From the Institute of Molecular Biotechnology e.V, Beutenbergstrasse 11, 07745 Jena, Germany
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ABSTRACT |
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A new mild experimental approach for isolation of
peptide membrane receptors and subsequent analysis of
post-translational modifications is described. Endothelin receptors A
and B were isolated on oligo(dT)-cellulose using
N-(-maleimidocaproyloxy)succinimide endothelin coupled
to a protected (dA)-30-mer. This allowed a one-step isolation of the
receptor from oligo(dT)-cellulose via variation solely of salt
concentration. The identity of the receptor was confirmed by direct
amino acid sequencing of electroblotted samples or by using antibodies
against ETA and ETB receptors. The method used
here is very fast, requires only very mild elution conditions and, for
the first time, gave both ETA and ETB receptors concurrently in very good yield. Following enzymatic in-gel digestion, MALDI, and electrospray ion trap mass spectrometric analysis of the
isolated endothelin B receptor showed phosphorylation at Ser-304, -418, -438, -439, -440, and -441. Further phosphorylation at either Ser-434
or -435 was observed. The endothelin B receptor is also palmitoylated
at Cys residues 402 and 404. Phosphorylation of Ser304 may play a role in Hirschsprung's
disease.
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INTRODUCTION |
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Although it is clear that post-translational modifications of G-protein-coupled receptors are intimately involved in the physiological function of these signal transduction systems, there is as yet relatively little direct evidence for the specific modifications and the relationship of the different modifications to signal transduction processes. These types of processes are not easily amenable to analysis by genomic methods, i.e. there is a need for efficient methods to directly analyze these processes at the proteome level. We report here new, efficient methods for rapid isolation of the endothelin B receptor and for highly sensitive analysis of its post-translational modifications via mass spectrometry.
Endothelin, the strongest vasoconstrictor yet known, is a 21-amino acid peptide with physiological effects on cellular development, differentiation, vasoconstriction, and mitogenesis (1, 2). There are 3 different endothelin isoforms, ET-1,1 ET-2, and ET-3 with different affinity for the two different endothelin receptor subtypes, A and B (3-6). Both receptors are members of the G-protein-coupled receptor superfamily (3-6, 7). The consensus ET receptor topology includes three extracellular domains, three intracellular loops, and a cytoplasmic COOH-terminal tail, separated by seven hydrophobic helical regions thought to span the lipid bilayer. In addition it has been presumed that ET receptors are post-translationally modified by glycosylation of the NH2 terminus and by phosphorylation and palmitoylation of the cytoplasmic surface. Based on homology with other G-protein-coupled receptors (8-10), there has been speculation regarding possible structures, functional regions, and sites of post-translational modifications for endothelin receptors (11-16). However, as yet there is very little direct evidence for attributes such as the sites and the roles of glycosylation, palmitoylation, and phosphorylation, the location of the endothelin-binding site and the basis for discrimination among the three different endothelin isoforms.
A role of endothelin in disease has recently been demonstrated by the finding that mutated ETB receptor is associated with Hirschsprung's disease (17-21). In addition, mice lacking the ET-1 gene display severe malformation of large blood vessels, stressing the importance of endothelin during development (22). Endothelin has been shown to be a mitogenic agonist in different cell types (23, 24). The signaling pathway by which ET-1 promotes cell proliferation involves activation of intracellular kinase cascades and transcription factor stimulation. Recently it was suggested that the cytoplasmic tail of ETB receptor is involved in activation of three distinct mitogen-activated signal transduction pathways requiring extracellular-regulated kinase, c-Jun N-terminal kinase, and p38 kinases (25). Studies conducted by site-directed mutagenesis of ETA receptor suggested that the third intracellular loop and the COOH-terminal tail are also important for receptor-G-protein coupling (26, 27).
We report here a new method for isolation of endothelin receptor using oligo(dA) covalently linked to endothelin via a specially developed bifunctional cross-linker. Affinity chromatography has been carried out using oligo(dT) columns with mild elution using only changes in salt concentration analogous to methods used for isolation of eukaryotic mRNA. In-gel digestion of electrophoretically purified receptor, subsequent peptide mass fingerprinting by MALDI-TOF or electrospray ion trap mass spectrometry, and fragment analysis by tandem (MS/MS) mass spectrometry have been used to characterize post-translational modifications of this receptor.
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EXPERIMENTAL PROCEDURES |
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Materials
Fresh bovine lungs were obtained at a local slaughterhouse
and immediately frozen with liquid N2. Digitonin,
pepstatin, leupeptin, soybean trypsin inhibitor,
tosyl-L-phenylalanyl chloromethyl ketone, triethylammonium
acetate, 1,4-dithiothreitol (Microselect for deprotecting the modified
oligonucleotide), tetrabutylammoniumhydrogensulfate (Bu4NHSO3), and chymostatin were from Fluka
Chemie; 1,10-phenanthroline was from Merck; Hepes,
N-(-maleimidocaproyloxy)succinimide, phenylmethylsulfonyl fluoride, Chaps, Ellman's reagent, bacitracin, bovine serum albumin, and MES were from Sigma; the Aquapore RP-300A HPLC column and chemicals
for sequencing were from Applied Biosystems; human
125I-endothelin-1 (74 TBq/mmol) was from Amersham Corp.;
DNase-free RNase A was from Boehringer Mannheim; (dA)30-homomer with
and without 5
-modification with 1-O-dimethoxytrityl
hexyldisulfide was from BioTeZ (Berlin). Other chemicals (Merck and
Roth) were of the best grade available. Trypsin was from Promega
(Madison, WI). Endothelin-1 was synthesized with Fmoc chemistry as
described previously (28-31).
Membrane Preparations
Bovine lung (1.4 kg) was homogenized in a Heavy Duty Blender in
3 volumes of 20 mM Tris-HCl, pH 7.4, which contained 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride,
10 µg/ml pepstatin, and 10 µg/ml leupeptin (buffer A). The
homogenized lungs were centrifuged at 5000 × g for 20 min at 4 °C. The supernatants were discarded, the membrane pellets
were suspended in 3 volumes of buffer A and washed 3 times by
centrifugation. Afterward the membranes were washed twice in 20 mM phosphate buffer, pH 7.4, which contained 500 mM NaCl, 20 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, and the same amount of protease
inhibitors as in buffer A (buffer B). The membrane preparations were
stored at 70 °C.
Ligand Binding Assays
The binding activity of the solubilized endothelin receptor was
tested with 125I-endothelin-1. 10-20 µg of total protein
were incubated for 2 h at 20 °C with
125I-endothelin-1 (15,000 cpm) in a total volume of 100 µl of NaCl/Pi, 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4, containing 1 mM
EDTA and 2 mM phenylmethylsulfonyl fluoride. Binding was terminated by gel filtration through Whatman GF/B glass filter sheets
precoated with 0.3% (w/v) polyethyleneimine. The filters were washed
three times with 4 ml of NaCl/Pi buffer in a filtration device (Hoeffer) and transferred to polystyrene tubes. The filter-bound radioactivity was measured in a -counter (Packard).
Synthesis of
(dA)30-5-S-N-(
-Maleimidocaproyloxy) succinimide Endothelin
((dA)30-5
-S-EMC-ET)
Synthesis of N-(-Maleimidocaproyloxy)succinimide
endothelin (EMC-ET)--
1 mg (400 nmol) of endothelin-1 was dissolved
in 200 µl of 35% acetonitrile in 0.05% trifluoroacetate and further
diluted with 50 mM sodium borate, 0.015% Triton X-305, pH
8.2. A 10-fold excess (4 µmol) of the heterobifunctional cross-linker
N-(
-maleimidocaproyloxy)succinimide dissolved in 200 µl
of acetonitrile, was added in 5 portions at 10-min intervals, followed
by intensive mixing. After incubation for 2.5 h at room
temperature the pH of the reaction mixture was adjusted to 6.0 with 140 mM potassium dihydrogen phosphate to protect the sensitive
maleimido group of the cross-linker from hydrolysis (32).
Synthesis of (dA)30-5-S-EMC-ET--
To (dA)30-5
-SS-R
1-O-dimethoxytrityl hexyldisulfide in 45 µl of 100 mM triethylammonium acetate, 10 µl of freshly prepared 1 M dithiothreitol in 200 mM Tris/HCl, pH 8.0, were added. The solution was mixed briefly then incubated for 3 h
at room temperature to achieve quantitative reduction of the disulfide
to the free thiol. For complete removal of the dithiothreitol, a gel
filtration on PC 3.2/10 column was performed under isocratic conditions
(50 mM MES, pH 6.0, 5 mM EDTA). The fraction
with the deprotected oligonucleotide (about 200 µl) was immediately
mixed with 15 nmol of EMC-ET, which had previously been concentrated in
a Speed-vac, and adjusted to pH 6.0 in a volume of 150 µl with 200 mM MES. The reaction mixture was incubated overnight under
argon to prevent dimerization of the thiol-containing oligonucleotides.
The extent of the reaction was monitored by gel-retardation on SDS gels
of the modified oligonucleotide, compared with the monomer and the dimer of the thiol-modified oligonucleotide.
Purification of ET Receptor on Oligo(dT)-cellulose using Oligo(dA)-coupled Endothelin-1
Frozen membranes (100 g) were thawed and suspended in 2 volumes
of 20 mM potassium phosphate, pH 7.4, containing 0.40%
digitonin, 0.25% Chaps, 500 mM NaCl, 20 mM
EDTA, 1 µg/ml RNase, and the same protease inhibitors as buffer A
(buffer C). The mixture was gently stirred at 4 °C for 2 h,
filtered through 3 layers of miracloth and the filtrate was centrifuged
at 100,000 × g for 1 h at 4 °C. The
supernatants (85 ml) were incubated with 0.5 nmol of
(dA)30-5-S-EMC-ET for 3 h at room temperature, 100 mg of
oligo(dT)-cellulose was added and the suspension was gently agitated at
4 °C for 12 h. Oligo(dT)-cellulose was pelleted by
centrifugation (4 °C, 1,000 × g, 5 min) and packed
in a micro column. The column was washed with 10 ml of buffer C at
4 °C and afterward eluted with 10 mM Tris/HCl, pH 7.4, 0.40% digitonin, 0.25% Chaps, 1 mM EDTA, 10 µg/ml
leupeptin, 10 µg/ml pepstatin. 200-µl fractions were collected. Fractions containing endothelin receptor were identified by SDS-PAGE and by immunoblot analysis.
SDS-Polyacrylamide Gel Electrophoresis
20 µl of each fraction from the oligo(dT)-cellulose column
were 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 250 mM 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 (34). Marker proteins (Bio-Rad) were phosphorylase b (95.0 kDa), bovine serum albumin (68.0 kDa), ovalbumin
(45.0 kDa), carbonic anhydrase (30.0 kDa), soybean trypsin inhibitor (20.1 kDa), and -lactalbumin (14.4 kDa). The resolved proteins were
visualized by silver staining (Sigma Rapid Silver Staining Kit).
Immunoblot Analysis
Immunoblot analysis followed the procedure described by Hagiwara et al. (35). 5 µl of the protein sample were loaded per gel lane. Proteins from SDS-PAGE were transferred electrophoretically to a nitrocellulose membrane (Scleicher & Schuell). The blot was incubated with anti-ETB receptor serum at a 1:5,000 dilution in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20, at room temperature for 1 h. The receptor-antibody complexes were treated with alkaline phosphatase-conjugate goat anti-rabbit immunoglobulin G antiserum (Sigma) at 1:2.000 dilution. The immunoprecipitate was stained with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (ready made solution from Böhringer, Mannheim).
In-gel Tryptic Protein Digestion
After visualization, the gel was destained with a solution of 25 mM ammonium bicarbonate, 50% acetonitrile. The proteins
were digested in the gel according to the modified procedure of Hellman et al. (36). The resulting peptides were separated using a
Hewlett-Packard 1090 HPLC on a Aquapore RP 300 A (2.1 × 250 mm)
column in 0.05% trifluoroacetic acid, 3% acetonitrile, 7%
1-propanol, 0.1% (w/v) octyl--glucoside using a gradient of 0.05%
trifluoroacetic acid, 30% acetonitrile, 70% 1-propanol, 0.1% (w/v)
octyl-
-glucoside from 0 to 60% in 50 min and 60-100 in 15 min. The
flow rate was 0.4 ml/min and the fractions were monitored at 215 and
280 nm.
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 onto a target disk and
allowed to air dry. Subsequently, 0.3 µl of matrix solution (1% w/v
-cyano-4-hydroxysuccinnamic 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
time-of-flight mass spectrometer. MS/MS analyis was carried out using
Finnigan Mat (San Jose, CA) LCQ ion trap mass spectrometer. For the
interpretation of MS and MS/MS spectra of protein digests we used the
Sherpa software (37) and the MS-Fit program available at the www site
at the University of California at San Francisco
(http://rafael.ucsf.edu/cgi-bin/msfit).
Protein Sequencing of the ETB Receptor
Proteins were electroblotted onto poly(vinylidenedifluoride) membrane, stained with Coomassie Blue, and directly used for protein sequencing. The protein sequence analyses were performed on an Applied Biosystems Procise 494 4-cartridge sequencer (ABI, Foster City). The sequencing results were analyzed using a Macintosh based 610A data system.
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RESULTS |
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Isolation of ETB Receptor Using a New
"Fishhook"--
We have designed a heterobifunctional
cross-linking reagent which in addition to a
N-hydroxysuccinimide-activated ester also possesses a
thiol-selective maleimido group. This linker was first attached to
endothelin-1 via the -amino group of Lys9. The identity
of the N-(
-maleimidocaproyloxy)succinimide endothelin (Fig. 1A) was checked by MALDI
mass spectrometry and protein sequencing. Following HPLC purification,
the thiol selective maleimido group was used to attach 30-mer(dA).
After purification (Fig. 1B), the final product,
(dA)30-5
-S-EMC-ET, was subsequently added to a suspension of bovine
lung membranes solubilized in digitonin/Chaps. The ET receptors were
purified in a manner similar to that used for isolation of eukaryotic
mRNA: absorbtion on oligo(dT)-cellulose, washing under conditions
which favor formation of a (dA·dT) double helix (high salt
concentration) and elution with very low salt concentrations that
destabilize a (dA·dT) double helix.
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MALDI-TOF Mass Spectrometry--
From the SDS-PAGE gels, about 4 pmol of Coomassie Blue-stained ETB receptor were subjected
to in-gel tryptic digestion. We took precautions during gel
electrophoresis to avoid formation of acrylamide adducts and used only
the best and purest chemicals and solvents available throughout the
entire purification process. MALDI mass spectrometry was used for
initial analysis of the entire mixture of tryptic peptides. This gave
predominantly singly charged fragments, which allows easier
interpretation of masses observed for peptide mixtures than is the case
for spectra generated by electrospray mass spectrometry. From the MALDI
mass spectra (Fig. 3), it was possible to
identify the entire NH2-terminal extracellular region and
peptides from the second and third extracellular regions. From the
cytoplasmic region, peptides from the second, third, and COOH-terminal
loops were observed. Peptides containing transmembrane helical regions
joined to loop regions could also be observed (Fig.
4, Table
I). Half of the total tryptic digest
(about 2 pmol) was submitted to HPLC separation using
octyl--glycoside as a detergent during elution. The separated
peptides were collected and subjected to MALDI analysis. As summarized
in Fig. 4 and Table I, analysis of the original peptide mixture and the
HPLC-separated peptides allowed the observation of peptides
corresponding to the entire protein sequence including all
transmembrane helices. Due to incomplete tryptic cleavage, which is
often observed for membrane receptors, two membrane helices with the
intervening loop were often recovered in a single peptide, presumably
because the attached loops decrease the overall hydrophobicity of the fragment (Fig. 4). The mass measurements were sufficiently accurate (better than 0.1% of the calculated mass) to give clear indications of
which peptides were post-translationally modified (Table I). We
observed phosphorylation for peptides 31, 45, 48, and 49. Peptides 31, 45, and 48 showed an increased mass of 80 Da, which is characteristic for peptides with single phosphorylation sites. For peptide 49, masses
consistent with mono, di-, tri-, and tetra-phosphorylated peptide were
observed. For peptide 43, palmitoylation at two sites was suggested by
an increase of mass of 476 Da (2 × 238 Da). Although endothelin B
receptor contains two consensus glycosylation sites at residues Asn-60
and -118, the masses of peptide 2a-9 (residues 27-100), peptide 4 (50-65), and peptide 10-15 (101-164) were consistent with no
glycosylation at these sites.
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Electrospray Ion-trap Mass Spectrometry-- To confirm the identity of the peptides and to determine the attachment sites of the post-translational modifications, we performed electrospray ion trap mass spectrometry of the unseparated peptide mixture with subsequent MS/MS analysis of selected fragments of interest. We observed 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. This has previously been reported for MALDI-ion trap mass spectrometry of phosphorylated peptides (41). In accordance with this, we observed that phosphopeptides 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.
Peptide 31 provides an example of the identification of a phosphorylation site. There were two pairs of peaks separated by 98 Da (m/z 1407.0 and 1307.6 Da). In addition the MS/MS (collision induced dissociation) spectrum of a peptide 31 showed a y ion series, y12, y11, and y10, which was sufficient to identify Ser304 in the amino acid sequence 304S(Pi)GMQIALNDHLK315 (Fig. 5A) as the site of phosphorylation. We also confirmed the phosphorylation of peptide 45 which appeared as a double peak of m/z 658.3 and 560.2. MS/MS analysis generated a clear set of y5, y4, y3, y2 and b5*, b4*, b3* (bn* are bn ions minus H3PO4), which confirmed that the only Ser in peptide 45, 417QSCLK421, namely the Ser418, is phosphorylated. Two peaks of m/z 515.2 Da and 417.8 Da clearly indicated a single phosphorylated residue in peptide 48, 434SSNK437. However, while the MS/MS spectra of this peptide confirmed the identity of the peptide, the spectra were not sufficient to identify the phosphorylation site as Ser434 or Ser435.
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Palmitoylation of ETB Receptor-- The increase of 476 Da in the mass of peptide 43 indicated that among the four Cys residues clustered in this peptide, two are sites for palmitoylation. The MS/MS analysis generated y132+, y122+, and y112+ (m/z 1021.6, 978.5, and 926.0 Da) peaks corresponding to a doubly charged palmitoylated peptide, thereby showing that Cys399 in peptide 398SCLCCWCQSFEEK410 is not palmitoylated (Fig. 5B). Fragments y72+ and y92+ (m/z 555.6 and 819.1) showed that Cys402 and Cys404 are palmitoylated, but not Cys401. Confirmation of palmitoylation of Cys404 was obtained from the set of doubly charged b ions, in particular ions b62+ of m/z 467.2 and b72+, m/z 638.3 Da. The difference of 170 Da between these two doubly charged b ions (1/2 of 341 Da for palmitoylcysteine) confirms that of the 3 Cys residues in the sequence 398-403, only one is palmitoylated.
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DISCUSSION |
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In the work reported here the special fishhook,
(dA)30-5-S-EMC-ET, allowed for a rapid, very mild single step
isolation of ETB receptor by absorbtion to
oligo(dT)-cellulose and elution in a small volume of very low salt
buffer. The fishhook has been constructed in such a way that it should
be applicable to the isolation of other peptide hormone receptors,
which we are currently testing. After this single step procedure, the
receptor is identified by SDS-gel electrophoresis and the protein from
the gel band is subjected to tryptic fragmentation followed by analysis
of the peptide mixture by MALDI and electrospray mass spectrometry.
Mass fingerprinting of tryptic peptides is becoming a standard
procedure for rapid identification of proteins in proteome analysis
(42, 43). The essence of this method is that treatment with trypsin
produces a limited number of peptides and that the identification of a
surprisingly small number of such peptides suffices to identify the
protein in sequence data banks without the need for detailed analysis
of potentially very complex mass spectroscopic fragmentation patterns.
In the present work we have found that mass fingerprinting of tryptic
fragments of ETB receptor by MALDI mass spectroscopy also
provided an efficient method to screen the entire protein sequence for
the presence of post-translational modifications. Peptides covering the
entire receptor sequence could be detected (Fig. 4) with adequate mass
accuracy (better than 0.1%, Table I) to identify sequential regions
containing post-translational modifications. Although it would in
principle be possible to use only electrospray ion trap mass
spectrometric analysis of the peptide mixture with subsequent MS/MS
sequencing of each peptide, the prior use of MALDI mass spectrometry to
identify the singly charged peptide masses of interest resulted in a
dramatically simplified interpretation of the data. It should be
stressed that in the present study, as is the general case with mass
spectroscopy of peptides and proteins, a large number of spectra were
acquired under different ionization/fragmentation conditions to obtain adequate mass information on all peptides. In this regard, we also
found that MALDI analysis of membrane peptides was facilitated by
appropriate HPLC of these peptides. Inefficient peptide separation due
to aggregation and the loss of very hydrophobic peptides from integral
membrane proteins in RP-HPLC is a well known problem. We used
octyl--glycoside in separation solvents, which definitively helped
in the recovery of transmembrane peptides, but most of the peptides
were found in several HPLC fractions. This clearly means that
noncovalent aggregation of peptides is still a problem during reverse
phase-HPLC. However, this was of no major concern in the present work
since MALDI showed that such aggregates dissociated into their peptide
components. The presence of octyl-
-glycoside to keep the membrane
peptides soluble resulted in much better signal intensity for these
peptides.
Our analysis of peptides from ETB receptor showed that we were able to recover peptides from the entire sequence of the receptor. MALDI, electrospray ion trap mass spectrometry, and MS/MS analysis revealed that ETB receptor is phosphorylated at Ser304, Ser418, Ser438, Ser439, Ser440, and Ser441. Furthermore, another phosphorylation at Ser434 or Ser435 was observed. The ETB receptor is also palmitoylated at two sites identified to be at Cys402 and Cys404. A few peptides were observed both with and without phosphorylation. Given the distinctive fragmentation patterns of these post-translational modifications during mass spectroscopy, we believe this reflects the existence of species of ETB receptor with different patterns of post-translational modifications. At the present state of knowledge about G-protein-coupled receptors, the large number of post-translational modifications observed for the ETB receptor 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.
Phosphorylation and palmitoylation at carboxyl-terminal sites are known to influence the signal transduction in some G-protein-coupled receptors (8-14). The COOH-terminal tail in all known ETB receptors is identical and with the exception of a few amino acid exchanges, all known ETA receptors also have identical COOH-terminal tails. However, there is almost no homology in the COOH-terminal region between ETA and ETB receptors (3, 4, 7, 44-46). Among the very few strongly conserved amino acids are Ser418 and Ser439 as well as Cys402 and Cys404 which we found to be phosphorylated and palmitoylated, respectively. Those positions may be post-translationally modified in the ETA receptor as well. However, phosphorylation of Ser304 and Tyr438 is possible only for the ETB receptor since these positions are replaced by Gly or Asp, respectively, in the sequence of ETA receptor. It seems likely that different post-translational modifications of ETA and ETB receptors will correlate with different signal transduction pathways and we are therefore currently analyzing post-translational modifications of the ETA receptor.
We suggest that phosphorylation at Ser304 of ETB receptor may be particularly important in the development of Hirschsprung's disease (17-21). Hirschsprung's disease is characterized by the absence of autonomic ganglion cells in the terminal bowel. It is the most common cause of congenital obstruction with an incidence of 1 in 5000 live births. It has been shown that the endothelin-induced signaling pathway is crucial for the development of enteric ganglia and that this pathway is genetically compromised in Hirschsprung's disease. The analysis of the ETB receptor gene in patients with Hirschsprung's disease demonstrated two mutations which resulted in stop codons producing truncated and nonfunctional ETB receptor (20) and a single site mutant that replaced Ser304, which is phosphorylated (this work) and strongly conserved in all known ETB receptors, with Asn (47). This suggests that phosphorylation of Ser304 may play a particularly important role in receptor-mediated signal transduction.
The site-directed mutagenesis of 2- and
2-adrenoreceptors and endothelin receptor A as well as
mass spectrometric investigations of rhodopsin showed that those
receptors are palmitoylated at Cys residues in the COOH-terminal tails
(14, 8, 27, 48). It is believed that the covalently bound palmitic acid
residue becomes intercalated in the membrane bilayer. Prevention of
palmitoylation of the
-adrenergic receptor produced functional
uncoupling of the receptor from the adenylyl cyclase pathway, rapid
desensitization in response to its ligand, and increased basal
phosphorylation (49). For
2-adrenergic receptor the
signal transduction pathways were not affected, but prevention of
palmitoylation caused a decrease of ligand-promoted down-regulation of
the receptor. In the case of nonpalmitoylated bovine rhodopsin, an
increase in signal transduction activity was observed (50).
Site-directed mutagenesis of ETA receptor (27) has shown
that non-palmitoylated ETA receptor shows no change in
ligand binding affinity or stimulation of adenylyl cyclase. However,
the lack of palmitoylation was reported to affect phosphatidylinositol
hydrolysis by phospholipase C activation after stimulation by
endothelin-1. Furthermore, it was observed that the mutated
ETA, in contrast to wild type ETA, failed to show a ligand-induced transient increase in cytoplasmatic calcium concentration. We have obtained direct evidence that ETB
receptor is palmitoylated at Cys402 and Cys404.
The cluster of Cys residues, 399CLCCWC404, is
highly conserved in endothelin receptors of the B type. Together with
the above results on other receptors, this suggests that palmitoylation
plays a role in the regulation of the ETB receptor, although no experimental evidence has so far been reported.
The isolation and direct analysis of the chemical structure of membrane receptors has traditionally been a notoriously difficult task. Known difficulties have included low available amounts, limitations in tryptic or other digestion methods, poor peptide separations, very poor recovery of membrane helical fragments, difficulties in obtaining reliable information on post-translational modifications by protein chemical sequencing, etc. As a consequence, the previously available direct protein analyses of membrane receptors were generally limited to proteins that were readily available in larger quantities. Apart from pioneering work on rhodopsin (11, 12), there are only a very few scattered reports in the literature on direct observation of post-translational modifications of G-protein-coupled membrane receptors (13-15). The present results indicate that with the development of new methods for rapid, mild isolation of as little as 1-2 pmol of membrane receptors on gels and the combination of highly sensitive MALDI and electrospray ion trap mass spectrometry, direct evaluation of the sites and types of post-translational modifications of membrane receptors is poised to become a routine characterization. The next challenge will be to correlate different patterns of post-translational modifications with different functional states.
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ACKNOWLEDGEMENTS |
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We thank Prof. Hagiwara of the Tokyo Institute of Technology, Japan, for a generous gift of antisera against bovine ETB receptor. We thank Drs. M. Görlach and S. Fischer-Frühholz for helpful discussions and E. Nyakatura for excellent technical help.
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FOOTNOTES |
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* This work was supported by Deutsche Forchungsgemeinschaft Grant Go-639/1-2.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.
To whom correspondence should be addressed: Institut für
Molekulare Biotechnologie e.V, Beutenbergstra
e 11, 07745 Jena, Germany. Tel.: 49-3641-656430; Fax: 49-3641-656431; E-mail:
jzimm@imb- jena.de.
1
The abbreviations used are: ET, endothelin;
ETA, endothelin A receptor; ETB, endothelin B
receptor; EMC-ET, N-(-maleimidocaproyloxy)succinimide endothelin; (dA)30-5
-S-EMC-ET,
(dA)30-5
-S-N-(
-maleimidocaproyloxy)succinimide endothelin; MS/MS, tandem mass spectrometry; TOF, time-of-flight; Bu4NHSO3, tetrabutylammoniumhydrogensulfate;
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid
chromatography; PAGE, polyacrylamide gel electrophoresis; MALDI,
matrix-assisted laser desorption ionization.
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REFERENCES |
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