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Role of the Conserved DRY Motif on G Protein Activation of Rat Angiotensin II Receptor Type 1A
Biochemical and Biophysical Research Communications 292, 362–367 (2002) doi:10.1006/bbrc.2002.6670, available online at http://www.idealibrary.com on
Role of the Conserved DRY Motif on G Protein Activation of Rat Angiotensin II Receptor Type 1A Kenji Ohyama,* ,1 Yoshiaki Yamano,† Tomoaki Sano,* Yoshiko Nakagomi,* Manabu Wada,‡ and Tadashi Inagami§ *Department of Clinical Nursing and Pediatrics, Yamanashi Medical University, Yamanashi 409-3898, Japan; †Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan; ‡JCR Pharmaceuticals, Hyogo 651-2241, Japan; and §Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received February 25, 2002
To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp–Arg–Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT 1A) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP 3 production, and the acidification of the medium in response to Ang II were determined. The effects of GTP␥S on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP 3 production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP 3 and acidify medium comparable with wild type AT 1A. These results indicate as follows; Asp 125 is essential for intracellular signal transduction involving G protein coupling, Arg 126 is essential for coupling of G q protein but not other G proteins, and Tyr 127 is not important for G protein coupling. © 2002 Elsevier Science (USA)
Key Words: angiotensin II; angiotensin II receptor; site-directed mutagenesis; signal transduction; extracellular acidification; G protein; G protein-coupled receptor; IP 3.
Angiotensin II (Ang II) receptor type 1A (AT 1A) has seven putative transmembrane domains and is a G protein-coupled receptor (1, 2). We previously reported that the highly conserved triplet of amino acid sequence Asp–Arg–Tyr (DRY) of the second intracellular 1 To whom correspondence should be addressed at Department of Clinical Nursing and Pediatrics, Yamanashi Medical University, 1110, Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, Japan. Fax: ⫹81-55-273-6605. E-mail: [email protected].
0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
loop (ICL2) and YFL of the cytosolic C-tail region were essential for G q coupling (3, 4). Importance of DRY for G-protein activation has been reported in several G-protein coupled receptors. For example, mutagenesis studies demonstrated that Asp residue in the  2adrenergic (5, 6), m1-muscarinic (7), and ␣2Aadrenergic (8) receptors was essential for activation of the respective effectors. The Arg residue in the V 2 vasopressin receptor (9), gonadotropin-releasing hormone (GnRH) receptor (10), and m1-muscarinic receptor (11) was important for the agonist-induced activation. On the other hand, mutation of the Tyr residue in the DRY motif of m1-muscarinic receptor (11) or the Ser residue in DRS motif of the GnRH receptor (10, 12) did not impair signal transduction. Recently, constitutive activation of receptors was reported in several mutant receptors with substitution of DRY motif (6, 13–15). Moreover, agonist binding affinity was reduced by Asp mutation (D130A) of CB 2 cannabinoid receptor (16) and was increased by Arg mutation (R131H) of  2-adrenergic receptor (9). In the present study, we focused on the triplet of amino acid sequence, Asp 125– Arg 126–Tyr 127 in ICL2 of the rat AT 1A. By constructing five substitutional mutants of rat AT 1A, we examined their ligand binding, InsP 3 production, and proton egression in response to Ang II with the aim of defining regions essential for the coupling and activation of G-proteins. MATERIALS AND METHODS Rat AT 1A mutagenesis. Site directed mutagenesis was performed by the procedure of Kunkel (17). Sites of substitution are shown in Fig. 1. Wild type AT 1A and its mutant cDNA in the expression vector pCDNA1 were transfected into and stably expressed in CHO-K1 cells. Binding assay. AT 1A-expressing CHO-K1 cells were grown in Ham’s F12 medium with 10% FCS in 24-well plates. They were washed with Hank’s balanced salt solution (HBSS) and incubated for
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FIG. 1. Schematic representation of the rat AT 1A receptor. The illustration shows the amino acid sequence in the N-terminal portion of the second intracellular loop. The sites of substitution are indicated by outline font.
90 min at 37°C in 250 l of Ham’s F12 medium with 2% FCS. The cells were incubated with [ 125I-Sar 1–Ile 8]-Ang II (DuPont) in varying concentrations from 0.3 nM to 10 nM in this medium for determination of the specific binding. After the incubation, cells were immediately placed on ice, washed three times with ice-cold HBSS, then solubilized with 250 l of 0.5 N NaOH. Radioactivity was measured in a ␥ counter. Specific [Sar 1–Ile 8]-Ang II binding was determined as a difference between the total binding of [ 125I-Sar 1–Ile 8]-Ang II in the absence and presence of 1 M unlabeled [Sar 1–Ile 8]-Ang II. Nonspecific binding was below 15% of the total binding. The dissociation constant (Kd) for [Sar 1–Ile 8]-Ang II binding was determined by Scatchard analysis. Inositol trisphosphate (InsP 3) determination. CHO-K1 cells transfected with mutated AT 1A cDNA were grown in 35 mm dishes. Confluent cells were washed with 1.0 ml of 20 mM HEPES buffer, preincubated in 0.5 ml of 20 mM HEPES buffer containing 0.1% bovine serum albumin (BSA) for 20 min at 37°C, then in 0.5 ml of HEPES buffer with 0.1% BSA and 10 mM LiCl for 10 min. Cells were then incubated in the same HEPES buffer with or without 1 M Ang II for 10 s at 37°C. Generated InsP 3 was extracted with a 1.5 ml mixture of chloroform/methanol/12N-HCl (1/2/0.05, V/V). A 0.4 ml mixture of chloroform and distilled water (1:1, V/V) was added to the extract, and centrifuged. The supernatant was washed with 0.8 ml of chloroform, and centrifuged. The supernatant was dried in a vacuum centrifuge (Speed vac). The dried extract was redissolved with 150 l of distilled water, sonicated for 30 min, and centrifuged. InsP 3 in 100 l of the supernatant was measured by competitive receptor binding using an InsP 3 assay kit (NEN). Effect of GTP␥S on Ang II binding. Transfected CHO-K1 cells were grown in 10 cm dishes, washed with HBSS, scraped, and collected by centrifugation at 1500 ⫻ g for 5 min. The plasma membrane fraction was prepared by the published method (18). Membranes thus obtained were suspended to a protein concentration of 250 g/ml in 50 mM Tris buffer (pH 7.4) containing 200 mM NaCl, 10 mM MgCl 2, 1 mM EDTA, 0.1% BSA and 100 g/ml phenylmethanesulfonyl fluoride, and used as the membrane preparation. Suspended membranes were incubated with 0.1 nM 125I-Ang II at 37°C for 60 min in the presence of varying concentrations of GTP␥S. The membrane-bound radioligand was separated from the free radioligand by filtration over glass filters (GF/B) using a cell harvester (Millipore). Radioactivity was measured in a ␥ counter. Measurement of proton secretion. CHO-K1 cells expressing wildtype and mutant receptors were seeded in 12-mm diameter disposable polycarbonate cell capsules (Molecular Devices, Germany) at a cell density of 7.5 ⫻ 10 4 cells/well and cultured for 12 h in F-12
medium containing 10% FCS and 200 g/ml G418. The cell capsules were then loaded into a cytosensor microphysiometer (Molecular Devices), in which a light addressable potentiometric sensor continuously measured the rate at which the cells acidified their environment. The measuring chambers of the microphysiometer were intermittently perfused by peristaltic pumps with a medium of low buffer concentration (0.92 mM phosphate) to increase the sensitivity of the system. A pumping cycle of 120 s consisted of a flow period of 80 s, followed by a flow-off period of 40 s. During flow-off periods, protons released from CHO-K1 cells accumulated in the measuring chamber and the rate of proton release was quantified by fitting the sensor data to a straight standard line by the least-squares procedure. The slope of this line represents the acidification rate. Numerically, a slope of 1 V/s is close to 1 mpH unit/min. At the end of a measurement period, flow was resumed and the next pumping cycle began, washing out the protons that had accumulated in the previous measuring cycle. Each measurement chamber was continuously supplied with two alternating media (low buffered RPMI including 10% FCS with or without 10 ⫺7 M of Ang II) and a switch from one to the other was done by an electromagnetic valve at a flow rate of 100 l/min. Statistical analysis. Statistical analysis of the results of proton egression study was performed by unpaired Student’s t test.
RESULTS Binding Affinity of Mutant Receptors As shown in Table 1 the dissociation constants (Kd) and the Bmax of the wild type AT 1A and its mutants determined by Scatchard analysis were very similar, indicating that the specific ligand binding affinity of the mutants was unaltered. Mut DRY-GGA stands for the triple mutant, D 125–R 126–Y 127 3 G 125–G 126–A 127, and is the one we reported previously (3). Effects of a Stable GTP Analog As shown in Fig. 2 and 3, binding of 125I-Ang II to wild type AT 1A, Mut R126A, Mut R126G, and Mut Y127A was dose-dependently decreased by GTP␥S, whereas the effects of GTP␥S (shift from a high affinity state to a low affinity form) were practically abolished in Mut D125A, Mut D125G, and Mut DRY-GGA.
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Binding Affinity of [Sar –Ile 8]-Ang II Binding for Rat AT 1A Wild Type Receptor and Mutant Receptors
Wild type Mut D125A Mut D125G Mut R126A Mut R126G Mut Y127A Mut DRY-GGA
Kd (nM)
Bmax (fmol/5 ⫻ 10 5 cells)
1.8 ⫾ 0.2 2.2 ⫾ 0.2 2.3 ⫾ 0.2 2.0 ⫾ 0.1 2.4 ⫾ 0.3 2.3 ⫾ 0.3 2.4 ⫾ 0.2
9.2 ⫾ 1.0 9.7 ⫾ 1.0 10.5 ⫾ 1.1 9.9 ⫾ 1.0 8.2 ⫾ 1.2 10.6 ⫾ 1.4 9.5 ⫾ 1.3
Note. Data represent results of three identical series of binding isotherms followed by Scatchard analysis. Results are presented as mean ⫾ SD.
Effects on InsP 3 Formation
FIG. 3. Effects of the stable GTP analog GTP␥S on 125I-Ang II binding to membrane preparations from CHO-K1 cells expressing wild type AT 1A and its mutants. Each point represents the mean ⫾ SD obtained from three separate experiments.
Binding of Ang II to AT 1A activates a PLC via G q resulting in rapid stimulation of InsP 3 formation. Thus, increased InsP 3 formation by Ang II can be considered to indicate effective coupling to G q of the mutants. In wild type AT 1A, InsP 3 concentration was significantly increased from 0.80 ⫾ 0.5 pmol/dish of unstimulated control to 6.5 ⫾ 1.0 pmol/dish in 10 s during Ang II stimulation. Similar results were obtained in Mut Y127A. By contrast, in Mut D125A (G), Mut R126A (G), and Mut DRY-GGA responses of InsP 3 production to Ang II stimulation were markedly reduced compared to wild type AT 1A (Fig. 4).
with Ang II (10 ⫺9 to 10 ⫺6 M) evoked a transient (2–3min) ⫹30% elevation in the rate of acidification (Fig. 5). In Mut R126A and Mut Y127A, stimulation with 10 ⫺7 of Ang II significantly increased the rates of acid secretion and the maximum rates were approximately ⫹15 to ⫹20%. By contrast, in CHO-K1 cells not expressing AT 1A or expressing Mut D125A, the rates of acid release were not influenced by the presence of Ang II (Figs. 6 and 7). These results are summarized and presented in Table 2.
Effects on Proton Egress
DISCUSSION
The acidification rate of the extracellular incubation medium in the cytosensor microphysiometer was typically in the range of 20 –200 mV/s. Time-courses of acidification and the maximum acidification rates are shown in Fig. 5 to 7. In wild type AT 1A, stimulation
The present study addresses the functional role of the conserved triplet amino acid sequence Asp 125– Arg 126–Tyr 127 in rat AT 1A. Specific roles of the DRY sequence have been elusive and no universally recognized common denominator of its actions has been identified.
FIG. 2. Effects of the stable GTP analog GTP␥S on 125I-Ang II binding to membrane preparations from CHO-K1 cells expressing wild type AT 1A and its alanine mutants. Each point represents the mean ⫾ SD obtained from three separate experiments.
FIG. 4. Increment of inositol trisphosphate (InsP 3) produced upon stimulation of transfected CHO-K1 cells by 1 M of Ang II. Each point represents the mean ⫾ SD obtained from three separate experiments in triplicate.
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FIG. 5. Time-course changes in acidification rate produced upon stimulation of transfected CHO-K1 cells by various concentration of Ang II.
We have constructed substitutional mutants of each three residues in Asp–Arg–Tyr in AT 1A, and showed diversity of actions of these residues by measuring three different functional responses. The measurement of acidification rate by the cytosensor microphysiometer has been shown to be a reliable method in experiment using transfected CHO cells expressing NK 3 receptor (19) and secretin receptor (20). The advantage of this system is that the cellular response to ligand stimulation can be promptly registered and continuously monitored in real-time. Mut D125A had no responses in three assays on GTP effect on agonist binding, InsP 3 production, and effect of proton egression causing extracellular acidification. Additionally, Mut D125G and Mut DRY-GGA had no responses on GTP effect and InsP 3 production, suggesting that Asp 125 is essential for G-protein dependent and independent intracellular signal transduction in AT 1A. Previous mutagenesis studies demonstrated that the Asp residue of the DRY triplets in the G s-coupled 2adrenergic (5), G q-coupled m1-muscarinic (7), and G i-
FIG. 7. Maximum increment of acidification rate produced upon stimulation of nontransfected and transfected CHO-K1 cells by 100 nM of Ang II. Each bar represents the mean ⫾ SD of three independent experiments. # denotes p ⬍ 0.05 vs untransfected CHO-K1 cells.
coupled ␣2A-adrenergic (8) receptors and corresponding acidic residue Glu in rhodopsin (21) were essential for activation of the respective effectors. Moreover, constitutive activation of the Asp-mutated receptors was reported in histamine H (2) receptor (9) and 2adrenergic receptor (6). The present findings that D125A and D125G mutants impair coupling to G q are in general agreement with these data indicating importance of Asp in DRY motif on receptor activation. On the other hand, mutation of Asp of the DRY motif in the GnRH receptor did not impair signal transduction (10). Thus the importance of the acidic residue of the acidic-arginine-aromatic triplet in ICL2 for signal transduction seems to vary from receptor to receptor. Effects of the variation do not appear to depend on the type of the coupled G-protein. In our previous study (4, 22), synthetic peptide with an amino acid sequence corresponding to the N-terminal portion of ICL2 including the DRY motif of rat AT 1A (P-1 in Fig. 1) did not activate G q (Fig. 8), G i and G o (22) in contrast to P-3 in ICL3 and P-5 in the cytoplasmic C-terminal region. These data indicate that the DRY motif alone is not sufficient to activate G-proteins directly, but is necessary presumably playing a role in G-protein binding (6). TABLE 2
Summary of the Results
Wild type Mut D125A Mut D125G Mut R126A Mut R126G Mut Y127A Mut DRY-GGA CHO-K1 FIG. 6. Time-course changes in acidification rate produced upon stimulation of transfected CHO-K1 cells by 100 nM of Ang II. 365
GTP effect
InsP 3 production
Acidification
⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺
⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺
⫹ ⫺ ND ⫹ ND ⫹ ND ⫺
Note. ND: not determined.
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FIG. 8. Effects of synthetic peptides on [ 35S] GTP␥S binding to G q protein. Purified G q (10 nM) was incubated with 100 nM of [ 35S] GTP␥S at room temperature in the absence (control) or presence of a synthetic peptide 100 M for 10 min. A 50-l aliquot of reaction mixture was subjected to the analysis of [ 35S] GTP␥S binding. The basal binding (control) was obtained as [ 35S] GTP␥S bound to G q in the absence of a synthetic peptide. The 100% (maximum) binding is set as [ 35S] GTP␥S binding in the presence of 2.5 mM Mg 2⫹. Each bar represents the mean ⫾ SD of three independent experiments (4, 22).
Mut R126A and R126G underwent a shift to the low-affinity form in response to the stable analog of GTP, GTP␥S and increased the rate of medium acidification by Ang II. However, they did not product InsP 3. These results suggest that Arg 126 in the DRY motif is essential for G q/11␣-coupled activation of phospholipase C but not for coupling with other G-proteins, G o and G i. The Arg 126 residue in the DRY motifs of the G 1/11␣-coupled receptors, GnRH receptor (10), m1muscarinic receptor (11), and N-formyl peptide receptor (23), was reported to be essential for the agonistinduced activation. In a recent report, mutation of R108A in A (3) adenosine receptor was found to be constitutively active, and the degree of the activation was more pronounced for the adenylate cyclase signaling pathway than phospholypase C pathway (13). These results suggest that the Arg in DRY motif is essential for G-protein coupling, mainly G q-protein. Although all of these receptors are coupled to G q, the DRY motif is preserved widely in G s and G i coupled receptors. R126G mutant of N-formyl peptide receptor was found not to bind G-protein but to become phosphorylated and subsequently internalized (24). Thus, the importance of Arg in DRY motif for signal transduction seems to vary from receptor to receptor as well as Asp residue. Mut Y127A had similar responses as wild type AT 1A receptor in the present study; Mut Y127A was able to produce InsP 3 and acidify medium comparable with the wild type. Mutation of the Tyr residue in the DRY motif of m1-muscarinic receptor (11) or Ser residue in the DRS motif of GnRH receptor (10) did not impair signal transduction. These concordant results indicate that Tyr 127 in the DRY motif is not important for G q coupling.
Using chimeric human AT 1 receptor mutant Wang et al. reported that the seven amino acid residues 219 – 225 in the N-terminal portion of ICL3 were important for G q coupling (25). Hunyady et al. reported using deletional mutants of ICL3 of rat AT 1A that the N-terminal half of the ICL3 (amino acid residues 215– 226) was an essential region of G q coupling (26). In our previous study (4, 22), a synthetic peptide corresponding to the N-terminal portion of ICL3 of rat AT 1A also elicited an activation of G q, G o, G i1, and G i2. Likewise, a peptide with the structure of the N-terminal portion of the cytoplasmic-tail in rat AT 1A activated G q, G o, G i1, and G i2. Using site-directed mutagenesis, we also reported that the C-terminal portion of ICL2, the C-terminal region of ICL3, and the N-terminal portion of cytosolic-tail were involved in G protein coupling (3, 4). Cloning studies on receptors belonging to the G protein coupled superfamily has revealed that most of the receptors for peptidic ligands possess relatively short ICL3 as compared with those for small ligands (27). It seems that many members of the G protein coupled receptor superfamily with short ICL3 require more than one intracellular region for optimal G protein binding and activation. Site-directed mutagenesis studies of the -adrenergic receptor defined that amino acids in the carboxyl-terminal portion of the ICL3 and the amino-terminal region of the cytoplasmic tail proximal to the seventh transmembarne domain were essential for G S activation (28). Probst et al. speculated that regions of ICL3 and the carboxyl terminus anchored to plasma membrane by a cysteine residue in the C-terminal tail, adjacent to the seventh transmembrane domain, may form clustered amphipathic ␣-helices (27). These helices, along with charged intracellular loops (i.e., DRY in ICL2), may cooperatively interact in efficient binding to and activation of G proteins. The activation of G q in AT 1A seems to require not only ICL3 and the N-terminal portion in the C-tail but also Asp–Arg in ICL2. In summary, the present study presents evidence that a G q coupling site in AT 1A receptor should be Asp 125 and Arg 126 in ICL2 but not Tyr 127. ACKNOWLEDGMENTS We thank the Japanese Cancer Research Resources Bank (JCRB)Cell for the gift of CHO-K1 cells. This study was supported in part by NOVARTIS Foundation (Japan) for the Promotion of Science, the Novo Nordisk Fund for Pediatric Study Group of Molecular Endocrinology, and the Child Health Support Association in Japan.
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Role of the Conserved DRY Motif on G Protein Activation of Rat Angiotensin II Receptor Type 1A Kenji Ohyama,* ,1 Yoshiaki Yamano,† Tomoaki Sano,* Yoshiko Nakagomi,* Manabu Wada,‡ and Tadashi Inagami§ *Department of Clinical Nursing and Pediatrics, Yamanashi Medical University, Yamanashi 409-3898, Japan; †Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan; ‡JCR Pharmaceuticals, Hyogo 651-2241, Japan; and §Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received February 25, 2002
To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp–Arg–Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT 1A) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP 3 production, and the acidification of the medium in response to Ang II were determined. The effects of GTP␥S on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP 3 production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP 3 and acidify medium comparable with wild type AT 1A. These results indicate as follows; Asp 125 is essential for intracellular signal transduction involving G protein coupling, Arg 126 is essential for coupling of G q protein but not other G proteins, and Tyr 127 is not important for G protein coupling. © 2002 Elsevier Science (USA)
Key Words: angiotensin II; angiotensin II receptor; site-directed mutagenesis; signal transduction; extracellular acidification; G protein; G protein-coupled receptor; IP 3.
Angiotensin II (Ang II) receptor type 1A (AT 1A) has seven putative transmembrane domains and is a G protein-coupled receptor (1, 2). We previously reported that the highly conserved triplet of amino acid sequence Asp–Arg–Tyr (DRY) of the second intracellular 1 To whom correspondence should be addressed at Department of Clinical Nursing and Pediatrics, Yamanashi Medical University, 1110, Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, Japan. Fax: ⫹81-55-273-6605. E-mail: [email protected].
0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
loop (ICL2) and YFL of the cytosolic C-tail region were essential for G q coupling (3, 4). Importance of DRY for G-protein activation has been reported in several G-protein coupled receptors. For example, mutagenesis studies demonstrated that Asp residue in the  2adrenergic (5, 6), m1-muscarinic (7), and ␣2Aadrenergic (8) receptors was essential for activation of the respective effectors. The Arg residue in the V 2 vasopressin receptor (9), gonadotropin-releasing hormone (GnRH) receptor (10), and m1-muscarinic receptor (11) was important for the agonist-induced activation. On the other hand, mutation of the Tyr residue in the DRY motif of m1-muscarinic receptor (11) or the Ser residue in DRS motif of the GnRH receptor (10, 12) did not impair signal transduction. Recently, constitutive activation of receptors was reported in several mutant receptors with substitution of DRY motif (6, 13–15). Moreover, agonist binding affinity was reduced by Asp mutation (D130A) of CB 2 cannabinoid receptor (16) and was increased by Arg mutation (R131H) of  2-adrenergic receptor (9). In the present study, we focused on the triplet of amino acid sequence, Asp 125– Arg 126–Tyr 127 in ICL2 of the rat AT 1A. By constructing five substitutional mutants of rat AT 1A, we examined their ligand binding, InsP 3 production, and proton egression in response to Ang II with the aim of defining regions essential for the coupling and activation of G-proteins. MATERIALS AND METHODS Rat AT 1A mutagenesis. Site directed mutagenesis was performed by the procedure of Kunkel (17). Sites of substitution are shown in Fig. 1. Wild type AT 1A and its mutant cDNA in the expression vector pCDNA1 were transfected into and stably expressed in CHO-K1 cells. Binding assay. AT 1A-expressing CHO-K1 cells were grown in Ham’s F12 medium with 10% FCS in 24-well plates. They were washed with Hank’s balanced salt solution (HBSS) and incubated for
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FIG. 1. Schematic representation of the rat AT 1A receptor. The illustration shows the amino acid sequence in the N-terminal portion of the second intracellular loop. The sites of substitution are indicated by outline font.
90 min at 37°C in 250 l of Ham’s F12 medium with 2% FCS. The cells were incubated with [ 125I-Sar 1–Ile 8]-Ang II (DuPont) in varying concentrations from 0.3 nM to 10 nM in this medium for determination of the specific binding. After the incubation, cells were immediately placed on ice, washed three times with ice-cold HBSS, then solubilized with 250 l of 0.5 N NaOH. Radioactivity was measured in a ␥ counter. Specific [Sar 1–Ile 8]-Ang II binding was determined as a difference between the total binding of [ 125I-Sar 1–Ile 8]-Ang II in the absence and presence of 1 M unlabeled [Sar 1–Ile 8]-Ang II. Nonspecific binding was below 15% of the total binding. The dissociation constant (Kd) for [Sar 1–Ile 8]-Ang II binding was determined by Scatchard analysis. Inositol trisphosphate (InsP 3) determination. CHO-K1 cells transfected with mutated AT 1A cDNA were grown in 35 mm dishes. Confluent cells were washed with 1.0 ml of 20 mM HEPES buffer, preincubated in 0.5 ml of 20 mM HEPES buffer containing 0.1% bovine serum albumin (BSA) for 20 min at 37°C, then in 0.5 ml of HEPES buffer with 0.1% BSA and 10 mM LiCl for 10 min. Cells were then incubated in the same HEPES buffer with or without 1 M Ang II for 10 s at 37°C. Generated InsP 3 was extracted with a 1.5 ml mixture of chloroform/methanol/12N-HCl (1/2/0.05, V/V). A 0.4 ml mixture of chloroform and distilled water (1:1, V/V) was added to the extract, and centrifuged. The supernatant was washed with 0.8 ml of chloroform, and centrifuged. The supernatant was dried in a vacuum centrifuge (Speed vac). The dried extract was redissolved with 150 l of distilled water, sonicated for 30 min, and centrifuged. InsP 3 in 100 l of the supernatant was measured by competitive receptor binding using an InsP 3 assay kit (NEN). Effect of GTP␥S on Ang II binding. Transfected CHO-K1 cells were grown in 10 cm dishes, washed with HBSS, scraped, and collected by centrifugation at 1500 ⫻ g for 5 min. The plasma membrane fraction was prepared by the published method (18). Membranes thus obtained were suspended to a protein concentration of 250 g/ml in 50 mM Tris buffer (pH 7.4) containing 200 mM NaCl, 10 mM MgCl 2, 1 mM EDTA, 0.1% BSA and 100 g/ml phenylmethanesulfonyl fluoride, and used as the membrane preparation. Suspended membranes were incubated with 0.1 nM 125I-Ang II at 37°C for 60 min in the presence of varying concentrations of GTP␥S. The membrane-bound radioligand was separated from the free radioligand by filtration over glass filters (GF/B) using a cell harvester (Millipore). Radioactivity was measured in a ␥ counter. Measurement of proton secretion. CHO-K1 cells expressing wildtype and mutant receptors were seeded in 12-mm diameter disposable polycarbonate cell capsules (Molecular Devices, Germany) at a cell density of 7.5 ⫻ 10 4 cells/well and cultured for 12 h in F-12
medium containing 10% FCS and 200 g/ml G418. The cell capsules were then loaded into a cytosensor microphysiometer (Molecular Devices), in which a light addressable potentiometric sensor continuously measured the rate at which the cells acidified their environment. The measuring chambers of the microphysiometer were intermittently perfused by peristaltic pumps with a medium of low buffer concentration (0.92 mM phosphate) to increase the sensitivity of the system. A pumping cycle of 120 s consisted of a flow period of 80 s, followed by a flow-off period of 40 s. During flow-off periods, protons released from CHO-K1 cells accumulated in the measuring chamber and the rate of proton release was quantified by fitting the sensor data to a straight standard line by the least-squares procedure. The slope of this line represents the acidification rate. Numerically, a slope of 1 V/s is close to 1 mpH unit/min. At the end of a measurement period, flow was resumed and the next pumping cycle began, washing out the protons that had accumulated in the previous measuring cycle. Each measurement chamber was continuously supplied with two alternating media (low buffered RPMI including 10% FCS with or without 10 ⫺7 M of Ang II) and a switch from one to the other was done by an electromagnetic valve at a flow rate of 100 l/min. Statistical analysis. Statistical analysis of the results of proton egression study was performed by unpaired Student’s t test.
RESULTS Binding Affinity of Mutant Receptors As shown in Table 1 the dissociation constants (Kd) and the Bmax of the wild type AT 1A and its mutants determined by Scatchard analysis were very similar, indicating that the specific ligand binding affinity of the mutants was unaltered. Mut DRY-GGA stands for the triple mutant, D 125–R 126–Y 127 3 G 125–G 126–A 127, and is the one we reported previously (3). Effects of a Stable GTP Analog As shown in Fig. 2 and 3, binding of 125I-Ang II to wild type AT 1A, Mut R126A, Mut R126G, and Mut Y127A was dose-dependently decreased by GTP␥S, whereas the effects of GTP␥S (shift from a high affinity state to a low affinity form) were practically abolished in Mut D125A, Mut D125G, and Mut DRY-GGA.
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Binding Affinity of [Sar –Ile 8]-Ang II Binding for Rat AT 1A Wild Type Receptor and Mutant Receptors
Wild type Mut D125A Mut D125G Mut R126A Mut R126G Mut Y127A Mut DRY-GGA
Kd (nM)
Bmax (fmol/5 ⫻ 10 5 cells)
1.8 ⫾ 0.2 2.2 ⫾ 0.2 2.3 ⫾ 0.2 2.0 ⫾ 0.1 2.4 ⫾ 0.3 2.3 ⫾ 0.3 2.4 ⫾ 0.2
9.2 ⫾ 1.0 9.7 ⫾ 1.0 10.5 ⫾ 1.1 9.9 ⫾ 1.0 8.2 ⫾ 1.2 10.6 ⫾ 1.4 9.5 ⫾ 1.3
Note. Data represent results of three identical series of binding isotherms followed by Scatchard analysis. Results are presented as mean ⫾ SD.
Effects on InsP 3 Formation
FIG. 3. Effects of the stable GTP analog GTP␥S on 125I-Ang II binding to membrane preparations from CHO-K1 cells expressing wild type AT 1A and its mutants. Each point represents the mean ⫾ SD obtained from three separate experiments.
Binding of Ang II to AT 1A activates a PLC via G q resulting in rapid stimulation of InsP 3 formation. Thus, increased InsP 3 formation by Ang II can be considered to indicate effective coupling to G q of the mutants. In wild type AT 1A, InsP 3 concentration was significantly increased from 0.80 ⫾ 0.5 pmol/dish of unstimulated control to 6.5 ⫾ 1.0 pmol/dish in 10 s during Ang II stimulation. Similar results were obtained in Mut Y127A. By contrast, in Mut D125A (G), Mut R126A (G), and Mut DRY-GGA responses of InsP 3 production to Ang II stimulation were markedly reduced compared to wild type AT 1A (Fig. 4).
with Ang II (10 ⫺9 to 10 ⫺6 M) evoked a transient (2–3min) ⫹30% elevation in the rate of acidification (Fig. 5). In Mut R126A and Mut Y127A, stimulation with 10 ⫺7 of Ang II significantly increased the rates of acid secretion and the maximum rates were approximately ⫹15 to ⫹20%. By contrast, in CHO-K1 cells not expressing AT 1A or expressing Mut D125A, the rates of acid release were not influenced by the presence of Ang II (Figs. 6 and 7). These results are summarized and presented in Table 2.
Effects on Proton Egress
DISCUSSION
The acidification rate of the extracellular incubation medium in the cytosensor microphysiometer was typically in the range of 20 –200 mV/s. Time-courses of acidification and the maximum acidification rates are shown in Fig. 5 to 7. In wild type AT 1A, stimulation
The present study addresses the functional role of the conserved triplet amino acid sequence Asp 125– Arg 126–Tyr 127 in rat AT 1A. Specific roles of the DRY sequence have been elusive and no universally recognized common denominator of its actions has been identified.
FIG. 2. Effects of the stable GTP analog GTP␥S on 125I-Ang II binding to membrane preparations from CHO-K1 cells expressing wild type AT 1A and its alanine mutants. Each point represents the mean ⫾ SD obtained from three separate experiments.
FIG. 4. Increment of inositol trisphosphate (InsP 3) produced upon stimulation of transfected CHO-K1 cells by 1 M of Ang II. Each point represents the mean ⫾ SD obtained from three separate experiments in triplicate.
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FIG. 5. Time-course changes in acidification rate produced upon stimulation of transfected CHO-K1 cells by various concentration of Ang II.
We have constructed substitutional mutants of each three residues in Asp–Arg–Tyr in AT 1A, and showed diversity of actions of these residues by measuring three different functional responses. The measurement of acidification rate by the cytosensor microphysiometer has been shown to be a reliable method in experiment using transfected CHO cells expressing NK 3 receptor (19) and secretin receptor (20). The advantage of this system is that the cellular response to ligand stimulation can be promptly registered and continuously monitored in real-time. Mut D125A had no responses in three assays on GTP effect on agonist binding, InsP 3 production, and effect of proton egression causing extracellular acidification. Additionally, Mut D125G and Mut DRY-GGA had no responses on GTP effect and InsP 3 production, suggesting that Asp 125 is essential for G-protein dependent and independent intracellular signal transduction in AT 1A. Previous mutagenesis studies demonstrated that the Asp residue of the DRY triplets in the G s-coupled 2adrenergic (5), G q-coupled m1-muscarinic (7), and G i-
FIG. 7. Maximum increment of acidification rate produced upon stimulation of nontransfected and transfected CHO-K1 cells by 100 nM of Ang II. Each bar represents the mean ⫾ SD of three independent experiments. # denotes p ⬍ 0.05 vs untransfected CHO-K1 cells.
coupled ␣2A-adrenergic (8) receptors and corresponding acidic residue Glu in rhodopsin (21) were essential for activation of the respective effectors. Moreover, constitutive activation of the Asp-mutated receptors was reported in histamine H (2) receptor (9) and 2adrenergic receptor (6). The present findings that D125A and D125G mutants impair coupling to G q are in general agreement with these data indicating importance of Asp in DRY motif on receptor activation. On the other hand, mutation of Asp of the DRY motif in the GnRH receptor did not impair signal transduction (10). Thus the importance of the acidic residue of the acidic-arginine-aromatic triplet in ICL2 for signal transduction seems to vary from receptor to receptor. Effects of the variation do not appear to depend on the type of the coupled G-protein. In our previous study (4, 22), synthetic peptide with an amino acid sequence corresponding to the N-terminal portion of ICL2 including the DRY motif of rat AT 1A (P-1 in Fig. 1) did not activate G q (Fig. 8), G i and G o (22) in contrast to P-3 in ICL3 and P-5 in the cytoplasmic C-terminal region. These data indicate that the DRY motif alone is not sufficient to activate G-proteins directly, but is necessary presumably playing a role in G-protein binding (6). TABLE 2
Summary of the Results
Wild type Mut D125A Mut D125G Mut R126A Mut R126G Mut Y127A Mut DRY-GGA CHO-K1 FIG. 6. Time-course changes in acidification rate produced upon stimulation of transfected CHO-K1 cells by 100 nM of Ang II. 365
GTP effect
InsP 3 production
Acidification
⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺
⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺
⫹ ⫺ ND ⫹ ND ⫹ ND ⫺
Note. ND: not determined.
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FIG. 8. Effects of synthetic peptides on [ 35S] GTP␥S binding to G q protein. Purified G q (10 nM) was incubated with 100 nM of [ 35S] GTP␥S at room temperature in the absence (control) or presence of a synthetic peptide 100 M for 10 min. A 50-l aliquot of reaction mixture was subjected to the analysis of [ 35S] GTP␥S binding. The basal binding (control) was obtained as [ 35S] GTP␥S bound to G q in the absence of a synthetic peptide. The 100% (maximum) binding is set as [ 35S] GTP␥S binding in the presence of 2.5 mM Mg 2⫹. Each bar represents the mean ⫾ SD of three independent experiments (4, 22).
Mut R126A and R126G underwent a shift to the low-affinity form in response to the stable analog of GTP, GTP␥S and increased the rate of medium acidification by Ang II. However, they did not product InsP 3. These results suggest that Arg 126 in the DRY motif is essential for G q/11␣-coupled activation of phospholipase C but not for coupling with other G-proteins, G o and G i. The Arg 126 residue in the DRY motifs of the G 1/11␣-coupled receptors, GnRH receptor (10), m1muscarinic receptor (11), and N-formyl peptide receptor (23), was reported to be essential for the agonistinduced activation. In a recent report, mutation of R108A in A (3) adenosine receptor was found to be constitutively active, and the degree of the activation was more pronounced for the adenylate cyclase signaling pathway than phospholypase C pathway (13). These results suggest that the Arg in DRY motif is essential for G-protein coupling, mainly G q-protein. Although all of these receptors are coupled to G q, the DRY motif is preserved widely in G s and G i coupled receptors. R126G mutant of N-formyl peptide receptor was found not to bind G-protein but to become phosphorylated and subsequently internalized (24). Thus, the importance of Arg in DRY motif for signal transduction seems to vary from receptor to receptor as well as Asp residue. Mut Y127A had similar responses as wild type AT 1A receptor in the present study; Mut Y127A was able to produce InsP 3 and acidify medium comparable with the wild type. Mutation of the Tyr residue in the DRY motif of m1-muscarinic receptor (11) or Ser residue in the DRS motif of GnRH receptor (10) did not impair signal transduction. These concordant results indicate that Tyr 127 in the DRY motif is not important for G q coupling.
Using chimeric human AT 1 receptor mutant Wang et al. reported that the seven amino acid residues 219 – 225 in the N-terminal portion of ICL3 were important for G q coupling (25). Hunyady et al. reported using deletional mutants of ICL3 of rat AT 1A that the N-terminal half of the ICL3 (amino acid residues 215– 226) was an essential region of G q coupling (26). In our previous study (4, 22), a synthetic peptide corresponding to the N-terminal portion of ICL3 of rat AT 1A also elicited an activation of G q, G o, G i1, and G i2. Likewise, a peptide with the structure of the N-terminal portion of the cytoplasmic-tail in rat AT 1A activated G q, G o, G i1, and G i2. Using site-directed mutagenesis, we also reported that the C-terminal portion of ICL2, the C-terminal region of ICL3, and the N-terminal portion of cytosolic-tail were involved in G protein coupling (3, 4). Cloning studies on receptors belonging to the G protein coupled superfamily has revealed that most of the receptors for peptidic ligands possess relatively short ICL3 as compared with those for small ligands (27). It seems that many members of the G protein coupled receptor superfamily with short ICL3 require more than one intracellular region for optimal G protein binding and activation. Site-directed mutagenesis studies of the -adrenergic receptor defined that amino acids in the carboxyl-terminal portion of the ICL3 and the amino-terminal region of the cytoplasmic tail proximal to the seventh transmembarne domain were essential for G S activation (28). Probst et al. speculated that regions of ICL3 and the carboxyl terminus anchored to plasma membrane by a cysteine residue in the C-terminal tail, adjacent to the seventh transmembrane domain, may form clustered amphipathic ␣-helices (27). These helices, along with charged intracellular loops (i.e., DRY in ICL2), may cooperatively interact in efficient binding to and activation of G proteins. The activation of G q in AT 1A seems to require not only ICL3 and the N-terminal portion in the C-tail but also Asp–Arg in ICL2. In summary, the present study presents evidence that a G q coupling site in AT 1A receptor should be Asp 125 and Arg 126 in ICL2 but not Tyr 127. ACKNOWLEDGMENTS We thank the Japanese Cancer Research Resources Bank (JCRB)Cell for the gift of CHO-K1 cells. This study was supported in part by NOVARTIS Foundation (Japan) for the Promotion of Science, the Novo Nordisk Fund for Pediatric Study Group of Molecular Endocrinology, and the Child Health Support Association in Japan.
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