Endothelin-B receptors activate Galpha 13

Kenichiro Kitamura1, Naoki Shiraishi2, William D. Singer3, Mary E. Handlogten2, Kimio Tomita1, and R. Tyler Miller2

2 Division of Nephrology, University of Florida, Gainesville, Florida 32610; 3 Department of Pharmacolgy, University of Texas Southwestern Medical School, Dallas, Texas 75235; and 1 Fourth Department of Internal Medicine, Kumamoto University, Kumamoto, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelin (ET) receptors activate heterotrimeric G proteins that are members of the Gi, Gq, and Gs families but may also activate members of other families such as Galpha 12/13. Galpha 13 has multiple complex cellular effects that are similar to those of ET. We studied the ability of ET receptors to activate Galpha 13 using an assay for G protein alpha -chain activation that is based on the fact that an activated (GTP-bound) alpha -chain is resistant to trypsinization compared with an inactive (GDP-bound) alpha -chain. Nonhydrolyzable guanine nucleotides and AlMgF protected Galpha 13 from degradation by trypsin. In membranes from human embryonic kidney 293 cells that coexpress ETB receptors and alpha 13, ET-3 and 5'-guanylylimidodiphosphate [Gpp(NH)p] increased the protection of alpha 13 compared with Gpp(NH)p alone. The specificity of ETB receptor-alpha 13 coupling was documented by showing that beta 2 receptors and isoproterenol or ETA receptors and ET-1 did not activate alpha 13 and that a specific antagonist for ETB receptors blocked ET-3-dependent activation of alpha 13.

G protein; cell signaling; G protein-coupled receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN (ET) is a peptide ligand for two homologous G protein-coupled receptors, ETA and ETB receptors. These receptors regulate multiple intracellular signaling processes, not all of which have been clearly associated with an individual G protein. ET's effects include inhibition of adenylyl cyclase, presumably through activation of a Galpha i; activation of phospholipase C, presumably through activation of members of the Gq/11 family of alpha -chains; stimulation of tyrosine phosphorylation by an undetermined mechanism; and activation of the mitogen-activated protein (MAP) kinases [extracellular signal-regulated protein kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38] (1, 34). ET has a variety of tissue-specific biological effects. In vascular smooth muscle, ET is a potent vasoconstrictor and stimulus for smooth muscle growth, where it stimulates endothelial PGI2 and NO synthesis. In myocardium, it is a positive inotrope and stimulates myocardial hypertrophy. In the adrenal cortex ET stimulates aldosterone secretion, in fibroblasts it stimulates Na+/H+ exchange (NHE1), and in epithelial cells it stimulates Na+/H+ exchange by a different NHE isoform (NHE3) (7, 34, 42).

The actions of ET may not be explained fully by its activation of G proteins of the alpha i or alpha q families. To determine whether some of the biological effects of ET could be due to coupling of ETB receptors to Galpha 13, a member of a distinct family of G protein alpha -chains, we studied the ability of ETB receptors to activate Galpha 13 in a transient cell expression system. Galpha 13 is activated by thrombin, thromboxane, thyroid-stimulating hormone (TSH), and ANG II AT1A receptors (23, 25, 31). Like ET, thrombin, TSH, and ANG II have complex biological actions that involve activation of multiple G protein alpha -chains. Galpha 13 is capable of transforming cells, stimulating JNK activity, activating immediate early response genes, and stimulating Na+/H+ exchange in a variety of cell types and appears to be involved in regulation of cell motility (32). ET and alpha 13 share a number of effects on cells, including stimulation of JNK and Na+/H+ exchange activities (7-9, 12, 27, 34, 41, 42, 44). Consequently, alpha 13 could mediate some of the biological effects of ET.

Ligand-bound receptors activate G proteins by catalyzing conformational changes and guanine nucleotide exchange. Many assays for specific G protein-receptor coupling are based on the measurement of conformational changes or guanine nucleotide exchange. Activation is characterized by reduced affinity for beta gamma subunits and receptors and sequestration of a trypsin cleavage site that is in the approximate center of the molecule (4, 15, 26-28). Resistance of the conformation-sensitive trypsin site to cleavage by trypsin is a sensitive and specific assay for the active conformation. Trypsin protection has been used to study conformational changes and receptor activation of native, expressed, and recombinant alpha s and transducin (14, 16, 26, 28). We determined the trypsin cleavage pattern for activated and inactivated purified, recombinant Galpha 13. We then overexpressed alpha 13 and ETB receptors in human embryonic kidney 293 (HEK-293) cells and measured activation of alpha 13 in membranes by trypsin protection. We demonstrate that ETB receptors activate Galpha 13.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

HEK-293 cells were obtained from the American Type Culture Collection. Tissue culture medium and serum were obtained from GIBCO; trypsin from Worthington or Sigma; guanine nucleotides from Boehringer Mannheim; ET-1, ET-3, and BQ-788 from Calbiochem; and the chemiluminescence system from Amersham. The cDNA encoding the human ETB and ETA receptors in the expression vector pMEhETB (driven by the SRalpha promoter) was a generous gift form Masashi Yanigasawa (University of Texas Southwestern, Dallas, TX) (35, 38).

Expression and Purification of Recombinant alpha 13

Recombinant alpha 13 (ralpha 13) was expressed in Sf9 cells and purified as reported (36). Briefly, Sf9 cultures were infected with viruses encoding the alpha 13, beta 2, and gamma 2 G protein subunits. The cells were harvested, and membranes were prepared and extracted with sodium cholate in the presence of 0.1 mM GDP; alpha 13 was purified using sequential phenyl-Sepharose, Mono Q, and beta gamma -agarose column chromatography (36).

Cell Culture and Transfection

HEK-293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 25 mM HEPES, pH 7.5, in a 10% CO2-90% air incubator. Ten-centimeter dishes of cells were transfected with the cDNA constructs by the Ca-PO4 coprecipitation method. Each transfection contained 5 µg of DNA. One microgram of each expression construct was used (alpha 13 in pCMV5, or ETA or ETB in pMHe) with 3 µg of pBluescript. If one or no mammalian expression construct was used, the mammalian expression vector pCMV5 was used to bring the total amount of expression vector to 2 µg.

Preparation of Cell Membranes

Cells were washed with phosphate-buffered saline and scraped in 1 ml of membrane buffer containing 20 mM HEPES, pH 8, 1.5 mM MgSO4, and 1 mM EDTA. The cells were disrupted by Dounce homogenization, nuclei were removed by centrifugation at 1,000 g for 3 min, and membranes were prepared by centrifugation of the supernatant at 15,000 g for 20 min. The pellets were resuspended in membrane buffer containing 10% glycerol and frozen at -70°C. All steps were carried out on ice or at 4°C.

Trypsin Protection Assay

Purified ralpha 13. The ralpha 13 protein was exchanged into buffer containing 20 mM Na-HEPES, pH 8, 1 mM EDTA, 1 mM dithiothreitol (DTT), 100 mM NaCl, and 0.1% polyoxyethylene 10 lauryl ether (Lubrol) by gel filtration through Sephadex G-50 resin. The ralpha 13 (0.3 µM) was incubated at 30°C for 60 min in 24 µl of a solution containing either 10 µM GDP and 0.5 mM MgCl2, 10 µM GDP, 50 µM AlCl3, 10 mM MgCl2, and 10 mM NaF or 2 µM-1 mM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) and 0.5 mM MgCl2. Aliquots of the samples (10 µl) were treated with 2 µl H2O or N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) trypsin (0.2-5 µg/ml final concentration) for 20 min at 20°C. Digestions were stopped by adding 3 µl of 1 mg/ml soybean trypsin inhibitor. The digestion products were separated by SDS-PAGE on 9% polyacrylamide gels and immunoblotted with rabbit antisera specific for the carboxy terminus of alpha 13 (B860) (36). The blots were visualized with the enhanced chemiluminescence system (Amersham).

alpha 13-Subunit in cell membranes. Membranes (15 µg per reaction) were suspended in buffer containing 0.05% polyoxyethylene 10 lauryl ether (Lubrol), 20 mM HEPES, pH 8.0, 1.5 mM MgSO4, and 1.5 mM EDTA. Where indicated, GDP and 5'-guanylylimidodiphosphate [Gpp(NH)p] were present at a concentration of 50 µM; AMF was 50 µM AlCl3, 10 mM MgCl2, and 10 mM NaF; and ET-3 and BQ-788 (ETB receptor antagonist) were present at 10 nM. Reactions began with the simultaneous addition of guanine nucleotide analogs, AMF, and ET agonists or antagonists. The reactions were incubated at 30°C for 5 min. At that time, 2.5 µl of TPCK trypsin (0.5 mg/ml in the same buffer as above) was added for 2 min. The reactions were stopped by removing a 10-µl aliquot to a tube containing 2.5 µl of 5× SDS gel loading buffer and 5 mg/ml soybean trypsin inhibitor. The digestion products were separated by SDS-PAGE on 9% polyacrylamide gels and immunoblotting with rabbit antisera specific for the carboxy terminus of alpha 13 (B860) (36). The blots were visualized with the enhanced chemiluminescence system (Amersham).

Labeling of G13 and Gq With Azidoanilido [32P]GTP and Immunoprecipitation

Membranes (20 µg) were prepared as described above and suspended in buffer containing 0.1% polyoxyethylene 10 lauryl ether (Lubrol), 50 mM HEPES, pH 7.4, 30 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, and 1.0 mM benzamidine on ice. The agonists (as indicated) were added on ice, and the tubes were warmed to 30°C for 3 min. The reactions were started with the addition of 5 µl of azidoanilido [32P]GTP (10 nM or ~5 × 106 cpm, total reaction volume 60 µl) and incubated at 30°C for 5 min. The reactions were placed on ice and exposed to ultraviolet light (254 nm) for 20 s. An equal volume of immunoprecipitation buffer (final concentrations 1% Nonidet P-40, 1% deoxycholate, 0.5% SDS, 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin) was added to each sample. The reactions were vortexed and centrifuged for 10 min at 13,000 g. The supernatants were transferred to new tubes, and 1 µl affinity-purified antibodies (B860 for alpha 13, WO87 for alpha q and alpha s) were added (17, 36). The reactions were incubated at 0.4°C for 2 h with constant rocking. Protein A-Sepharose (10 µl) was then added, and the incubation continued for 2 h. The reactions were centrifuged and the pellets were washed three times by resuspension in immunoprecipitation buffer and centrifugation. The immunoprecipitated products were then separated on a 9% polyacrylamide gel, and the gel was dried and exposed to X-ray film (31, 39).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We established the trypsin cleavage pattern for Galpha 13 in the active and inactive conformations using purified, recombinant protein. We found that, like the other G proteins studied to date (transducin and Galpha s), binding of a GTP analog or activation of alpha 13 with GDP and AMF protects a conformation-sensitive trypsin site that is in the approximate center of the protein (26-28). As shown in Fig. 1, trypsinization of the 43-kDa GDP-bound pure protein produces two transient peptides of ~41 and 39 kDa that are seen in lower concentrations of trypsin (0.2 and 1 µg/ml). These peptides are both completely degraded in the presence of 5 µg/ml trypsin. In contrast, in the presence of AMF, a 39-kDa peptide is preserved even after digestion with 5 µg/ml of trypsin. This same 39-kDa peptide is also protected by GTPgamma S in a dose-dependent fashion over the concentration range of 2 µM-1 mM GTPgamma S. Protection is not as effective as that seen with AMF, possibly because the spontaneous rate of guanine nucleotide exchange by purified alpha 13 is slow, whereas activation of alpha -chains by AMF does not require guanine nucleotide exchange (36). These studies demonstrate that activation of alpha 13 by AMF or GTP analogs produces a 39-kDa peptide that is protected from degradation by trypsin.


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Fig. 1.   Trypsin cleavage pattern of recombinant, purified Galpha 13 in presence of GDP, AlMgF (AMF), and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). Approximately 7 pmol of Galpha 13 protein were incubated with guanine nucleotides and AMF (GDP: 20 µM; AMF: 10 µM GDP, 50 µM AlCl3, 10 mM NaF, and 10 mM MgCl2; GTPgamma S: 2 µM-1 mM as shown) for 60 min at 30°C. Aliquots (~3 pmol) were removed and treated with trypsin (0-5 µg/ml) as shown for 20 min at 20°C. Peptides were size-fractionated on 9% gels, transferred to nitrocellulose, and immunoblotted with an antibody to Galpha 13 (B860, 1:1,000) and visualized with chemiluminescence (36). Positions of 43-kDa peptide, intact Galpha 13, and 39-kDa protected fragment are shown at left.

To determine whether Galpha 13 could be activated to produce a trypsin-resistant conformation in membranes, we activated alpha 13 with AMF in membranes prepared from HEK-293 cells that had been transfected with alpha 13. Figure 2 shows that, like the pure protein, alpha 13 expressed in cell membranes is degraded by trypsin in the presence of GDP alone but that a protected band of ~39 kDa is found in the presence of AMF. In contrast to the studies with the pure protein, the protected 39-kDa band is not stable over time.


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Fig. 2.   Trypsin cleavage pattern of Galpha 13 expressed in human embryonic kidney 293 (HEK-293) cells in presence of AMF. Fifteen micrograms of membrane protein from HEK-293 cells were incubated with GDP (50 µM) and AMF (50 µM AlCl3, 5 mM MgCl2, and 10 mM NaF) for 5 min. Trypsin (final concentration 37.5 µg/ml) was then added for 2 min at 37°C, and the reactions were stopped by addition of 5× Laemmli sample buffer and boiling. Trypsin was omitted in lanes 1 and 2 to establish size and amount of intact alpha 13. Peptides were size-fractionated on 9% SDS gels and identified as in Fig. 1. Positions of molecular mass markers are shown at right, and positions of intact alpha 13 (43 kDa) and protected fragment of alpha 13 (39 kDa) are shown at left. Densitometric ratios of 39-kDa protected bands to 43-kDa bands are 0.35, 0.32, and 0.15 at 30, 60, and 120 s, respectively.

In membranes from HEK-293 cells that express both alpha 13 and ETB receptors, trypsinization in the presence of Gpp(NH)p results in a protected 39-kDa band, and the amount of protection is enhanced approximately twofold by the addition of ET-3 (Fig. 3A). The ratio of the protected 39-kDa band to the 43-kDa band was 0.33 for Gpp(NH)p alone and 0.78 for Gpp(NH)p with ET. We used Gpp(NH)p instead of GTP because the rate of hydrolysis of GTP by alpha 13 is rapid compared with the time required for trypsinization in this assay. In studies not shown, neither ET-1 nor ET-3 affected trypsin protection of G13 in membranes in the absence of expressed receptors. To demonstrate that ligand-dependent coupling of ETB receptors to alpha 13 is specific for ETB receptors, we coexpressed beta 2-adrenergic receptors and alpha 13 in HEK-293 cells to determine whether a receptor that has different biological activities from ETB receptors could enhance trypsin protection of the 39-kDa band of alpha 13 (Fig. 3B). Incubation of membranes containing expressed beta 2-adrenergic receptors and alpha 13 with isoproterenol did not enhance protection of the 39-kDa band of alpha 13 compared with similar lanes without isoproterenol. We further explored the specificity of the interaction of ETB receptors with Galpha 13 by comparing the ability of ET-1 to increase protection of Galpha 13 in cells that express either ETA or ETB receptors and Galpha 13. Figure 4 shows that ET-1 (an agonist with equal potency for ETA and ETB receptors) increases protection of Galpha 13 in a dose-dependent manner in membranes that express ETB receptors and Galpha 13, but not in membranes that express ETA receptors and Galpha 13 (34). In the membranes that express the ETB receptors, the ratio of the 39-kDa band increases from 0.68 [Gpp(NH)p alone] to 1.19 [Gpp(NH)p with 10-7 M ET-1]. However, in the membranes with ETA receptors and Galpha 13, the ratio remains relatively constant, ranging from 1.1 to 1.3. 


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Fig. 3.   Coupling of Galpha 13 to endothelin-B (ETB) and beta 2-adrenergic receptors expressed in HEK-293 cell membranes. Fifteen micrograms of membrane protein from HEK-293 cells that had been transfected with cDNAs coding for alpha 13 and ETB receptor (A) or alpha 13 and beta 2-adrenergic receptor (B) were incubated with 50 µM GDP [guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S)], 50 µM 5'-guanylylimidodiphosphate [Gpp(NH)p], 10 nM ET-3, and 1 µM isoproterenol (Iso) as indicated. Trypsinization and immunoblotting were carried out as described in Fig. 2. Positions of molecular mass markers are shown at right, and positions of intact alpha 13 (43 kDa) and protected fragment of alpha 13 (39 kDa) are shown at left. In A, densitometric ratio of 39-kDa protected bands to 43-kDa bands for Gpp(NH)p is 0.33 and for Gpp(NH)p and ET-3 is 0.78. In B, densitometric ratios of 39-kDa protected bands to 43-kDa bands for Gpp(NH)p are 0.25 for Gpp(NH)p and 0.15 for Gpp(NH)p and isoproterenol.


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Fig. 4.   Coupling of ETB or ETA receptors to Galpha 13. Aliquots of membranes from cells expressing either ETB or ETA receptors and alpha 13 were incubated with Gpp(NH)p (50 µM) and varying concentrations of ET-1 (0-10-7 M) for 3 min. Trypsinization and immunoblotting were carried out as described in Fig. 2. Position of molecular mass marker is shown at right, and positions of intact alpha 13 (43 kDa) and protected fragment of alpha 13 (39 kDa) are shown at left. Histogram was obtained by scanning films shown (representative experiment) and plotting integrated densities for protected 39-kDa bands in each sample. For ETB, the densitometric ratio of 39-kDa protected band to 43-kDa band for Gpp(NH)p is 0.68 and for 10-9, 10-8, and 10-7 M ET-1 ratios are 0.73, 1.22, and 1.19, respectively. For ETA, densitometric ratio of 39-kDa protected band to 43-kDa band for Gpp(NH)p is 1.1 and for 10-9, 10-8, and 10-7 M ET-1 ratios are 1.2, 1.13, and 1.3, respectively.

We used the specific ETB receptor antagonist BQ-788 as an additional test of specificity to confirm that the enhanced protection of expressed alpha 13 was due to ET-3 binding to the ETB receptor (13). As shown in Fig. 5, ET increases protection of the 39-kDa band of Galpha 13 by approximately twofold, whereas BQ-788 prevents protection of alpha 13 by ET-3. Additionally, BQ-788 reduces the basal level of protection in the presence of Gpp(NH)p. These studies demonstrate that protection of alpha 13 is a specific function of the ETB receptor.


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Fig. 5.   Inhibition of ET-3-dependent activation of Galpha 13 by BQ-788 (an ETB receptor antagonist) in membranes from HEK-293 cells that express alpha 13 and ETB receptor. Fifteen micrograms of membrane protein from HEK-293 cells that had been transfected with cDNAs coding for alpha 13 and ETB receptor were incubated with 50 µM GDP, 50 µM Gpp(NH)p, 10 nM ET-3, and 100 nM BQ-788 as indicated. Trypsin was omitted in lane 1 to establish size of intact alpha 13. Trypsinization and immunoblotting were carried out as described in Fig. 2. Positions of molecular mass markers are shown at right, and positions of intact alpha 13 (43 kDa) and protected fragment of alpha 13 (39 kDa) are shown at left. Densitometric ratios of 39-kDa protected bands to 43-kDa bands are 0.4 for Gpp(NH)p, 0.82 for Gpp(NH)p + ET, 0.48 for Gpp(NH)p + ET + BQ-788, and 0.24 for Gpp(NH)p + BQ-788.

To confirm that the trypsin protection assay reflects incorporation of GTP by the G protein alpha -chain, we documented stimulation of incorporation of azidoanilido [32P]GTP into alpha 13 and alpha q in membranes from HEK-293 cells that expressed ETB or ETA receptors and the alpha -chains. The alpha -chains were identified by immunoprecipitation with specific antisera (2, 31, 39). As shown in Fig. 6A, ET-1 stimulates incorporation of azidoanilido [32P]GTP into alpha 13 and alpha q in membranes from cells that express ETB receptors. In cells that express ETA receptors, ET-1 stimulates incorporation of azidoanilido [32P]GTP into alpha q but not alpha 13 (Fig. 6B). As a further test of specificity, we determined whether ETA or ETB receptors could activate alpha s. Figure 6C shows that neither ETB nor ETA receptors with ET-3 are able to activate Galpha s. However, as a positive control, beta 2 receptors and isoproterenol increase photolabeling of Galpha s. These results are similar to those obtained in Figs. 3-6.


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Fig. 6.   Receptor-stimulated labeling of Galpha q, Galpha 13, or Galpha s (45-kDa form) in membranes by azidoanilido [32P]GTP. Membranes were prepared from HEK-293 cells expressing ETA receptors and alpha q or alpha 13 (A); ETB receptors and alpha q or alpha 13 (B); or alpha s with ETA receptors and ET-3, ETB receptors and ET-3, beta 2-adrenergic receptors (beta -AR), or beta 2-adrenergic receptors and isoproterenol (C). Membranes were incubated with azidoanilido [32P]GTP and ET-3 or isoproterenol as indicated and exposed to ultraviolet light, and alpha -chains were immunoprecipitated with specific antisera for Galpha q, Galpha 13, and Galpha s (17, 36). Immunoprecipitates were size-fractionated on SDS gels, and gels were dried and exposed to X-ray film. Results shown are representative of 2 experiments. MW, molecular weight.

Figure 7 is a summary of the data presented above and represents a comparison of receptor-dependent protection of the 39-kDa band of alpha 13 in cells that express alpha 13 and the ETB receptor, the ETA receptor, or the beta 2-adrenergic receptor. Individual experiments were analyzed by calculating the ratio of the protected 39-kDa band to the 43-kDa band in the membranes with Gpp(NH)p alone and comparing that ratio with the ratio in the membranes with receptor agonist. ET treatment generally resulted in a twofold increase in this ratio. In separate experiments with different membrane preparations, the ratios varied considerably, but ET treatment still resulted in a twofold increase in the ratio. Therefore, to compare multiple experiments using different membrane preparations, we calculated the multiple increase in the ratio of the protected 39-kDa band to the 43-kDa band in the experimental membranes (ET, BQ-788 + ET, isoproterenol) over the membranes with Gpp(NH)p alone. ET consistently increased protection of the 39-kDa band (2.13 ± 0.15-fold, P < 0.05, ANOVA). BQ-788 inhibited the ET-induced protection (1.3 ± 0.1-fold for ET + BQ-788 vs. 2.13 ± 0.15-fold for ET alone, P < 0.05, ANOVA). The other comparisons were not statistically different from control [Gpp(NH)p alone].


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Fig. 7.   Summary of trypsin protection data for Galpha 13. Films were scanned with a Umax Vista 12 scanner and analyzed with Photoshop and National Institutes of Health image analysis software. On each film, ratio of 39-kDa protected band to 43-kDa band was determined, and value for Gpp(NH)p alone was divided into values for experimental conditions [ET, BQ-788 (BQ) + ET, ETA receptors, or beta 2 receptors (Beta) + isoproterenol]. These values were then averaged to produce values shown ± SE (ET-3, n = 6; BQ-788 + ET-3, n = 3; ETA, n = 4; beta 2 receptors + isoproterenol, n = 4). Cont, control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrated that ETB receptors activate Galpha 13 when they are coexpressed in HEK-293 cells. The specificity of the interaction of ETB receptors with Galpha 13 was demonstrated by the facts that ETA and beta 2-adrenergic receptors did not increase the protection of G13 from degradation by trypsin and that the specific ETB antagonist BQ-788 blocked the ability of ET-3 to protect Galpha 13. Similar results were obtained using an independent technique, photolabeling of alpha -subunits and immunoprecipitation with specific antisera (Fig. 6). Studies by others have shown that, depending on the cell type, ET receptors may activate members of the alpha s, alpha i, and alpha q/11 families (34). Our results demonstrate that ETB receptors can also activate a member of a fourth family of G protein alpha -chains, Galpha 13 (37). These results indicate that, in some tissues, the complex biological effects of ET are due to activation of Galpha 13 and the intracellular signaling systems it controls.

Several other receptors, those for thrombin, thromboxane, TSH, and ANG II, have been shown to activate Galpha 13 (23, 25, 31). We studied the activation of alpha 13 by ETB receptors by coexpressing the two proteins in HEK-293 cells. Others have used essentially the same technique to study portions of signaling pathways including receptor-G protein interactions and demonstrated specific interactions in this type of system (5, 6, 14, 27, 48).

The rGalpha 13 and Galpha 13 expressed in HEK-293 cells behaved similarly in that both were degraded by trypsin in the presence of GDP alone and protected by AMF and nonhydrolyzable guanine nucleotides. However, these proteins differed in their spontaneous rates of guanine nucleotide exchange. This rate is slow for the purified protein, as demonstrated by the relatively poor protection of rGalpha 13 by GTPgamma S. In contrast, the rate of spontaneous guanine nucleotide exchange [Gpp(NH)p] was substantial in cells that expressed ETB receptors with alpha 13 or with alpha 13 alone. In cells that coexpressed the ETB receptor and alpha 13, the rate of guanine nucleotide exchange was further stimulated by addition of the ligand (ET-3) for the receptor. These results suggest that even without ligand (ET-3) or ETB receptors, guanine nucleotide exchange is catalyzed by some factor in the membranes. We cannot determine whether the spontaneous rate of guanine nucleotide exchange seen in the membrane preparation was due to the presence of other receptors that activate alpha 13 or whether some other protein interacts with the alpha -chain to facilitate guanine nucleotide exchange.

The magnitude of receptor-stimulated guanine nucleotide exchange for receptors and G protein alpha -chains found by other investigators in membrane preparations is comparable to that which we found with ETB receptors and Galpha 13. Offermanns et al. (31) and Laugwitz et al. (23) measured thrombin and TSH receptor-simulated photoaffinity labeling of G13 with azidoanilido GTP. They found that the activated receptors increased photolabeling of Galpha 13 by 82 and 123%, respectively, over the basal, or spontaneous, rate of guanine nucleotide exchange (no receptor ligand). In studies using other receptors and alpha -chains, they demonstrated that agonist-stimulated receptors stimulated alpha -chain labeling by 95-320% (24, 33). Our trypsin protection studies (Fig. 7) demonstrated a 2.13 ± 0.15-fold, or ~113%, increase in the amount of active Galpha 13 in response to ETB receptor stimulation. Using proteins expressed in a baculovirus expression system with [35S]GTPgamma S and immunoprecipitation, Barr et al. (2) demonstrated that stimulation of 5-HT1A receptors resulted in an approximately twofold increase in guanine nucleotide exchange compared with receptor alone. In all of these systems, the basal rate of guanine nucleotide exchange is substantial, so that when agonist-stimulated and "spontaneous" guanine nucleotide exchange rates are compared, the difference is on the order of two- to fourfold. Trypsin protection studies in which rhodopsin and transducin were coexpressed in COS cells demonstrated a greater multiple increase in protection of transducin by light-activated rhodopsin because the spontaneous rate of guanine nucleotide exchange in this system is much lower than for other receptor-G protein combinations (14).

ET acts through ETA and ETB receptors. These receptors have different affinities for their ligands (ETA: ET-1 >=  ET-2 > ET-3; ETB: ET-1 = ET-2 = ET-3) and patterns of tissue and cell-type distribution (34). The differences between these two receptors and the intracellular signaling pathways they activate have not been clearly established in all tissues. Most studies to date rely on selectivity of ET-1 or ET-3 agonists and antagonists for the different receptors. Some of the apparent differences may be due to cell-specific expression of downstream signaling molecules. ETA and ETB receptors share the ability to increase intracellular Ca2+ through activation of a member of the Galpha q/11 family of G proteins as well as regulating Ca2+ entry by undefined mechanisms (34). They also appear to activate phospholipase A2 and phospholipase D, which also may be involved in Ca2+ signaling. ETB and ETA receptors are capable of inhibiting cAMP production (7, 29, 34). In one report, ETA receptors acting through alpha s stimulated adenylyl cyclase (11). ETB receptors are capable of activating three members of the MAP kinase family, ERK, JNK, and p38 (1). In several studies, Galpha 13 activated the JNK pathway, so ETB receptors may act through alpha 13 to activate JNK in these systems (8, 41, 44).

ETB receptors stimulate NHE1 activity in fibroblasts (the Na+/H+ exchanger isoform expressed in most cells) and NHE3 in renal epithelial cells (7, 34, 42). These transporters are regulated by similar extracellular signals under a number of circumstances, but the precise intracellular mechanisms that regulate them are not fully defined (30, 40). The mechanisms may involve changes in intracellular Ca2+ levels, protein kinase C activity, protein kinase C-independent mechanisms, and tyrosine kinase activity (3, 30, 34). Expression studies with two G protein alpha -chains, Galpha q and Galpha 13, showed that they activate NHE1 in part through increasing mRNA and protein levels (20). Therefore, because alpha q and alpha 13 activate NHE1 as does ET through ETB receptors, ETB receptors may activate these alpha -chains. Galpha q activates phospholipase C and may stimulate Na+/H+ exchange activity by that mechanism, but the mechanisms by which Galpha 13 acts are not defined. Preliminary studies indicate that ETB receptors and Galpha 13 stimulate an NHE1 promoter-reporter construct (Shiraishi and Miller, unpublished observations). Consequently, Galpha 13 is a candidate for one of the G proteins that is activated by ETB receptors and that regulates Na+/H+ exchange.

Galpha 13 appears to activate a number of downstream signaling molecules. Two recent reports show that it activates p115, an RGS protein, that is also a guanine nucleotide exchange factor for the small GTP binding protein Rho A (18, 22). Other molecules may also be targets of Galpha 13. Galpha 13 contributes to regulation of hormone-stimulated Ca2+ influx and intercellular Ca2+ by other mechanisms that are independent of phospholipase C, K channels, the small GTP binding protein cdc42, and the JNK pathway (19, 20, 25, 41, 46, 47, 49). These and possibly other signaling pathways contribute to cell transformation, induction of inducible NO synthase, and activation of Na+/H+ exchange (7, 10, 20, 21, 42, 43, 45).

Our studies demonstrate that ETB receptors activate Galpha 13 in HEK-293 cells. Our studies do not address which of the potential signaling proteins are downstream of ETB receptors and alpha 13 in HEK-293 cells, but they do demonstrate that the complex biological effects of ET derive in part through signaling by ETB receptors though an additional family of G protein alpha -chains.


    ACKNOWLEDGEMENTS

K. Kitamura and N. Shiraishi contributed equally to this work.


    FOOTNOTES

The authors thank Paul Sternweis and Shmuel Muallem for valuable discussions.

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41726, funds from the American Heart Association with funds from the Florida affiliate (R. T. Miller), Grants GM-15359 and GM-31954, and funds from the Robert A. Welch foundation (P. C. Sternweis), and a training grant from the National Kidney Foundation (to K. Kitamura and N. Shiraishi).

Address for reprint requests and other correspondence: R. T. Miller, Division of Nephrology, Univ. of Florida, Box 100224 JHMHC, 1600 SW Archer Rd., Gainesville, FL 32610 (E-mail: tmiller{at}nervm.nerdc.ufl.edu).

Received 10 July 1997; accepted in final form 25 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aquila, E., A. Whelchel, H. J. Knot, M. Nelson, and J. Posada. Activation of multiple mitogen-activated protein kinase signal transduction pathways by the endothelin B receptor requires the cytoplasmic tail. J. Biol. Chem. 271: 31572-31579, 1996[Abstract/Free Full Text].

2.   Barr, A. J., L. F. Brass, and D. R. Manning. Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. J. Biol. Chem. 272: 2223-2229, 1997[Abstract/Free Full Text].

3.   Bourne, H. R. Signal transduction. Team blue sees red. Nature 376: 727-729, 1995[Medline].

4.   Bourne, H. R., D. A. Sanders, and F. McCormick. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348: 125-132, 1990[Medline].

5.   Buhl, A. M., B. J. Eisfelder, G. S. Worthen, G. L. Johnson, and M. Russell. Selective coupling of the human anaphylatoxin C5a receptor and alpha 16 in human kidney 293 cells. FEBS Lett. 323: 132-134, 1993[Medline].

6.   Chabre, O., B. R. Conklin, S. Brandon, H. R. Bourne, and L. E. Limbird. Coupling of the alpha 2 adrenergic receptor to multiple G proteins. J. Biol. Chem. 269: 5730-5734, 1994[Abstract/Free Full Text].

7.   Chu, T. S., Y. Peng, A. Cano, M. Yanagisawa, and R. J. Alpern. EndothelinB receptor activates NHE-3 by a Ca2+-dependent pathway in OKP cells. J. Clin. Invest. 97: 1454-1462, 1996[Abstract/Free Full Text].

8.   Coso, O. A., M. Chiariello, J.-C. Yu, H. Teramoto, P. Crespo, N. Xu, T. Miki, and S. J. Gutkind. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81: 1137-1146, 1995[Medline].

9.   Dhanasekaran, N., and J. M. Dermott. Signaling by the G12 class of G proteins. Cell. Signal. 8: 235-245, 1996[Medline].

10.   Dhanasekaran, N., M. V. V. S. VaraPrasad, S. J. Wadsworth, J. M. Dermott, and G. Van Rossum. Protein kinase C-dependent and -independent activation of Na+/H+ exchanger by Galpha 12 class of G proteins. J. Biol. Chem. 269: 11802-11806, 1994[Abstract/Free Full Text].

11.   Eguchi, S., Y. Hirata, M. Ihara, M. Yano, and F. Marumo. A novel ET-A antagonist (BQ 123) inhibits endothelin 1 induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells. FEBS Lett. 302: 243-246, 1992[Medline].

12.   Eiam-Ong, S., S. A. Hilden, A. J. King, C. A. Johns, and N. E. Madias. Endothelin-1 stimulates the Na+/H+ and Na+/HCO-3 transporters in rabbit renal cortex. Kidney Int. 42: 18-24, 1992[Medline].

13.   Fukuroda, T., T. Fujikawa, S. Ozaki, K. Ishikawa, M. Yano, and M. Nishikibe. Clearance of circulating endothelin-1 by ET-B receptors in rats. Biochem. Biophys. Res. Commun. 199: 1461-1465, 1994[Medline].

14.   Garcia, P. D., R. Onrust, S. M. Bell, T. P. Sakmar, and H. R. Bourne. Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells. EMBO J. 14: 4460-4469, 1995[Abstract].

15.   Gilman, A. G. G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56: 615-649, 1987[Medline].

16.   Graziano, M. P., and A. G. Gilman. Synthesis in Escherichia coli of GTPase-deficient mutants of Gsalpha . J. Biol. Chem. 264: 15475-15482, 1989[Abstract/Free Full Text].

17.   Gutowski, S., A. Smrcka, L. Nowak, D. Wu, M. I. Simon, and P. C. Sternweis. Antibodies to the alpha q subfamily of guanine nucleotide-binding regulatory protein alpha  subunits attenuate activation of phosphatidylinisitol 4,5-bisphosphate hydrolysis by hormones. J. Biol. Chem. 266: 20519-20524, 1991[Abstract/Free Full Text].

18.   Hart, M. J., X. Jiang, T. Kozasa, W. Roscoe, W. D. Singer, A. G. Gilman, P. C. Sternweis, and G. Bollag. Direct stimulation of the guanine nucleotide exchange activity of p115RhoGEF by Galpha 13. Science 280: 2112-2114, 1998[Abstract/Free Full Text].

19.   Hooley, R., C.-Y. Yu, M. Symons, and D. L. Barber. Galpha 13 stimulates Na+-H+ exchange through distinct Cdc42-dependent and rhoA-dependent pathways. J. Biol. Chem. 271: 6152-6158, 1996[Abstract/Free Full Text].

20.   Kitamura, K., W. D. Singer, A. Cano, and R. T. Miller. Galpha q and Galpha 13 regulate NHE-1 in a renal epithelial cell line. Am. J. Physiol. 268 (Cell Physiol. 37): C101-C110, 1995[Abstract/Free Full Text].

21.   Kitamura, K., W. D. Singer, R. A. Star, S. Muallem, and R. T. Miller. Induction of inducible nitric oxide synthase by the heterotrimeric G protein Galpha 13. J. Biol. Chem. 271: 7412-7415, 1996[Abstract/Free Full Text].

22.   Kozasa, T., X. Jiang, M. J. Hart, P. J. Sternweis, W. D. Singer, A. G. Gilman, G. Bollag, and P. C. Sternweis. p115 RhoGEF, a GTPase activating protein for Galpha 12 and Galpha 13. Science 280: 2109-2111, 1998[Abstract/Free Full Text].

23.   Laugwitz, K.-L., A. Allgeier, S. Offermanns, K. Spicher, J. Van Sande, J. E. Dumont, and G. Schultz. The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families. Proc. Natl. Acad. Sci. USA 93: 116-120, 1996[Abstract/Free Full Text].

24.   Laugwitz, K.-L., S. Offermanns, K. Spicher, and G. Schultz. µ- And delta -opiod receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells. Neuron 10: 233-242, 1993[Medline].

25.   Macrez-Lepretre, N., F. Kalkbrenner, J.-L. Morel, G. Schultz, and J. Mironneau. G protein heterotrimer Galpha 13beta 1gamma 3 couples the angiotensin AT1A receptor to increases in cytoplasmic Ca in rat portal vein myocytes. J. Biol. Chem. 272: 10095-10102, 1997[Abstract/Free Full Text].

26.   Masters, S. B., R. T. Miller, M.-H. Chi, F.-H. Chang, B. H. Beiderman, N. G. Lopez, and H. R. Bourne. Mutations in the GTP binding site of alpha s alter stimulation of adenylyl cyclase. J. Biol. Chem. 264: 15467-15474, 1989[Abstract/Free Full Text].

27.   Mazzoni, M. R., and H. E. Hamm. Interaction of transducin with light-activated rhodopsin protects it from proteolytic digestion by trypsin. J. Biol. Chem. 271: 30034-30040, 1996[Abstract/Free Full Text].

28.   Miller, R. T., S. B. Masters, K. A. Sullivan, B. Beiderman, and H. R. Bourne. A mutation that prevents GTP-dependent activation of the alpha -chain of Gs. Nature 334: 712-715, 1988[Medline].

29.   Nadler, S. P., J. A. Zimplemann, and R. L. Hebert. Endothelin inhibits vasopressin-stimulated water permeability in rat terminal inner medullary collecting duct. J. Clin. Invest. 90: 1458-1466, 1992[Medline].

30.   Noel, J., and J. Pouyssegur. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am. J. Physiol. 268 (Cell Physiol. 37): C283-C296, 1995[Abstract/Free Full Text].

31.   Offermanns, S., K.-L. Laugwitz, K. Spicher, and G. Schultz. G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc. Natl. Acad. Sci. USA 91: 504-508, 1994[Abstract].

32.   Offermanns, S., V. Mancino, J.-P. Revel, and M. I. Simon. Vascular system defects and impaired cell chemokinesis as a result of Galpha 13 deficiency. Science 275: 533-536, 1997[Abstract/Free Full Text].

33.   Offermanns, S., G. Schultz, and W. Rosenthal. Identification of receptor-activated G proteins with photoreactive GTP analog, [alpha -32P]GTP azidoanilide. Methods Enzymol. 195: 286-301, 1991[Medline].

34.   Rubanyi, G. M., and M. A. Polokoff. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol. Rev. 46: 325-415, 1994[Medline].

35.   Sakamoto, A., M. Yanagisawa, T. Sawamura, T. Enoki, T. Ohtani, T. Sakurai, K. Nakao, T. Toyo-aka, and T. Masaki. Distinct subdomains of human endothelin receptors determine their selectivity to endothelinA-selective antagonist and endothelinB-selective agonists. J. Biol. Chem. 268: 8547-8553, 1993[Abstract/Free Full Text].

36.   Singer, W. D., R. T. Miller, and P. C. Sternweis. Purification and characterization of the alpha  subunit of G13. J. Biol. Chem. 269: 19796-19802, 1994[Abstract/Free Full Text].

37.   Strathmann, M. P., and M. I. Simon. Galpha 12 and Galpha 13 subunits define a fourth class of G protein alpha  subunits. Proc. Natl. Acad. Sci. USA 88: 5582-5586, 1991[Abstract].

38.   Takebe, Y., M. Seiki, J.-I. Fujisawa, P. Hoy, K. Yokota, K.-I. Arai, M. Yoshida, and N. Arai. SRalpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of the human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8: 466-472, 1988[Medline].

39.   Thomas, R., and T. Pfeuffer. Photoaffinity labeling of GTP-binding proteins. Methods Enzymol. 195: 280-301, 1991[Medline].

40.   Tse, M., S. Levine, C. Yun, S. Brant, L. T. Counillon, J. Pouyssegur, and M. Donowitz. Structure/function studies of the epithelial isoforms of the mammalian Na/H exchanger gene family. J. Membr. Biol. 135: 93-108, 1993[Medline].

41.   VaraPrasad, M. V. V. S., J. M. Dermott, L. E. Heasley, G. L. Johnson, and N. Dhanasekaran. Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of Galpha 12 and Galpha 13. J. Biol. Chem. 270: 18655-18659, 1995[Abstract/Free Full Text].

42.   Vigne, P., A. Ladoux, and C. Frelin. Endothelins activate Na/H exchange in brain capillary endothelial cells via a high affinity endothelin-3 receptor that is not coupled to phospholipase C. J. Biol. Chem. 266: 5925-5928, 1991[Abstract/Free Full Text].

43.   Voyno-Yasenetskaya, T., B. R. Conklin, R. L. Gilbert, R. Hooley, H. R. Bourne, and D. L. Barber. Galpha 13 stimulates Na-H exchange. J. Biol. Chem. 269: 4721-4724, 1994[Abstract/Free Full Text].

44.   Voyno-Yasenetskaya, T., M. P. Faure, N. G. Ahn, and H. R. Bourne. Galpha 12 and Galpha 13 regulate extracellular signal-regulated kinase and c-jun kinase pathways by different mechanisms in COS-7 cells. J. Biol. Chem. 271: 21081-21087, 1996[Abstract/Free Full Text].

45.   Voyno-Yasenetskaya, T. A., A. M. Pace, and H. R. Bourne. Mutant alpha  subunits of G12 and G13 proteins induce neoplastic transformation of Rat-1 fibroblasts. Oncogene 9: 2559-2565, 1994[Medline].

46.   Wilk-Blaszczak, M. A., S. Gutowski, P. C. Sternweis, and F. Belardetti. Bradykinin modulates potassium and calcium currents in neuroblastoma hybrid cells via different pertussis toxin-insensitive pathways. Neuron 12: 109-116, 1994[Medline].

47.   Wilk-Blaszczak, M. A., W. D. Singer, S. Gutowski, P. C. Sternweis, and F. Belardetti. The G protein G13 mediates inhibition of voltage-dependent calcium current by bradykinin. Neuron 13: 1215-1224, 1994[Medline].

48.   Wong, Y. H. Gi assays in transfected cells. Methods Enzymol. 238: 81-94, 1994[Medline].

49.   Xu, X., K. Kitamura, K. Lau, S. Muallem, and R. T. Miller. Differential regulation of Ca release activated Ca influx (CRAC) by heterotrimeric G proteins. J. Biol. Chem. 270: 29169-29175, 1995[Abstract/Free Full Text].


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