wt (data not shown). No PLA2 activation was observed in mock-transfected or COSB2wt cells
indicating the absence of this transduction pathway in COS-7 cells.
Therefore we choose [3H]BK binding and IPs production to
exemplify the signaling properties of the recombinant human
B2 receptors expressed in COS-7 cells. No endogenous
B2 receptor was detected with [3H]BK binding,
PLC, or PLA2 assays in the mock-transfected CHO-K1 and HEK
293 cells.
Generation of Mutant B2 Receptors--
To study the
functional role of the COOH-terminal tail region corresponding to
intracellular domain ID-4 of the B2wt we produced three
truncation variants with progressive deletions of the COOH-terminal portion of the receptor. These truncation mutants are designated by
"t" followed by the name (single letter code) and position of their
carboxyl-terminal amino acid, i.e. tR351, tI334, and tY320;
they lack the COOH-terminal 14, 30, or 44 residues of the native
receptor (Fig. 1). Three deletion mutants
are designated by "del" followed by the positions delimiting the
deleted region, i.e. del[335-351], del[335-342], and
del[343-351] lacking 17, 8, and 9 residues, respectively, of the
COOH-terminal tail region (Fig. 1). We also created 7 receptor mutants
in which alanine (Ala) was substituted for serine (Ser) or threonine
(Thr) residue(s), singly or combined (Table
I). In the mutant B2 Expression and Characterization of Mutant B2
Receptors--
The mutant receptors were characterized for their
ligand binding and signal transduction properties. Ligand binding was
assessed by incubating transfected cells with a wide range of
[3H]BK concentrations at 1.25 × 10 Internalization of [3H]BK by Mutant B2
Receptors Expressed in COS-7 Cells--
In the COSB2wt
which were transfected with 15 µg of cDNA and challenged with 2 nM [3H]BK (Fig.
2), the internalization occurred with a
t1/2 of 12 min at 37 °C and represented 60.3 ± 2.5% (n = 22) of the bound [3H]BK at
70 min. The same internalization rate was observed in COS cells
transfected with 0.75, 1.5, 3, 5, or 10 µg of cDNA and displaying
a wide range of receptor density (from 0.9 to 5.0 pmol/mg of protein)
(data not shown). This is also the case for CHO cells which expressed
the B2wt stably at different density (0.3 to 2.5 pmol/mg
protein) (not shown) with the remark that the internalization was more
efficient (72.6 ± 1.5% (n = 7) of the bound
radioactivity) in CHO than in COS cells. Regarding the effects of the
receptor mutations, most of the data presented below were from COS-7
cells transfected with 15 µg of cDNA. In these cells, it was
apparent that the serial truncations of the COOH-terminal tail region
gave distinct phenotypes: tR351 which lacks the extreme 14 COOH-terminal residues had an unchanged internalization capacity
whereas tI334 displayed a markedly reduced capacity to internalize
[3H]BK, i.e. 35% of that of the
B2wt (100%). Further truncations of the receptor
exemplified by mutant tY320 did not result in a greater reduction of
the internalization capacity (Fig. 2). These findings suggest that the
COOH-terminal portion distal to Arg351 is dispensable for
receptor internalization while the segment spanning positions 335 to
351 comprising 17 residues appears to be of critical importance to
receptor sequestration. To further test this hypothesis we constructed
a deletion mutant, del[335-351] which selectively lacks segment
Gln335 to Arg351 and retains the portion distal
to Arg351 (Fig. 1). The internalization capacity of
del[335-351] was similar to that of tI334 demonstrating that the 13 most COOH-terminal residues of the B2 receptor (positions
352 to 364) cannot rescue this phenotype. In addition, the same
internalization rate was observed over a wide range of density of this
mutant receptor del[335-351] that was established by transfecting
cells with 0.75 to 15 µg of cDNA (not shown). To pinpoint the
region(s) important for internalization we generated mutants where the
proximal 8 residues of the critical Gln335
Because the segment of the COOH-terminal domain of the B2wt
that appears to interfere with receptor internalization contains 5 residues of serine/threonine that may be critical for receptor sequestration we constructed mutant receptors where a single residue was changed to alanine, i.e. S339A, T342A, and S348A, or two
adjacent residues were substituted at the same time, i.e.
T345A/S346A. The internalization capacity was reduced in the order
Thr342 > Thr345, Ser346 > Ser339 > Ser348 although the level (41.1 ± 4.5% to 88.3 ± 3.1% of B2wt) was not as dramatic
as for del[335-351] and tI334 (24.7 and 35.1% of B2wt, respectively) (Fig. 2). By contrast, the simultaneous replacement of
distant Ser/Thr residues, i.e. S339A/T342A and
S339A/T345A/S346A, produced a markedly reduced internalization
(27.6 ± 2.6 and 32.4 ± 1.8% of B2wt,
respectively) similar to that observed for del[335-351] and tI334
(Fig. 2). This increment was not further increased by the substitution
of all 5 Ser/Thr residues of the Gln335 Internalization of [3H]BK by Mutant B2
Receptors Expressed in CHO-K1 and HEK 293 Cells--
Additionally to
their characterization in the COS-7 cells, the wild type and some
mutant receptors were tested for their ability to internalize
[3H]BK when expressed in CHO-K1 cells (stably and
transiently) and in HEK 293 (transiently). The results summarized in
Fig. 3 demonstrated that in those cells
the truncation mutant tY320 and the deletion mutant del[335-351] as
well as the point mutation B2 Immunoprecipitation of Mutant Receptors--
The above
observations made it of interest to study the receptor phosphorylation,
especially to demonstrate whether the Ser and Thr residues discussed
above are implicated. To this end, transfected cells with the various
truncated, deleted and point mutated receptor mutants were labeled with
[35S]methionine or [32P]orthophosphate, and
incubated with or without 1 µM BK for 5 min prior to cell
lysis. For immunoprecipitation of the receptor we applied antiserum
AS346 which has been raised against a peptide derived from the COOH
terminus of the B2wt (positions 329 to 364) (21). In
35S labeling experiments (Fig.
4d), immunoprecipitation of
B2wt resulted in a diffuse band of 60-100 kDa that was
superimposable with the immunoreactive band found in 32P
labeling experiments (Fig. 4, a-c). All mutant receptors
including B2 Phosphorylation of Mutant Receptors--
Under basal conditions,
i.e. in the absence of BK, a significant phosphorylation was
observed for B2wt and mutant receptors except for
del[335-351], del[343-351], and S348A suggesting that Ser at
position 348 may represent a major target site for ligand-independent phosphorylation. BK dramatically increased the phosphorylation level of
B2wt as well as of T237A, S316A, and S331A where Ser and
Thr residues external to the Gln335
Interestingly, the treatment of COSB2wt with 0.1 µM okadaic acid (a potent phosphatase 2A/1 inhibitor) for
5 min prior to and during the 10-min incubation period with 2 nM [3H]BK (Fig.
5) resulted in a considerable increase in
the internalization rate: 64.6 ± 6.7% of the total
[3H]BK bound was internalized under okadaic acid
treatment compared with 34.4 ± 2.2% under control conditions
(p < 0.05). Okadaic acid also resulted in a marked
increase in the phosphorylation level of B2wt in the
presence of BK (Fig. 5). By contrast, the same treatment applied to the
cells expressing the mutant B2 Relationship between B2 Receptor Phosphorylation and
Internalization--
It has been reported that sucrose inhibits
receptor endocytosis through clathrin-coated vesicles and that
phosphorylated Ser/Thr residues in the COOH-terminal tail region of the
Post-stimulatory desensitization and cellular redistribution of
GPCRs are important mechanisms that regulate the availability and
signaling capacity of hormonal effector systems. For instance, ligand-induced internalization of the The present work demonstrates that a short sequence segment covering
positions 335 to 351 of the bradykinin B2 receptor
COOH-terminal tail plays a major role in the agonist-induced
internalization and phosphorylation of the receptor, but is not
necessary for PLC and PLA2 activation. Five amino acid
residues, namely Ser in positions 339, 346, and 348 and Thr in
positions 342 and 345, were shown to be involved in these functions and
their respective contribution was analyzed. Complementary approaches
for receptor modifications, i.e. truncation, deletion, and
mutation of single or multiple residues were used to get insights into
the structure-function relationships within the COOH-terminal tail.
Most of the mutations resulted in alteration in the receptor capacity
to undergo internalization and these alterations were consistent in
different cell types. A reduction was always observed, indicating that
the COOH-terminal tail possesses positive rather than negative motifs
for internalization; at maximum we observed almost 80% reduction in
the receptor sequestration in COS-7 cells. This contrasts with the
findings that the human The maximum reduction in receptor internalization was obtained with
three distinct types of mutations and the effect of these mutations was
consistent in the three different cell types tested COS-7, CHO-K1, and
HEK 293. Substitution of the 5 Ser/Thr residues in the
Gln335 The present study shows that B2 receptor internalization is
independent of the affinity for BK or of the efficiency of PLC and
PLA2 coupling as reported for the truncated human platelet activating factor receptor (40). The COOH-terminal tail segment downstream of Tyr320 as well as the proximal residues
Thr237, Ser316, and Ser331 do not
function in the coupling of the receptors to G proteins stimulating the
PLC pathway. These observations are in agreement with those of Faussner
et al. (7) who reported that truncations either at
Gly327 or Lys315 diminished the capacity to
internalize BK but left the capacity to activate PLC unchanged. These
and our results are, however, inconsistent with a recent report (39)
claiming compromised ligand internalization associated with reduced PLC
activity for a rat B2 receptor mutant devoid of the
terminal 34 residues (corresponding to tR331 for the human receptor).
Differences in species (rat versus man) and expression
systems (Rat-1 versus COS-7, CHO-K1, and HEK 293) may help
explain some of the observed discrepancies.
Our work provides strong evidence that phosphorylation is the
triggering signal that enters the B2wt in the
internalization process. Several lines of evidence support this notion:
(i) BK stimulation induces internalization and homologous receptor
phosphorylation; (ii) the kinetics of receptor internalization and
recycling parallel those of receptor phosphorylation and
dephosphorylation in HF-15 cells (24); (iii) inhibition of phosphatase
activity which delays B2wt receptor dephosphorylation
increases [3H]BK internalization but neither alters the
internalization of [3H]BK by B2 The question whether BK-sensitive phosphorylation sites can serve as
recognition motifs for receptor interaction with clathrin-coated vesicles was not elucidated in the present work. Indeed, the mutant del[335-351] with reduced internalization capacity and lacking these
sites still exhibited a sensitivity to the clathrin-coated vesicle
disrupting sucrose (data not shown) and to the effect of co-expression
of dominant-negative forms of In conclusion, this work has allowed us to establish that optimal
internalization and phosphorylation of the B2wt require the
integrity of Ser339, Ser346,
Ser348, Thr342, and Thr345
residues, located in a short segment of 17 residues (Gln335
One limitation of the present approach is highlighted by the finding of
alternative phosphorylation of closely spaced Ser/Thr residues within
the putative phosphorylation cassette. Hence we cannot draw firm
conclusions as to relative importance, functional hierarchy, and/or
sequential modification of the critical Ser/Thr residues. Studies aimed
at the precise mapping of the phosphorylation sites in vivo
and at the elucidation of the sequence of phosphorylation events in the
human B2 bradykinin receptor are underway.
-arrestin and dynamin, suggesting the existence of additional
receptor structure(s) involved in the receptor sequestration through
clathrin-coated vesicles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin and dynamin mutants
documented a role of clathrin-coated pit pathway in receptor internalization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ST in which Ser339,
Thr342, Thr345, Ser346, and
Ser348 were simultaneously substituted by Ala. The mutant
S339A/T345A/S346A was obtained by combining the primers used for the
S339A and T345A/S346A mutants.
5 M
captopril, 0.08 unit/ml aprotinin, 0.1 mg/ml bacitracin), 0.1% fatty
acid-free bovine serum albumin (BSA), and [3H]BK (110 Ci/mmol, NEN, Leblanc Mesnil, France). For equilibrium studies and
determination of receptor density or apparent affinity for
[3H]BK, incubation was carried out at 4 °C for 6 h in the presence of 1.25 × 10
11 to 2.5 × 10
8 M [3H]BK (COS cells) or
10
11 to 10
7 M
[3H]BK (CHO cells). The cells were extensively washed
with HBSS and bound [3H]BK was determined in a liquid
scintillation counter. Internalization of [3H]BK was
studied by incubating the cells with 2 nM
[3H]BK at 37 °C for 2 to 70 min (4). Cells were washed
with HBSS before cell surface-bound radioligand was separated from
internalized radioactivity by an acidic washing step (27) with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. To study the
role of protein kinase C (PKC) activity, cells were preincubated with
10
7 M phorbol 12-myristate 13-acetate (PMA)
or 10
7 M staurosporine (Sigma,
St-Quentin-Fallavier, France) for 30 min before and during the
incubation with [3H]BK. To monitor the effect of
phosphatase inhibitor, cells were incubated with 10
7
M okadaic acid (Sigma) 5 min prior to [3H]BK
challenge, and internalization was followed for 10 min in the
continuous presence of the phosphatase inhibitor. In some internalization experiments, cells were treated with hypertonic saline
solution containing 0.4 M sucrose to study the contribution of clathrin-coated vesicle pathway (28). In addition, internalization was examined in cells co-transfected with the receptor and with rat
-arrestin cDNA or its inactive fragment (319-418) mutant in
pEGFP obtained from Bunnett and co-workers (29) and
pcDNA3 plasmids encoding for bovine
-arrestin
fragment (319-418) mutant and inactive human dynamin K44A both
obtained from Benovic and co-workers (30). Co-transfection was done
using 15 µg of each plasmid. For determination of specific binding,
each assay included measurements of nonspecific binding in the presence
of 1000-fold excess of unlabeled BK; nonspecific binding was
substracted from total binding determined in the absence of unlabeled
BK. In each culture plate, the protein content was determined according
to the manufacturer's instructions in three wells using BSA as the standard protein (Bio-Rad, München, Germany).
10 to 10
7 M).
To study the role of PKC, cells were treated with PMA
(10
7 M) or staurosporine (10
7
M) for 30 min before and during the incubation with BK.
Reactions were terminated by addition of 3% (w/v) ice-cold perchloric
acid and total [3H]inositol phosphate radioactivity was
isolated using AG1-X8 anion exchange column chromatography (formate
form, 100-200 mesh, Bio-Rad) after the radioactivity contained in
phospholipids had been extracted with chloroform (4). Results are
expressed as the ratio between the radioactivity measured in the
inositol phosphates and the total radioactivity incorporated into the
overall compounds labeled with
myo-[3H]inositol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ST we
exchanged 5 Ser/Thr residues located in the center portion of the
COOH-terminal domain, i.e. at positions 339, 342, 345, 346, and 348 (Fig. 1). We have also constructed 3 mutants where Ser/Thr
residues have been replaced by Ala in other regions of the receptor,
i.e. T237A, S316A, and S331A (not shown). The rationale for
the design of the various mutants is given below.
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Fig. 1.
Schematic representation of the COOH-terminal
tail of B2wt and mutant receptors. Top, mutated amino
acid residues are indicated by their position within the B2wt sequence;
the numbering is according to Hess et al. (39). Ser and Thr
residues which were replaced by Ala are marked by asterisks.
Center, cytoplasmic tail truncations were performed at 3 sites to generate tR351, tI334, and tY320. Bottom, deletion
of segments holding 17, 8, and 9 residues generated mutants
del[335-351], del[335-342], and del[343-351], respectively;
the deleted segments are identified by their positions (given in
brackets) and marked by bold lines.
[3H]BK binding and BK-induced PLC activation characteristics
of B2wt and mutant receptors in COS cells
11 to 2.5 × 10
8 M), before
determination of specific binding (see "Experimental Procedures").
Data were first plotted using Scatchard coordinates. Given the
curvilinear character of the plots obtained, binding values at the
three highest [3H]BK concentrations were used to estimate the
maximal binding capacity (Bmax). All binding values
were then plotted using Hill coordinates to estimate the
[3H]BK concentration (KDapp)
corresponding to half-saturation of binding sites. In each PLC
activation experiment out of the 14 performed, up to six mutants were
studied together with the B2wt as control. Cells at 72 h
after transfection were incubated at 37 °C for 15 min with
10
11 to 10
7 M BK and 10 mM
LiCl, before determination of IPs production (see "Experimental
Procedures"). For each mutant receptor, results are the mean ± S.E. of at least three independent experiments, each performed in
triplicate.
11
M to 2.5 × 10
8 M for COS-7
cells (Table I) and at 10
11 M to
10
7 M for CHO-K1 cells (Table
II). The incubation was carried out for
6 h under equilibrium conditions at 4 °C to suppress
internalization. In COS cells (Table I) the kinetics of
[3H]BK binding displayed negative cooperativity as
previously observed for the human B2wt in CHO-K1 cells (4).
The receptor density determined by Scatchard analysis (31) was similar
for the B2wt and mutant receptors except for tY320 and
B2
ST which were 10- and 5-fold less expressed,
respectively. The kinetics of [3H]BK binding estimated
from Hill analysis of the binding data (32) were similar for the
various receptors since half-maximum binding
(KD,app) occurred within a narrow range
(2.3 to 6.3 nM) of [3H]BK concentrations.
Regarding the signal transduction properties, the basal IPs production
was similar for B2wt and the mutant receptors demonstrating
that none of the mutations resulted in a constitutive activation of
PLC. All mutant receptors were able to trigger PLC activation in
response to 10
11 to 10
7 M BK
and responded to BK with maximum stimulation (4.1-8.5-fold) and
EC50 values (0.2-0.8 nM) in the same order of
magnitude as those observed with B2wt with the exception of
tY320 which had a higher EC50 value (2.2 ± 0.9 nM). Then, we choose to stably express in CHO-K1 cells the
mutant tY320 bearing the most pronounced structural change and also the
largest deleted mutant, del[335-351] lacking the 5 Ser and Thr
residues. The B2wt CHO cell line was obtained as described
previously (4). In these cells (Table II), tY320 also exhibited a
higher EC50 value for PLC activation, a greater
EC50 value for PLA2 activation and a lower
affinity for [3H]BK. By contrast, the mutant
del[335-351] exhibited a slightly higher affinity for
[3H]BK and exhibited a 30- and 50-fold lower
EC50 value for PLC and PLA2 activation,
respectively, compared with B2wt (Table II). These data
indicate that the COOH-terminal tail is not necessary for signal
transduction but that its conformation influences the efficiency of BK
binding and coupling to PLC and PLA2.
[3H]BK binding and BK-induced PLC and PLA2 activation
characteristics of B2wt and mutant receptors in CHO-K1 cells
11 to 10
7 M),
before determination of specific binding (see "Experimental
Procedures"). Data were obtained as explained in the legend of Table
I. In coupling experiments, cells were incubated at 37 °C with
10
11 to 10
7 M BK for 10 min before measurement of
PLA2 activation and 15 min in presence of 10 mM
LiCl before determination of IPs production (see "Experimental
Procedures"). For each mutant receptor, results are the mean ± S.E. of at least three independent experiments.
Arg351 segment are removed, del[335-342], or where the
distal 9 residues are deleted, del[343-351]. The effect of the
largest deletion mutant (del[335-351]) was almost quantitatively
reproduced by deletion mutant del[335-342] (24.7 ± 5.2 and
30.7 ± 5.2%, respectively, of B2wt). However,
del[343-351] also resulted in a reduced internalization capacity
(56.5 ± 4.5% of B2wt), suggesting that the adjacent
segments spanning positions 335 to 342 and 343 to 351 do not function
in a simple additive manner for receptor internalization.
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Fig. 2.
[3H]BK internalization in COS-7
cells transfected with B2wt and mutant receptors. Cells (used
72 h after transfection) were incubated with 2 nM
[3H]BK at 37 °C for the times indicated. Unbound
radioactivity was removed at 4 °C before the cell surface-associated
and the internalized radioactivities were separated and quantitated as
described under "Experimental Procedures." Specific binding was
calculated as the difference of total binding and nonspecific binding
in the presence of unlabeled BK (10 µM). Internalized
radioactivity represents the acid-resistant fraction (%) of the total
specific binding. Results are given as the percentage of the control,
i.e. B2wt internalization at 70 min representing
60.3 ± 2.5% of the [3H]BK bound (100%
corresponding to 0.18 ± 0.01 pmol/mg of protein). For point
mutations, the data for five representative mutants (out of 10) are
presented. Values are means ± S.E. from at least three
independent experiments each performed in duplicate.
TS345-356A, T345A/S346A.
Arg351 segment, i.e.
S339A/T342A/T345A/S346A/S348A (Fig. 2, note that this mutant
B2
ST represents a full-length receptor protein). Together these findings suggest that Ser and Thr residues within the
17-residue segment spanning positions 335 to 351 contribute differentially to receptor endocytosis and can very likely intervene in
this process in an alternative manner. Mutations of Ser/Thr residues
external to this segment, e.g. T237A, S316A, and S331A neither altered the binding capacity nor the internalization rate of
the resultant receptor mutants (data not shown).
ST receptor all exhibited a
markedly decreased ability to internalize [3H]BK compared
with the B2wt, like in COS-7 cells. However, the fraction
of the [3H]BK binding which was internalized differed
slightly from one cell type to another. For the control
B2wt, this fraction was of 72.6 ± 1.5% in CHO-K1,
57. ± 3.1% in HEK 293, and 60.3 ± 2.5% in COS-7. These
observations can be linked to the differential amount of GRKs and
arrestins expressed in these cell types reported by Menard et
al. (33). It should be pointed out that the deletion mutant
del[335-351] exhibited in every cell type the same reduced internalization rate than B2
ST supporting our hypothesis
that phosphorylation of Ser and Thr within the segment 335-351 is
crucial for an optimal internalization of the BK B2
receptor.
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Fig. 3.
[3H]BK internalization in HEK
293 and CHO cells with transient or stable expression of the
B2wt or mutant receptors. Cells cultured during
48 h after transfection for transient expression or stable CHO
cell lines at confluence were incubated with 2 nM
[3H]BK at 37 °C for 70 min. Data were obtained as
described in the legend of Fig. 2. Results (mean ± S.E.) are
given as the percent of the control, i.e. B2wt
internalization which represents 57.4 ± 3.1% of the
corresponding total specific binding in HEK 293 cells and 72.6 ± 1.5% and 89.6 ± 1.9% in transiently and stably expressing CHO
cells, respectively; ***, p < 0.005, test ANOVA.
ST were readily immunoprecipitated by the
antiserum except for deletion mutants del[343-351] and
del[335-351] which were precipitated at a 3-4-fold lower
efficiency, and truncation mutants tR351, tI334, and tY320 which failed
to react. These findings localize major immunogenic epitope(s)
recognized by antiserum AS346 to the extreme COOH-terminal receptor
portion distal of residue Arg351.
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Fig. 4.
BK-induced phosphorylation of B2wt and mutant
receptors. COS cells used 40 h after transfection were
radiolabeled with 35S-labeled amino acids (panel
d) or with [32P]orthophosphate and incubated for 5 min with (+) or without ( ) 1 µM BK. The cells were
lysed, and the receptors immunoprecipitated with antiserum AS346. The
radiolabeled proteins were analyzed by reducing 10% SDS-PAGE and
autoradiography. Relative molecular masses of standard proteins (not
shown) are indicated on the left. Data are representative of
three independent experiments with similar results.
TS345-346A, T345A/S346A;
S339T342A, S339A/T342A;
S339TS345-346A,
S339A/T345A/S346A.
Arg351
segment had been mutated (data not shown). In marked contrast, BK
failed to increase the phosphorylation level of del[335-351], B2
ST, and S339A/T345A/S346A (Fig. 4); these mutants are
characterized by a markedly reduced internalization capacity (Figs. 2
and 3). In all mutants exhibiting an "intermediate" internalization
phenotype such as del[335-342], del[343-351], S339A, T342A,
S348A, S339A/T342A, and T345A/S346A BK was still able to increase the
phosphorylation (Fig. 4). Together these results demonstrate that the
Ser and Thr residues phosphorylated upon ligand stimulation are located within the Gln335
Arg351 segment. The data
also point to a close relationship between internalization and
phosphorylation implying that BK promotes receptor internalization by
homologous phosphorylation. To further test this hypothesis, we
investigate with the B2wt whether PKC is involved in the
internalization and phosphorylation processes. It was apparent that
[3H]BK binding was not sensitive to application, before
and during the ligand incubation, of 0.1 µM PMA or
staurosporine, an activator and inhibitor of PKC, respectively (not
shown). Under the same conditions, there was also no change in
[3H]BK internalization and BK stimulation of PLC.
Concurrently, an immunoprecipitation of the B2wt showed
that, unlike BK, PMA did not increase the receptor phosphorylation (not
shown). Thus, PKC regulation of B2wt was not detected in
COS-7 cells like in CHO-K1 cells (4). As discussed elsewhere in detail
(4), this is in contrast with the reported role of PKC in
B2 receptor modulation in other cell types (17, 34-36) and
can be explained by a low PKC content of COS-7 and CHO-K1 cells facing
receptor overexpression.
ST had no effect on the
[3H]BK internalization rate and the phosphorylation level
(Fig. 5). These findings further support the hypothesis of a close
relationship between internalization rates and phosphorylation levels
of the B2 receptor.
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Fig. 5.
Effects of phosphatase inhibitor treatment on
B2wt and B2 ST receptor
internalization and phosphorylation. COS cells used 72 h
after transfection were (a) incubated with 0.1 µM okadaic acid for 5 min prior and during a 10-min
incubation period with 2 nM [3H]BK at
37 °C. Results are expressed in percent of internalized
radioactivity in untreated B2wt cells which represents
34.4 ± 2.2% of total bound radioactivity. b,
32P radiolabeled transfected cells were exposed to 0.1 µM okadaic acid in the presence or absence of 1 µM BK for 5 min before analysis of antiserum
AS346-immunoprecipitated proteins as described in the legend to Fig. 4;
*, p < 0.05, test ANOVA.
2-adrenergic receptor couple to the endocytic machinery via the
adaptor protein arrestin (5, 6, 33). We here wondered whether the
critical residues in the Gln335
Arg351
segment are essential for receptor endocytosis through clathrin-coated vesicles. For this purpose, we exposed cells expressing the
B2wt or the mutant receptor del[335-351] to a standard
solution or to hypertonic solution containing 0.4 M sucrose
for 30 min prior to and for 10 min after the addition of
[3H]BK, and measured the internalization of the
radioligand. Both the B2wt as well as the mutated receptor
showed reduction of [3H]BK internalization by roughly
70% (not shown). The same results were obtained whether receptors were
expressed in COS-7 cells or CHO-K1 cells. This suggests that the
clathrin-mediated, sucrose-sensitive endocytosis of the bradykinin
B2 receptor might not require the Ser/Thr phosphorylation
residues in the Gln335
Arg351 segment. To
further document this hypothesis, we tested the ability of wild type
and dominant-negative mutant
-arrestin and dynamin to influence the
sequestration of B2wt and the residual sequestration of
B2
ST receptors in COS-7 cells. Fig.
6 shows that unlike for the
-adrenergic receptor (33), the co-transfection with
-arrestin did
not increase the internalization of [3H]BK by either the
B2wt or B2
ST receptors, but co-expression of
-arrestin (319-418) or dynamin K44A mutants exhibited inhibited [3H]BK endocytosis for both receptors. One possibility to
explain the inability of the overexpression of wild type
-arrestin
to increase significantly the internalization rate of both receptors, is that the endogenous proteins may be sufficient to mediate maximal internalization of these receptors. In any case these observations indicate that although clathrin-mediated endocytosis may be important for B2 receptor internalization this mechanism does not
exclusively involve the phosphorylation of Ser/Thr residues in the
Gln335
Arg351 segment.
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Fig. 6.
Effects of
-arrestins or/and dynamin co-transfection on
B2wt and B2
ST receptor
internalization. COS cells were transfected with 15 µg of
expression vector for B2wt and B2
ST,
together with 15 µg of empty vector or wt rat
-arrestin, rat, or
bovine
-arrestin (319-418) mutants, or human dynamin-K44A mutant,
as indicated. Then, 72 h after the transfection, the cells were
incubated with 2 nM [3H]BK at 37 °C for 10 min. Results are given as the percent of the control, i.e.
B2wt or B2
ST-mediated internalization which
represents 26.8 ± 4.1 and 4.1 ± 0.8% of the total bound
radioactivity at 10 min, respectively. Data are the mean ± S.E.
of eight determinations. They were analyzed using unpaired
Student's t test (*, p < 0.05, when
compared with the corresponding B2wt and
B2
ST control cells).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptor allows the assembly of a scaffold of signaling factors of the
mitogen-activated protein kinase system on intracellular vesicles and
thus connects this receptor to the mitogenic pathways of the cell (37).
Accumulating evidence suggests that specific sequence motifs of the
intracellular loops and/or reversible modifications such as acylation
and phosphorylation of the cytoplasmic domains of the receptors play
critical roles in these processes (38). However, the precise molecular
mechanisms driving desensitization and internalization of GPCRs have
often remained unknown.
2-adrenergic receptor truncated
at position 365 was internalized to a greater extent than the wild type
receptor in these cells (14). Our data show that the COOH-terminal tail
contributes significantly to the internalization of the
B2wt, although other structures such as the intracellular
loops may also be involved, albeit with a reduced efficiency (39).
Arg351 segment (B2
ST)
produced a similar impairment in internalization as the deletion of the
entire segment (del[335-351]) or the truncation in position 334 (tI334). Furthermore, substitution of only two (Ser339 and
Thr342) or three (Ser339, Thr345,
and Ser346) of these residues resulted in the same effect.
This signifies that substitution of the critical Ser/Thr residues
rather than a global structural change was responsible for the observed
effects. Deletions which remove the proximal residues
Ser339 and Thr342 (del[335-342]) or the
distal residues Thr345, Ser346, and
Ser348 (del[343-351]) each gave a phenotype intermediate
to that of B2wt and del[335-351]. Intermediate
phenotypes were also observed for individual (S339A, T342A, and S348A)
or combined replacements (T345A/S346A). Of note, the sum of the effects
of partial deletions 335-342 and 343-351 exceeded the effect of the
full deletion 335-351. Likewise, the combined effects of individual
substitutions exceeded the effect of a simultaneous mutation of all 5 residues in B2
ST. This indicates that the contribution
of the non-mutated residues is smaller in the mutated receptors than in
the intact receptor and suggests that the Ser/Thr residues of the
Gln335
Arg351 segment do not operate in a
simple additive manner but operate in coordination to optimize receptor sequestration.
ST nor the
phosphorylation state of this mutant receptor; (iv) PMA and
staurosporine treatment which fail to change [3H]BK
internalization do not affect receptor phosphorylation; (v) mutations
tY320, del[335-351], B2
ST, and S339A/T345A/S346A that suppress homologous receptor phosphorylation impair receptor
internalization in all cell types tested; (vi) mutation of three
phosphorylation sites, i.e. Ser339,
Thr345, and Ser346, suffices to abolish
homologous receptor phosphorylation and to markedly reduce
internalization capacity of the human B2 receptor. Remarkably Ala substitution of 3 out of 14 potential phosphorylation sites (Ser, Thr, and Tyr) present in the 4 intracellular domains are
sufficient to abolish ligand-induced phosphorylation and to markedly
reduce the internalization capacity of the human B2
receptor studied in COS-7 cells. This observation points to the fact
that the remaining non-mutated Ser/Thr residues cannot rescue the
altered phosphorylation and internalization phenotype. On the contrary, the contributions of the non-mutated residues to internalization appear
to be curtailed in these mutants.
-arrestin and dynamin. The experiments
presented here are the first to address the involvement of arrestins
and dynamin in the internalization of the bradykinin B2
receptor. The magnitude of the inhibition of the B2
ST
internalization induced by co-expression of dominant-negative forms of
-arrestin or dynamin in COS-7 cells was of the same extent as that
observed for B2wt in the same cell line. This suggests that
the B2
ST receptor has a affinity for arrestins
comparable to those of the B2wt receptor and its residual
internalization still involves clathrin-coated vesicles. The
internalization that was not sensitive to
-arrestin and dynamin
mutants could reflect sequestration of the receptor in caveolae, like
that described for the B2 receptor in DDT1 MF-2 smooth
muscle cells by de Weerd et al. (25), and in A431 cells by
Haasemann et al. (41). Interestingly, the desensitization
that we have previously characterized in the CHO-K1 cells expressing
the B2wt (4) did not occur in COS-7 expressing either the
B2wt or mutant receptors, probably because of a
differential expression of the human B2 receptor
desensitization machinery between the two cell types. The lack of
desensitization in COS-7 cells, together with the decreased
internalization observed in some mutants, is, however, of interest
because it indicates that receptor internalization is not a
prerequisite for the desensitization, as reported by others (10).
Arg351) in the center portion of the COOH-terminal
tail. However, the mutation of these residues is not sufficient to
completely suppress the sequestration through clathrin-coated vesicles,
suggesting that the internalization of the
[3H]BK-B2 receptor complex proceeds from
different mechanisms involving distinct receptor structures. The
critical Ser/Thr residues are flanked by 2 acidic residues, Glu in
positions 337 and 350 delimiting a core sequence of 14 residues,
Glu337
Glu350. Previous in vitro
studies with the
-adrenergic receptor have pointed to the potential
importance of acidic residues juxtaposed to Ser/Thr residues (42).
Comparison of the human Glu337
Glu350
segment with the sequences of other vertebrate kinin receptors (17, 18,
43) demonstrates that this cassette is well conserved among the kinin
receptors: 9 of the 14 residues (65%) are invariant including all
serine and threonine residues (Ser339, Ser346,
Ser348, Thr342, and Thr345), a
centrally located positive residue of arginine (Arg344),
and the proximal acidic residue (Glu337) whereas the distal
acidic residue is conserved (Glu350 or Asp350).
By contrast the overall sequence identity of intracellular domain ID4
is poor (15/64 corresponding to 23%). Our notion that the
Glu337
Glu350 cassette plays a central role
for receptor phosphorylation and internalization is further
strengthened by the finding that the replacement of COOH-terminal tail
region of the wild-type human B1 receptor which fails to
undergo ligand-induced phosphorylation and internalization, by the
homologous region of the B2 receptor holding the
Glu337
Glu350 cassette confers the capacity
to the B1/B2 receptor chimera for ligand-induced internalization (7) and
phosphorylation.2
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Benovic for the generous gift of
pcDNA3 plasmids encoding for bovine -arrestin
fragment (319-418) mutant and inactive human dynamin K44A and Dr.
Bunnett for wt rat
-arrestin and its inactive fragment (319-418)
mutant in pEGFP.
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FOOTNOTES |
---|
* This work was supported by INSERM and grants from the Bristol-Meyers Squibb Institute for Medical Research (Princeton, NJ) and the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a grant from La Fondation Pour la Recherche Medicale. To whom correspondence should be addressed. Tel.: 33-1-45-87-61-00; Fax: 33-1-45-35-66-29; E-mail: pizard{at}ifm.inserm.fr.
Present address: Ludwig Institute for Cancer Research, P.O.
Box 595, S-75124 Uppsala, Sweden.
2 A. Faussner and A. Blaukat, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: GPCRs, G protein-coupled receptors; HBSS, Hank's buffered saline solution; BK, bradykinin; B2wt, human B2 BK wild-type receptor; COSB2wt, COS-7 cells overexpressing the B2wt; PLC, phospholipase C; PLA2, phospholipase A2; BSA, bovine serum albumin; IPs, inositol phosphates; [Ca2+]i, intracellular Ca2+; CHO, Chinese hamster ovary; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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