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INTRODUCTION |
Bradykinin, a member of the kinin family (1), is a nonapeptide
with diverse biological activities ranging from a role in the
inflammatory process to regulatory effects on vascular permeability, blood pressure, renal homeostasis, and pain generation (2, 3).
Bradykinin mediates its physiological effects by binding to and
activation of the bradykinin B2 receptor. Molecular cloning has revealed the primary structure of the B2 receptor (4)
and classified it as a member of the G protein-coupled receptor
superfamily. The consensus bradykinin receptor topology predicts four
extracellular domains
(ED11-4) and intracellular
domains (ID1-4), each separated by seven transmembrane helical regions
(TM1-7) spanning the lipid bilayer. B2 receptors are
post-translationally modified by glycosylation (5), phosphorylation
(6), and presumably by palmitoylation of the cytoplasmic surface.
Based on homology with other G protein-coupled receptors there have
been indications regarding possible structural features probed by
agonists, antagonists, and anti-idiotypic antibodies (5, 6), functional
regions (7), and sites of post-transitional modifications for the
B2 receptor (8). Site-directed mutagenesis indicated the
importance of Tyr residues and of ID4 for the signaling and the uptake
of the B2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9). However, as yet there is
still rather little direct evidence at the protein level for attributes
such as the precise sites and the roles of glycosylation, palmitoylation, and phosphorylation of bradykinin B2 or
other G protein-coupled receptors. Indeed phosphorylation sites
for few G protein-coupled receptors have been mapped to date (10-12), and most of these sites reflect the in vitro phosphorylation
of the isolated receptor.
We report here on the isolation of rat B2 receptor from
transfected Chinese hamster ovary (CHO) cells using oligo(dA)
covalently linked to bradykinin via a specially developed bifunctional
cross-linker. Affinity chromatography has been carried out under very
mild conditions using oligo(dT) columns analogous to methods used for
isolation of eukaryotic mRNA. In-gel digestion of
electrophoretically separated receptor, subsequent peptide mass
fingerprinting by matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) or electrospray ionization (ESI) and
fragment analysis by tandem (MS/MS) mass spectrometry have been used to
characterize post-translational modifications of this receptor and its
multiply phosphorylated species. The patterns of post-translational
modifications which have been observed for the receptor under in
vivo conditions show correlations among the various modifications
and provide new information on their possible role(s) in the functional
regulation of the bradykinin B2 receptor.
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EXPERIMENTAL PROCEDURES |
Materials--
Ham's F-12 nutrient medium was from Life
Technologies, Inc. Dithiothreitol Microselect, used for deprotecting
the modified oligonucleotide, tetrabutylammoniumhydrogensulfate, and
chymostatin were from Fluka. 1,10-Phenanthroline was from Merck. Hepes,
N-(
-maleimidocaproyloxy)succinimide (EMCS),
phenylmethanesulfonyl fluoride, CHAPS, Ellman's reagent, bacitracine,
bovine serum albumin, and MES were from Sigma. The Aquapore RP-300A
HPLC column and chemicals for peptide sequencing were from Applied
Biosystems. DNase-free RNase A was from Boehringer Mannheim.
(dA)30 homomer with and without 5'-modification by
1-O-dimethoxytritylhexyldisulfide was from BioTeZ (Berlin).
Met-Lys-bradykinin was from Bachem; trypsin was from Promega. Other
chemicals (from Merck and Roth) were of the best grade available.
Synthesis of Polyadenylated
Met-Lys-Bradykinin--
Met-Lys-bradykinin derivatized with EMCS at
the
-NH2 group of Lys2 was prepared by a
synthesis similar to that used recently to prepare an analogous
endothelin derivative (12). 1 mg (400 nmol) of Met-Lys-bradykinin was
dissolved in 400 µl of 35% acetonitrile, 0.05% trifluoroacetate,
further diluted with 50 mM sodium borate and 0.015% Triton
X-305, pH 8.2, and a 10-fold excess (4 µmol) of the
heterobifunctional cross-linker EMCS dissolved in 200 µl of
acetonitrile was added. Subsequent steps in the reaction were carried
out as described previously (12). The derivatized Met-Lys-bradykinin, N-(
-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin
(EMC-MKBK), was purified on an Aquapore RP-300 column using a gradient
of 0-70% acetonitrile in 0.1% trifluoroacetic acid. The purity of the fraction containing EMC-MKBK was checked by protein
sequencing to confirm derivatization at Lys2 and by
MALDI-TOF mass spectrometry (found MH+ 1513.9, expected
1513.6). (dA)30-5'-SS-R was used for attachment of 30-mer
(dA) to the EMC-MKBK according to the procedure in Ref. 12. The
(dA)30-5'-S-EMC-MKBK was purified on a Sephasil C18 column
by applying a linear gradient of 2-70% acetonitrile in 0.1 M triethylammoniumacetate and 2 mM tetrabutylammoniumhydrogensulfate.
Cell Culture and Membrane Preparation--
CHO cells transfected
with rat B2 receptor cDNA (4, 13) were grown in Ham's
F-12 medium containing 10% fetal calf serum and 50 µg/ml
streptomycin in a water-saturated, 5% CO2 atmosphere at
37 °C (13, 14). Cells grown to 80% confluence were harvested for
membrane preparation by scraping in 100 mM NaCl, 10 mM Pipes at pH 6.8. The cells obtained were washed twice
with the same buffer and subsequently resuspended in 2 mM
EDTA, 10 mM Pipes at pH 6.8. All subsequent steps were
carried out at 4 °C. Cell rupture was performed by Ultra-Turrax
(IKA-Labortechnik, Staufen, Germany) at 20,000 rpm for 2 min. Membranes
were collected by centrifugation at 30,000 × g for 10 min and resuspended in 100 mM NaCl, 2 mM EDTA,
10 mM Pipes at pH 6.8 to a final protein concentration of 2 mg/ml. The membrane suspension was stored at
80 °C.
Affinity Purification of Bradykinin B2
Receptor--
CHO membranes (600 µl) were suspended in 2 volumes of
20 mM potassium phosphate, pH 7.4, containing 4 mM CHAPS, 500 mM NaCl, 20 mM EDTA
(buffer A). The mixture was stirred gently at 4 °C for 1 h and
was then centrifuged at 100,000 × g for 1 h at
4 °C. The supernatants were incubated with 0.2 nmol of
(dA)30-5'-S-EMC-MKBK for 1 h at 4 °C, 30 mg of
oligo(dT)-cellulose was added, and the suspension was agitated gently
at 4 °C for 2 h. The oligo(dT)-cellulose was pelleted by
centrifugation (4 °C, 1,000 × g, 5 min) and packed into a microcolumn. The column was washed with 10 ml of buffer A at
4 °C and afterward eluted with 10 mM Tris/HCl, pH 7.4, 4 mM CHAPS, 1 mM EDTA. Fractions (50 µl each)
containing B2 receptor were identified by
SDS-polyacrylamide gel electrophoresis and immunoblot analysis.
Gel Electrophoresis and In-gel Tryptic Protein Digestion--
10
µl of each fraction from the oligo(dT)-cellulose column was mixed
with an equal volume of sample buffer containing 6% SDS, 10% (v/v)
2-mercaptoethanol, 20% (v/v) glycerol, 0.01% bromphenol blue, and
0.25 M Tris/HCl, pH 6.8, and brought to 95 °C for 2 min. Electrophoresis was performed in 10 or 12.5% polyacrylamide gels
in the presence of 0.1% SDS at a constant current of 40 mA for
1.5 h (15). The resolved proteins were visualized by Coomassie Blue staining. After visualization, the gel was destained with a
solution of 25 mM ammonium bicarbonate and 50%
acetonitrile. The band corresponding to the B2 receptor was
cut out and digested in the gel according to (16) as modified
previously (12).
Mass Spectrometric Analysis--
For MALDI mass spectrometry,
samples were dissolved in 5 µl of 50% acetonitrile, 0.1%
trifluoroacetic acid and sonicated for few minutes. Aliquots of 0.5 µl were applied to a target disk and allowed to air dry.
Subsequently, 0.3 µl of matrix solution (1% w/v
-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% v/v
trifluoroacetic acid) was applied to the dried sample and again allowed
to dry. Spectra were obtained using a Bruker Biflex MALDI-TOF mass
spectrometer. MS/MS analysis was carried out using a Finnigan MAT (San
Jose, CA) LCQ ion trap mass spectrometer coupled on-line with a HPLC
system (Hewlett Packard 1090). For the interpretation of MS and MS/MS
spectra of protein digests we used the Sherpa software (17), the
MS-Fit, MS-Tag, and MS-Product programs available at the UCSF web site
(http://rafael.ucsf.edu/cgi-bin/msfit), the PepSearch programs at the
EMBL web site
(http://www.mann.embl-heidelberg.de/Services/PeptideSearch/FR_peptideSearchForm.html), and the PepFrag program at the Rockefeller University web site (http://prowl.rockefeller.edu/).
 |
RESULTS |
Isolation of Rat Bradykinin B2 Receptor from CHO
Cells--
Rat bradykinin B2 receptor was isolated from
CHO cells using a "fishhook" strategy similar to that recently
applied successfully for isolation of another peptide receptor,
endothelin B receptor (12). The fishhook consists of the receptor
ligand (here MLBK) and a (dA)30 polynucleotide joined via
the heterobifunctional cross-linking reagent EMCS which, in addition to
a N-hydroxysuccinimide-activated ester, also possesses a
thiol-selective maleimido group. This linker was attached to MKBK via
the
-amino group of Lys2, and the thiol-selective
maleimido group was used to attach (dA)30. The final
product, (dA)30-5'-S-EMC-MKBK, was subsequently added to a
suspension of CHO cell membranes solubilized in the non-ionic detergent
CHAPS. B2 receptors bound to
(dA)30-5'-S-EMC-MKBK were purified in a manner similar to
that used for the isolation of eukaryotic mRNA: absorption on
oligo(dT)-cellulose, washing in the presence of high salt
concentrations, which favor the formation of a poly(dA·dT) double
helix, and elution with very low salt concentrations that destabilize a
poly(dA·dT) double helix. Recombinant B2 receptor is
highly expressed in the transfected CHO cells used for the present
experiments (1.3 pmol/mg of protein (13)) and shows a strong band at
about 42 kDa in SDS-polyacrylamide gel electrophoresis of total
membrane proteins (lane 1, Fig.
1). This band is strongly enriched
relative to other membrane proteins by the fishhook purification
strategy (lane 2, Fig. 1). Subsequent analysis of this band
(Fig. 1, lane 2) revealed that it contains highly pure
B2 receptor suitable for detailed analysis by mass spectrometry. By comparison with intensities from the unpurified extract of membrane proteins, we estimate that about 70% of cellular B2 receptor was recovered after fishhook purification.

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Fig. 1.
Purification of rat bradykinin B2
receptor from CHO cells with
(dA)30-5'-S-EMC-MKBK/oligo(dT)-cellulose. The elution
profile of the receptor protein from an oligo(dT)-cellulose column
monitored by 10% SDS-polyacrylamide gel electrophoresis with Coomassie
Blue staining is shown. Lane 1, total membrane proteins
prior to isolation of B2 receptor; lanes 2-4,
the first three fractions eluted from the oligo(dT) column; lane
5, molecular mass marker proteins. The molecular mass of the rat
bradykinin B2 receptor is indicated at the
left.
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Identification of Tryptic Fragments of Rat B2
Receptor--
Bands corresponding to Coomassie Blue-stained bradykinin
B2 receptor were cut out from the SDS-polyacrylamide gels
and subjected to in-gel tryptic digestion. MALDI mass spectrometry was
used for the initial analysis of the entire mixture of tryptic
peptides. From the MALDI spectra it was possible to identify peptides
from the extracellular domains ED3 as well as peptides from cytoplasmic regions ID2, ID3, and ID4. Peptides containing TM helical regions TM3-TM5 joined to the corresponding loop region(s) could also be
observed (Fig. 2 and Table
I). In the present studies we did not
observe peptides from domains ED1, TM1, ID1, TM2, and ED2 (Fig. 2)
presumably because this region of the B2 receptor has only
two internal sites for tryptic hydrolysis at positions 10 and 91 and
therefore would be expected to yield large, hydrophobic peptides except
for the extreme amino-terminal peptide of 10 residues.

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Fig. 2.
Peptides identified by MALDI-TOF mass
spectrometry of rat B2 receptor. Solid
lines denote tryptic peptides identified from the tryptic peptide
mixture. Filled black circles with white letters
indicate post-translationally modified amino acid residues. Amino acid
residues have been numbered according to the full-length B2
receptor identified recently (4, 43).
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Identification of Post-translationally Modified Peptides--
The
MALDI mass measurements gave clear indications of which peptides were
post-translationally modified (Table I). We observed phosphorylation
for peptides 6-7, 26-31, 28-31, and 32-33. Peptides 6-7, 26-31,
and 28-31 showed an increased mass of 80 Da, which is characteristic
for peptides with single phosphorylation sites. For peptide 32-33, a
mass consistent with a diphosphorylated peptide was observed. For
peptide 30-31, palmitoylation was suggested by an increase in mass of
238 Da. The identity of the peptides was confirmed by electrospray ion
trap mass spectrometry of the unseparated peptide mixture with
subsequent MS/MS analysis of selected fragments of interest. It is
known that in electrospray ion trap mass spectrometry the
phosphorylated peptides partially lose the
H3PO4 moiety in the mass spectrometer, thus
producing a pair of peaks separated by a mass difference of 98 Da (18). In accordance with this, we observed that phosphopeptides 6-7, 26-31,
and 28-31 could be identified by a pair of masses 80 Da higher (the
mass of H3PO4 minus H2O) and 18 Da
lower than expected based on the amino acid sequence. For the peptide
32-33 the loss of two H3PO4 moieties confirmed
the presence of two phosphorylation sites. We conclude that
intracellular domains ID2 and ID4 of the rat B2 receptor
are modified by phosphorylation (ID2, ID4) and palmitoylation (ID4).
Identification of Phosphorylation Sites--
For the peptide
26-31, a m/z of 1992.2 Da was observed in the
MALDI-TOF spectra. There were two pairs of peaks separated by 98 Da
(m/z 1992.2 and 1895.8 Da). In addition the MS/MS
ESI spectrum of peptide 26-31 (Fig.
3A) showed a y ion
series, y15, y14, y13, y12,
y9, y8, y7, and a b ion
series, b15, b14, b13, b12,
b11, b9, b8, and b7, which
was sufficient to identify Tyr352 and not
Ser348 in the amino acid sequence
344FRKKSREVY(Pi)QAICRK358 as the
site of phosphorylation. The presence of the w11 ion
confirmed that Ser348 is not phosphorylated. For peptide
6-7 an increase of 80 Da was observed, suggesting that the peptide
161YLALVKTMSMGR171 is phosphorylated at
Tyr161 or Ser168. MS/MS analysis generated
y12 and y11 ions as well as b12,
b11, b10, and b7 ions that were
sufficient to determine that Tyr161 is the phosphorylation
attachment site.

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Fig. 3.
Electrospray mass spectroscopic analysis of
tryptic peptides. Panel A, MS/MS spectra of
phosphopeptides 26-31,
344FRKKSREVY(Pi)QAICRK358
(M+H)+. Panel B, MS/MS spectra of
diphosphopeptide 32-33,
359GGCMGESVQMENSMGTLRTSISVDR383
(M+2H)2+. Inset, MS3 analysis of
peptide y12-pp. The fragment y2+25-pp with the
loss of one or two phosphate groups is indicated by the ions y25
( 49) and y25 ( 98), respectively. Panel C,
palmitoylated peptide 30-31,
350EVYQAIC(pal)RK358 (M+2H)2+. The
symbols p and pp denote fragment ions with one or
two attached phosphate groups, respectively.
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For the peptide 32-33,
359GGCMGESVQMENSMGTLRTSISVDR383, the MALDI-TOF
mass spectra gave a parent ion with an m/z of
2806.4 corresponding to this peptide with two phosphate groups
attached. No ions corresponding to this peptide with more or fewer than
two phosphate groups could be detected. Given that this peptide has six
potential phosphorylation sites, Ser at positions 365, 371, 378, and
380 and Thr at 374 and 377, this was a first indication that
phosphorylation of this peptide does not involve uncoordinated
phosphorylation of these sites. Further evidence for correlation among
the sites that are phosphorylated in this peptide has been obtained
from ion trap ESI mass spectra after fragmentation of this peptide
(Fig. 3B). Initial inspection of the MS/MS spectra clearly
indicated a very complicated phosphorylation pattern for this peptide.
From the y2+ and b2+
series of fragments, clear evidence of concurrent phosphorylation at
Ser365 and Ser371 was obtained from the
fragments y2+12 and
b2+14-pp (Fig.
4A, note that one or two
attached phosphate groups are indicated by -p or -pp in the following
text and in Fig. 4). Other fragments that would be consistent with this
phosphorylation pattern include y2+18-p,
y2+16-p, and y2+5 as well
as b2+19-pp, b2+17-pp,
and b2+12-p. Similarly, the fragment
y2+6-pp-98, which corresponds to the peptide
y2+6-pp with the loss of two phosphate groups,
gives clear evidence for concurrent phosphorylation at
Ser378 and Ser380. Other fragments that would
be consistent with concurrent phosphorylation of these two serines
include y2+18-pp,
y2+14-pp, y2+12-pp, and
y2+4-p as well as
b2+21-p. (Fig. 4). There are three fragments,
y2+7-p, y2+6-p, and
b2+18-p which cannot be consistent with the the
above two types of diphosphorylation and which require phosphorylation
at one of Ser365, Ser371, or Thr374
as well as at one of Thr377, Ser378, or
Ser380. Evidence for concurrent phosphorylation at
Thr374 and Ser380 was obtained by
MS3 analysis of the y series fragment
y2+12-pp (Figs. 3B, inset,
and 4B).

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Fig. 4.
Schematic of the analysis of phosphorylation
patterns for the diphosphorylated peptide
359GGCMGESVQMENSMGTLRTSISVDR383.
Panel A, ESI ion trap MS/MS fragmentation of the parent ion
m/z 2806.4. The standard numbering of the
b2+ series ions (above) and
y2+ series ions (below) is indicated on the
sequence of this peptide. The six potential phosphorylation sites are
indicated by the encircled serine and threonine residues and
the amino acid residue numbers at the top of the figure.
Starting either from the parent ion b2+25-pp or
the parent ion y2+25-pp,
b2+ series and y2+ series
fragment ions that were observed are shown above or below the peptide
sequence, respectively. The number of attached phosphate groups is
indicated by the label of the ion, e.g.
y2+18-p indicates the peptide fragment
VQMENSMGTLRTSISVDR with one attached phosphate group. Fragment ions
that can arise from diphosphorylation at Ser365 and
Ser371, at Ser378 and Ser380, and
at Thr374 and Ser380 are indicated by
solid, dash, and dot-dash lines,
respectively. Fragment ions that may arise from more than one of the
diphosphorylation patterns are indicated by dotted lines.
Open, gray, and solid circles denote
phosphorylation sites that, respectively: are not phosphorylated ( ),
belong to a group of potential phosphorylation sites where the actual
site of phosphorylation cannot be ascertained from the fragment ion
( ), or are phosphorylated ( ). For example, the ions
y2+25-pp and y2+18-p
indicate phosphorylation at Ser365 with a second
phosphorylation site at any one of Ser371,
Ser378, Ser380, Thr374, or
Thr377. Panel B, ESI ion trap MS3
analysis of peptide y2+12-pp. Panel
C, the three diphosphorylation patterns that are necessary and
sufficient to explain all MS results.
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The three concurrent phosphorylation patterns shown in Fig.
4C suffice to explain all observed fragments. Evidence that
other patterns of diphosphorylation probably do not exist was obtained by MS3 analysis of fragments
y2+16-p, which indicated that when
Ser365 is phosphorylated, the only concurrent
phosphorylation site is Ser371, and of y7-pm
which indicated that Thr377 is probably never
phosphorylated, at least under the conditions to which the CHO cells
were subjected in the present experiments. Given that diphosphorylation
of the five sites Ser365, Ser371,
Ser378, Ser380, and Thr374 could
occur in 10 different combinations, the present evidence that only
three of these combinations occur is strong evidence for coordinated
diphosphorylation. We confirmed these results with many more
NH2-terminal (b2+H2O,
b2+NH3,
b2+H3PO4), COOH-terminal
(y2+H2O,
y2+NH3,
y2+H3PO4), and internal
fragment ions, which for clarity have been omitted from further
discussion here, but were of considerable help during the course of
spectra analysis.
Palmitoylation of B2 Receptor--
The increase of 238 Da in the mass of peptide 30-31 indicated the presence of a palmitate
residue (Table I). The MS/MS analysis generated
y92+, y82+,
y72+, y62+,
y52+, y42+,
y32+, and b42+,
b52+, b62+,
b72+, b82+,
b92+ ions showing that Cys356 in
peptide 350EVYQAICRK358 is palmitoylated (Fig.
3C). MS/MS analysis of this peptide showed no
phosphorylation at Tyr352 (Fig. 3C). Conversely,
MS/MS analysis of peptide
344FRKKSREVY(Pi)QAICRK358
demonstrated phosphorylation at residue Tyr352 but not
palmitoylation at Cys356 (Fig. 3A). This result
was confirmed by MS/MS analysis of peptide 347KSREVY(Pi)QAICRK358 (not shown).
In the MALDI mass spectra we were able to find masses corresponding to
peptides 25-31, 26-33, and 28-32 (Table I) with no phosphorylation
at Tyr352 or palmitoylation at Cys356, but no
masses were detected which correspond to peptides with simultaneous
phosphorylation and palmitoylation of Tyr352 and
Cys356, respectively. Together these results indicate a
mutually exclusive palmitoylation at Cys356 or
phosphorylation at Tyr352 of intracelluar domain ID4 of the
rat B2 receptor.
 |
DISCUSSION |
Post-translational modifications such as phosphorylation are a
widespread mechanism thought to regulate the desensitization, internalization, and resensitization of G protein-coupled receptors. For two prototypic receptors, i.e. rhodopsin and
2-adrenergic receptor, the phosphorylation sites have
been studied in some detail (9-11, 19-21). Typically serine and
threonine residues located in the carboxyl-terminal domains of these
receptors are phosphorylated in response to light (rhodopsin) or ligand
(adrenergic receptors). Because of their extremely low abundance in the
cell, ligand-dependent G protein-coupled receptors have
been notoriously refractory to chemical analysis of phosphorylation
sites in vivo, and major discrepancies between the mapping
of phosphorylation sites in vitro and the effect of
corresponding mutations in vivo have been reported (22). In
the present work we took advantage of a highly sensitive technique,
i.e. mass fingerprinting (23, 24), to map
post-translationally modified peptides from a typical G protein-coupled peptide hormone receptor isolated from transfected CHO cells by a novel
fishhook technique. Tryptic peptides could be recovered from a large
part of the sequence of the B2 receptor including, except
for Thr92 and Thr95 of ID1, all those sequence
regions that might be expected to show post-translational modifications
by phosphorylation or fatty acid attachment. MALDI, ESI mass
spectrometry and MSn analysis revealed that the
B2 receptor is phosphorylated at Ser365,
Ser371, Ser378, Ser380, and
Thr374. Further, we observed phosphorylation at
Tyr161 and at Tyr352 and palmitoylation at
Cys356.
A number of previous studies on bradykinin B2 receptor
cloned into CHO cells (13, 25-28) have indicated that binding
affinities for bradykinin and physiological responses to cell
stimulation with bradykinin are similar to those observed in primary
cell types. Because the receptor was isolated from unstimulated cells, the observed phosphorylation sites most probably represent the "basal" pattern of phosphorylation caused by intrinsic receptor activity (22), although there might be some contribution from accidentally released ligands, e.g. from culture medium
supplemented with fetal calf serum, which is a rich source of
kininogens (29). However, basal phosphorylation in the absence of a
ligand, which was not inhibited by antagonists, has previously been
shown for human bradykinin B2 receptor in human foreskin
fibroblast cells (30), suggesting that such basal phosphorylation may
not be unusual.
Phosphorylation and palmitoylation are major mechanisms that modulate
signal transduction and internalization of many G protein-coupled receptors (31-35). Except for Ser365, all
post-translational modification sites observed in the present work are
strongly conserved in all known B2 receptors, and the present work provides some new information on the possible roles of
post-translational modification of these sites. For example, residue
Tyr161 is highly conserved in all known sequences of
B2 receptor with Tyr161 being a part of the DRY
motif that is conserved in the family of G protein-coupled receptors.
Because Tyr161 seems to play a significant role in G
protein coupling of the B2 receptor (9), our present
results suggest that phosphorylation of this critical residue may
modulate the signaling capacity of this receptor. Similar
phosphorylation of the DRY motif might be important for other G
protein-coupled receptors.
Out of 7 Ser/Thr residues present in the carboxyl-terminal domain ID4
of the B2 receptor, five phosphorylation sites at
Ser365, Ser371, Ser378,
Ser380, and Thr374 have been identified.
Previous work has demonstrated that truncation of 34 residues at the
carboxyl-terminal tail of the rat B2 receptor has a major
impact on receptor internalization (36) thus further supporting the
notion that phosphorylation and sequestration of the receptor may be
causally linked (9). An unexpected finding of this study is that
phosphorylation of region ID4 of the B2 receptor involves
three different molecular species with diphosphorylation at
Ser365 and Ser371, at Ser378 and
Ser380, and at Thr374 and Ser380.
The observations that only diphosphorylation apparently occurs and that
the sites are correlated has important implications for attempts to
analyze functional aspects of the bradykinin B2 receptor, e.g. it seems essential to know the patterns of correlated
phosphorylation prior to mutation of phosphorylation sites and the
measurement of resultant physiological responses. Indeed, the present
results suggest that in mutation experiments it is potentially possible not only to block phosphorylation at some sites, but perhaps also to
create novel phosphorylation patterns that do not exist in the natural
receptor, with potentially aberrant physiological responses (21).
Present results also allow a new suggestion for the role in
internalization of the Tyr352 and Cys356, both
of which are strongly conserved in bradykinin B2 receptors. Site-directed mutagenesis of rat-1 cells suggested that these residues
may define an important site in B2 receptor internalization but may
contribute to the specificity of the site by phenol ring itself or the
amino acid bulk rather than by phosphorylation (9). Our results showed
that rat B2 receptor is phosphorylated at Tyr352 in
CHO cells. The phosphorylated residue Tyr352 is
sequentially close to Cys356, which our results showed to
be palmitoylated.
Another unanticipated finding of this study is the apparently mutually
exclusive phosphorylation at Tyr352 and palmitoylation at
Cys356. Because these residues are sequentially juxtaposed
one may speculate that in the nonacylated form of the B2
receptor residue Tyr352 is available to tyrosine kinase(s),
whereas anchoring of the corresponding region in the membrane in the
palmitoylated form prevents their access. This scenario is reminiscent
of observations made for the
2-adrenergic receptor (37)
where a complete desensitization of the nonpalmitoylated receptor was
observed because of hyperphosphorylation of two residues
(Ser345/Ser346) located next to the critical
Cys341 residue used for fatty acid derivatization (38).
Because of the many divergent roles that have been postulated for
receptor palmitoylation (39-42), the direct demonstration in this work
of fatty acid derivatization of the bradykinin B2 receptor
at Cys356 suggests that further investigation of the role
of palmitoylation in the kinin receptor is needed.
It is clear from the present results that it is not sensible to
speak of "the basal state" of bradykinin receptor in terms of
a single molecular species. It is highly likely that the same applies
to "the stimulated state" and that, as is beginning to be evident
with other receptors (9-11, 19-21), a very complex set of temporal
and spatial changes involving a substantial number of correlated
post-translational modifications of the receptor will have to be
elucidated in order to relate such modifications to physiological
pathways. The present results demonstrate that such modifications can
be detected and directly characterized for receptors modified
under in vivo conditions and provide indications of the
kinds of directed mutagenesis experiments that are likely to be helpful
in resolving the probably very complex relationships between
post-translational modifications and physiological pathways for
bradykinin and probably other G protein-coupled receptors.
At the present state of knowledge about G protein-coupled receptors,
the large number of post-translational modifications observed for the
B2 receptor, and particularly the correlation of
modifications at different sites, was unanticipated and suggests that
the number and diversity of post-translational modifications of such
receptors, together with concomitant roles in signal transduction pathways, may be much more complex than presently realized. Taken together with recent analogous results on endothelin B receptor (12),
it appears that the types of isolation procedures and mass spectrometry
analyses established in the present work for direct analysis of
modifications in in vivo systems at the protein level will
be suitable, perhaps essential, for relating specific post-translational modifications to functional states of the bradykinin receptor. In this context, it may be noted that we have recently established "functional proteomics" methods for observing complex, time-dependent phosphorylation/dephosphorylation responses
for large numbers of downstream proteins following receptor stimulation (44, 45). Together with the present methods, this potentially allows
correlation under in vivo conditions of specific
post-translational modifications of bradykinin receptor with downstream responses.