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 ET_B 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 ET_B 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 ET_A 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.

	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/P_i, 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/P_i 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-ET_B 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 ET_B 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.

	RESULTS

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Isolation of ET_B 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.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Preparation of the (dA)30-5-S-EMC-ET fishhook. A, purification of EMC-ET. The purification was carried out on a Sephacel C18 column using a gradient of 5-70% acetonitrile in 0.1% trifluoroacetate. Inset, MALDI mass spectrometry of EMC-ET; B, purification of ((dA)30-5-S-EMC-ET) on a Sephacel C18 column by applying a linear gradient of 2-70% acetonitrile in 100 mM triethylammonium acetate, 2 mM Bu4NHSO3. Inset, structure of (dA)30-5-S-EMC-ET.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Purification of ETB receptor from bovine lung using the (dA)30-5-S-EMC-ET/oligo(dT)-cellulose purification system. A, the elution profile of the receptor protein from an oligo(dT)-cellulose column monitored by 10% SDS-PAGE with silver staining. Elution fractions are represented by the numbered lanes. Immunoblotting (not shown) revealed that the bands at 34 and 49 kDa correspond to ETB receptor, while the band at about 54 kDa corresponds to ETA receptor. b, the effect of externally added endothelin-1 on the purification of ETB receptor. The protein fractions were analyzed on 12.5% SDS-PAGE. Lane 1, immunoblot of the purified ETB receptor contained in fraction 2 from the oligo(dT)-cellulose column. Lane 2, immunoblot of material obtained with the identical purification procedures except for the presence 1 mM endothelin-1 as a specific competitor. C, the effect of (dA)30-5-S-EMC-ET concentration on the yield of purified ETB receptor. Immunoblots of ETB receptor show the purification of ETB receptors performed as described under "Experimental Procedures" except that the concentration of (dA)30-5-S-EMC-ET was variable. Concentration of (dA)30-5-S-EMC-ET: 0 (lane 1), 0.37 nM (2), 1.5 nM (3), 6.0 nM (4), and 24.0 nM (5).

MALDI-TOF Mass Spectrometry-- From the SDS-PAGE gels, about 4 pmol of Coomassie Blue-stained ET_B 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.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   MALDI-TOF mass spectrometry of peptides obtained by in-gel trypsin digestion of ETB receptor. The peptides correspond to (M + H)+ masses except for peptides 25, 19-20, 8, and 31, which correspond to (M + 2H)2+ masses. Where the same masses are shown, the same peptide is indicated in only one spectra. The insets show spectra of partially purified peptides obtained from HPLC.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Peptides identified by MALDI-TOF mass spectrometry of bovine ETB receptor. Solid lines denote tryptic peptides identified from peptide mixture, dotted lines denote tryptic peptide identified after HPLC separation using octyl--glycoside in the elution solvent. Solid black circles indicate post-translationally modified amino acid residues. Squares indicate phosphorylation at Ser434 or Ser435.

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.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Post-translational modifications of tryptic peptides 31 and 43. A, MS/MS spectrum of phosphopeptide 31, S(Pi)GMQIALNDHLK (M + H)+. The notation b* denotes corresponding bn ions minus 98 (H3PO4), which confirms the phosphopeptide. B, MS/MS spectra of palmitoylated peptide 43, SCLCC(Pal)WC(Pal)QSFEEK (M + 2H)2+.

Palmitoylation of ET_B 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.

	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 ET_B 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 ET_B 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 ET_B 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 ET_B receptor is phosphorylated at Ser³⁰⁴, Ser⁴¹⁸, Ser⁴³⁸, Ser⁴³⁹, Ser⁴⁴⁰, and Ser⁴⁴¹. Furthermore, another phosphorylation at Ser⁴³⁴ or Ser⁴³⁵ was observed. The ET_B 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 ET_B 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 ET_B 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 ET_B receptors is identical and with the exception of a few amino acid exchanges, all known ET_A receptors also have identical COOH-terminal tails. However, there is almost no homology in the COOH-terminal region between ET_A and ET_B 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 ET_A receptor as well. However, phosphorylation of Ser³⁰⁴ and Tyr⁴³⁸ is possible only for the ET_B receptor since these positions are replaced by Gly or Asp, respectively, in the sequence of ET_A receptor. It seems likely that different post-translational modifications of ET_A and ET_B receptors will correlate with different signal transduction pathways and we are therefore currently analyzing post-translational modifications of the ET_A receptor.

We suggest that phosphorylation at Ser³⁰⁴ of ET_B 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 ET_B receptor gene in patients with Hirschsprung's disease demonstrated two mutations which resulted in stop codons producing truncated and nonfunctional ET_B receptor (20) and a single site mutant that replaced Ser³⁰⁴, which is phosphorylated (this work) and strongly conserved in all known ET_B 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 ET_A receptor (27) has shown that non-palmitoylated ET_A 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 ET_A, in contrast to wild type ET_A, failed to show a ligand-induced transient increase in cytoplasmatic calcium concentration. We have obtained direct evidence that ET_B 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 ET_B 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.