INTRODUCTION

Angiotensin II (Ang II)¹ receptor type 1A (AT_1A) is a seven-transmembrane, G protein-coupled receptor (1, 2). Ang II activates G_q, G_i, and G_o proteins through the AT_1A receptor (3-5). The GTP·G_q complex stimulates phospholipase C (PLC) resulting in inositol trisphosphate (InsP₃) generation (6, 7).

Not a single consensus structure has yet been identified within the G protein-coupled superfamily that uniquely defines the G protein binding function. Mutagenesis studies on adrenergic receptors and rhodopsin indicate that the C-terminal domains of the third intracellular loop (ICL3) and the N-terminal region of the cytosolic tail are essential for coupling to G proteins (8-10). By contrast, the regions of the thyrotropin receptor essential for intracellular signal transduction appear to be the first intracellular loop (ICL1) and the C-terminal regions of the second intracellular loop (ICL2) and the third intracellular loop (ICL3) (11). Four isoforms of prostaglandin E receptor subtype EP3, which differ only at their C-terminal tails and are produced by alternative splicing, couple to different G proteins. Thus the C-terminal tail of EP3 determines G protein specificity (12).

Studies by Wang et al. (13) using chimeras of human AT₁ and AT₂ suggested that the N-terminal portion of ICL3 was important for G_q coupling. We reported observations suggesting that the acidic-arginine-aromatic (DRY) triplet of ICL2, the C-terminal portion of ICL2, the C-terminal region of ICL3, and the cytosolic C-terminal tail region were involved in G protein coupling. Our data from transient transfection of the AT_1A receptor in COS7 cells showed that the last 50 amino acid residues (beyond Phe³⁰⁹) were also important for G_q coupling (14). Thomas et al. (15) reported that truncation of the last 45 amino acid residues of the rat AT_1A beyond Leu³¹⁴ was not important for efficient coupling to the G protein. Thus, we focused on the amino acid sequence between Lys³¹⁰ and Leu³¹⁴, constructed five deletional mutants and four substitutional mutants of rat AT_1A, and examined their InsP₃ production and ligand binding. We also synthesized nine peptides based on the amino acid sequence of the cytosolic region and examined their G_q activation with the aim of defining a region in the C-tail essential for the coupling to G_q in rat AT_1A.

MATERIALS AND METHODS

The entire coding region of rat kidney AT_1A was cloned into EcoRI site of a plasmid pUC19 (16). A KpnI-EcoRI fragment was subcloned into polylinker sites of the plasmid vector pBluescript II KS+, and single-stranded DNA was prepared using helper phage R 408 (Stratagene). Site-directed mutagenesis was performed by the procedure of Kunkel (17). Sites of truncation and substitution are shown in Fig. 1. The mutated DNA sequences were confirmed by Sanger's dideoxynucleotide sequencing method (18). The mutated AT_1A cDNA was excised with enzymes BamHI and XhoI and introduced into the expression vector pCDNA1.

Forty µg of plasmid constructs containing the wild type or mutated rat AT_1A cDNA were co-transfected with 1 µg of pSV-G1-Neo (Green Cross Corp.) into 5 × 10⁶ of Chinese hamster ovary (CHO-K1) cells in 500 µl of phosphate buffer using a gene pulser (Bio-Rad). Native CHO-K1 cells do not express Ang II receptor.

Transfected CHO-K1 cells were cultured for 2 days in 10-cm dishes in Ham's F12 medium (Life Technologies, Inc.) containing 10% fetal calf serum. Then the culture medium was changed to selection medium containing 400 µg/ml Geneticin (G418, Life Technologies, Inc.). When individual colonies emerged 10-14 days after the transfection, 60 sufficiently separated colonies were isolated and inoculated into 200 µl of selection medium in 96-well plates. Each of these colonies was scaled up independently to 24-well plates, and the binding assay was performed using ¹²⁵I-Ang II (NEN Life Science Products). The binding assay was repeated three times for each clone. Two colonies were selected after the binding assay, and 300 single cells isolated from the colonies were cultured in the selection medium in 96-well plates. Two or three weeks later, each of these clones was scaled up independently to 24-well plates, and binding assay was performed again. The clone expressing the highest specific binding of ¹²⁵I-Ang II was selected.

AT_1A-expressing CHO-K1 cells were grown in Ham's F12 medium with 10% fetal calf serum in 24-well plates. They were washed with Hank's balanced salt solution and incubated for 90 min at 37 °C in 250 µl of Ham's F12 medium with 2% fetal calf serum. Varying concentrations of [¹²⁵I-Sar¹,Ile⁸]Ang II (NEN Life Science Products) from 0.3 to 10 nM were incubated in this medium for determination of the specific binding. After the incubation, cells were immediately placed on ice, washed three times with ice-cold Hanks' balanced salt solution, and then solubilized with 250 µl of 0.5 N NaOH. Radioactivity was measured by a gamma counter. Specific [Sar¹,Ile⁸]Ang II binding was determined as the difference between the total binding of [¹²⁵I-Sar¹,Ile⁸]Ang II in the absence and presence of 1 µM [Sar¹,Ile⁸]Ang II. Nonspecific binding was below 15% of the total binding. The dissociation constant (K_d) for [Sar¹,Ile⁸]Ang II binding was determined by Scatchard analysis.

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. Then the cells were incubated in the same HEPES buffer with or without 1 µM Ang II for 10 s at 37 °C. InsP₃ was extracted with a 1.5-ml mixture of chloroform/methanol, 12 N 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 Speedvac. The dried extract was redissolved with 150 µl of distilled water, sonicated for 30 min, and centrifuged. InsP₃ in 100 µl of the supernatant was measured by competitive receptor binding using an InsP₃ assay kit (NEN Life Science Products).

Effect of GTPS on Ang II Binding

Transfected CHO-K1 cells were grown in 10-cm dishes, washed with Hanks' balanced salt solution, scraped, and collected by centrifugation at 1500 × g for 5 min. The plasma membrane fraction was prepared by a published method (19). Membranes obtained were suspended at a protein concentration of 250 µg/ml in 50 mM Tris buffer (pH 7.4) containing 200 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 0.1% BSA, and 100 µg/ml phenylmethanesulfonyl fluoride and used as the membrane preparation.

Dose response was determined as follows. Suspended membranes were incubated with 0.1 nM ¹²⁵I-Ang II at 25 °C for 60 min in the presence of varying concentrations of GTPS. For studying the time course of ligand binding, membranes were incubated with 0.1 nM ¹²⁵I-Ang II for 60 min at 37 °C to attain binding equilibrium and unlabeled Ang II (1 µM) or GTPS (10 µM) or both of them were added to the mixture and incubated for another 60 min. 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 gamma counter.

The amino acid sequences of the peptides used in this study are shown in Fig. 1. They were synthesized by the solid-phase method and purified to 95-99% homogeneity by high performance liquid chromatography using a Nucleosil 5 C18 column eluted with a linear concentration gradient (0-60%) of CH₃CN containing 0.1% trifluoroacetic acid. The lyophilized synthetic peptide was dissolved in water. Heterotrimeric forms of G_q proteins from bovine liver were purified to homogeneity as published (20).

GTPS Binding Assay

[³⁵S]GTPS binding to 10 nM purified heterotrimeric G_q promoted by synthetic peptides was measured in 25 mM HEPES-NaOH buffer (pH 7.4) containing 120 µM MgCl₂, 100 µM EDTA, and 100 nM [³⁵S]GTPS in the absence of phospholipids as described by Okamoto et al. (9). Briefly, G_q was incubated at 37 °C for 10 min in the absence (control) or presence of a synthetic peptide (100 µM). The incubation was terminated by addition of 10 volumes of ice-cold stopping buffer containing 100 mM Tris-HCl (pH 8.0), 25 mM MgCl₂, 100 mM NaCl, and 20 µM GTP. After a 50-µl aliquot of the reaction mixture was rapidly filtered through a nitrocellulose filter (pore size, 0.45 µm) and washed three times with the stopping buffer, the filter was counted in a liquid scintillation counter. The maximal binding of [³⁵S]GTPS to G_q was measured in the presence of 1 µM GTPS and 25 mM Mg²⁺ at room temperature by the method of Northup et al. (21) as a positive control.

The results of experiments with the synthetic peptide study was examined by unpaired Student's t test. p values less than 0.05 were considered significant.

RESULTS

As shown in Table I the dissociation constants (K_d) and B_max values of the wild type AT_1A and its mutants determined by Scatchard analysis were similar, indicating that the mutants possessed similar ligand binding affinity and sites of comparable magnitude. In this study [¹²⁵I-Sar¹,Ile⁸]Ang II was used as ligand. Scatchard plots indicated single high affinity sites. When ¹²⁵I-Ang II was used as ligand results indicated similar single high affinity sites. The possible presence of low affinity sites was practically undetectable.

Table I. Binding affinity of [Sar1-Ile8]Ang II binding for rat AT1A wild type receptor and mutant receptors Data represent results of three identical series of binding isotherms followed by Scatchard analysis. Results are presented as means ± S.D. Kd Bmax nM fmol/mg protein Wild type 1.6  ± 0.1 18.8  ± 1.4 Mut 310-del 2.4  ± 0.2 16.0  ± 1.1 Mut 312-del 2.4  ± 0.2 17.8  ± 1.6 Mut 313-del 2.0  ± 0.3 15.2  ± 1.8 Mut 314-del 1.8  ± 0.2 15.6  ± 1.6 Mut 318-del 2.5  ± 0.3 17.8  ± 1.8 Mut Y312A 2.4  ± 0.4 16.6  ± 1.8 Mut F313A 2.6  ± 0.4 15.5  ± 1.9 Mut L314A 2.8  ± 0.3 14.8  ± 1.2 Mut K310,311Q 2.7  ± 0.3 18.3  ± 1.4

As shown in Fig. 2, the binding of ¹²⁵I-Ang II to wild type AT_1A, Mut 318-del, and Mut K310,311Q receptors were dose-dependently decreased by GTPS, whereas the effect of GTPS (shift from a high affinity state to a low affinity form) was practically abolished in Mut 310-del, Mut 312-del, Mut 313-del, Mut 314-del, Mut Y312A, Mut F313A, and Mut L314A receptors.

Effects of the stable GTP analog GTPS on ¹²⁵I-Ang II binding to membrane preparations expressing wild type AT_1A and its mutants.

Time-related changes in dissociation of ¹²⁵I-Ang II from the wild type and mutated receptors are shown in Fig. 3. The binding of ¹²⁵I-Ang II to the receptors in membrane preparations reached a plateau in 60 min. In wild type AT_1A and all of its mutants, the receptor-bound ¹²⁵I-Ang II was displaced by 1 µM unlabeled Ang II to similar extents (the range of half-life time of dissociation was 19.5 to 21.4 min). GTPS (10 µM) markedly shortened the half-life time of the spontaneous dissociation in the wild type AT_1A, and Mut 318-del, and Mut-K310,311Q receptors (3.5 to 4.6 min), whereas the binding of ¹²⁵I-Ang II remained unchanged for 60 min in Mut 310-del, Mut 312-del, Mut 313-del, and Mut 314-del. Moreover, although the half-life times in the wild type AT_1A and Mut 318-del were shortened in the presence of both Ang II and GTPS (0.9 to 1.2 min), those in other deletion mutants were similar to the half-life time in the presence of Ang II alone (17.0 to 20.1 min).

Time course study on the dissociation of ¹²⁵I-Ang II binding in the presence of excessive concentration of Ang II and/or GTPS in the wild type AT_1A (a) and its mutants (b, Mut 310-del; c, Mut 312-del, d, Mut 313-del; e, Mut 314-del; and f, Mut 318-del).

Binding of Ang II to AT_1A activates a PLC via G_q resulting in stimulation of InsP₃ formation. Thus, increased InsP₃ formation by Ang II can be considered to indicate effective coupling to G_q of the mutants. In unmutated AT_1A, InsP₃ production was significantly increased from 2.52 ± 0.05 pmol/dish of unstimulated control to 16.55 ± 1.88 pmol/dish at 10 s after Ang II stimulation. Similar results were obtained in Mut 318-del and Mut K310,311Q. By contrast, in Mut 310-del, Mut 312-del, Mut 313-del, Mut 314-del, Mut Y312A, Mut F313A, and Mut L314A responses of InsP₃ to Ang II stimulation were abolished (Fig. 4).

G_q was incubated for 10 min in the presence of [³⁵S]GTPS with peptides representing domains in the cytoplasmic segments of native AT_1A (P-1 to P-5) or mutated peptides of P-5. As shown in Fig. 5, Peptides P-3 and P-5 activated G_q as well as positive control. The G_q-activating function was attenuated to 25% relative to intact P-5 in Mut P-5 (Tyr³¹², Phe³¹³, and Leu³¹⁴ were replaced by alanine). The uptake of [³⁵S]GTPS was significantly lower in Mut Y, Mut F (p < 0.01) and Mut L (p < 0.05) than in P-5.

Effect of synthetic peptides on [³⁵S]GTPS binding to of G_q protein.

DISCUSSION

The cytoplasmic C-terminal (C-tail) region was shown to play an essential role in agonist-induced receptor internalization (15). However, its role in the G protein-coupled phospholipase activation has been controversial. Now in three independent approaches using five deletion mutants, four alanine substitution mutants, and synthetic peptides with native and mutated amino acid sequences corresponding to an N-terminal region of C-tail, we were able to identify the tripeptide region Tyr³¹²-Phe³¹³-Leu³¹⁴ (Fig. 5) as a domain essential for G_q activation.

Different experimental approaches produce results leading to different and sometimes contradicting conclusions. Wang et al. (13) using chimeric human AT₁ with a grafted AT₂ C-tail that shows G_q activation concluded that the major determinant of G_q coupling specificity is in the ICL-3, and the C-tail has little role in the activation of PLC. However, we had shown that deletion beyond Phe³⁰⁹ (Mut 310-del) abolished the G_q-coupled inositol 1,4,5-trisphosphate formation (14). Since both of these modifications could introduce additional factors such as conformational changes or the effect of ICL3 not directly related to the action of deleted or replaced residues, multiple approaches had to be taken. Loss of G_q coupling in Mut 310-del and complete recovery of the G_q activation in Mut 318-del narrowed the G_q coupling domain to residues 310-317 (Figs. 1 and 4) (14). The observation of a robust activity with Mut 315-del by Thomas et al. (15) further narrowed it to a region between residues 310 and 314. Almost complete loss of the activity with Mut 312-del, 313-del, 314-del, and single residue alanine mutation Y312A, F313A, and L314A and the preservation of a full PLC activity with the double mutant K310Q,K311Q indicated that Tyr³¹²-Phe³¹³-Leu³¹⁴ is the essential domain required for PLC activation. Its essential role in G_q coupling was also determined by loss of the well known GTPS-induced shift to a low affinity state for agonist binding in these mutants (Figs. 2 and 3). Further evidence for the essential role of the tripeptide sequence for the G protein coupling was obtained by a third and completely independent approach in which peptides with the amino acid sequences of the native and alanine-substituted C-tail (residues 307-320) were allowed to interact with purified heterotrimeric G_q, and binding to [³⁵S]GTPS was examined. Again, alanine substitution of Tyr³¹²-Phe³¹³-Leu³¹⁴ singly or three together significantly reduced GTPS binding. It is interesting to note that, whereas the triple mutant Mut P-5 lost almost the entire binding ability, other mutants, particularly Mut L (L314A), retained recognizable binding, although the conformation of the C-tail domain of AT_1A and the shorter synthetic segment may have a different conformation. These results suggest synergism of the three residues and some difference in the role of Tyr³¹² and Leu³¹⁴ in G_q activation and GTPS binding.

The G protein coupling sites seem to vary from receptor to receptor, and no definitive rules or consensus sequences seem to exist. For example the N-formyl peptide receptor uses ICL2 (22), and G protein specificity of PGE₂ receptor isoforms (EP3) is determined by the C-tail region. More than a single domain could participate in the interaction. The possibility of cooperation of ICL3 and amphipathic -helical structure of the N-terminal region of the C-tail has been proposed by Probst et al. (23). The -adrenergic receptor uses the C-terminal region of ICL3 and the N-terminal region of C-tail for G_s activation (10).

The present finding that the synthetic 16-mer peptide P-3 with the amino acid sequence of the N-terminal region of ICL3 and the C-tail peptide (P-5) activated purified G_q just as well as the C-tail peptide (P-5) (Fig. 5) supports the observation of Hunyady et al. (24) that the deletion of a sequence (215-226) in this domain abolished G_q activation. It also supports the observation of Wang et al. (13) that in chimeras of AT₁ and AT₂, ICL3 plays dominant roles in G_q coupling. Shirai et al. (25) showed the same ICL3 domain activates G_i1, G_i2, and G_o by using the synthetic peptide P-3. These results indicate that AT_1A may use and require the tripeptide sequence of C-tail in collaboration with ICL3 in G_q activation. Interesting information revealed by the activation of G proteins by these peptides are that the same peptides (P-3 and P-5) are capable of activating G_i, G_o, and G_q. Mechanisms by which a receptor selects the type of G proteins are yet to be clarified. On the other hand, peptides with unrelated sequences like P-1, P-2, and P-4 which did not show the activation may be considered as controls and indicate that activation by P-3 and P-5 is specific to their sequences.

B_max values of mutated receptors expressed in each cell line were at levels comparable to that of the wild type. Hence, the decrease in InsP₃ formation in Mut 310-del, Mut 312-del, Mut 313-del, Mut 314-del, Mut Y312A, Mut F313A, and Mut L314A should be due to the receptor mutation rather than a decrease in expression of each mutant receptor. Our previous study using substitutional mutations of basic polar amino acid residues in ICL2 and ICL3 indicated that ICL2 and the C-terminal domain of ICL3 would be important for G_q coupling (14). These mutations targeted at domains with dense electrical charges probably caused nonspecific conformational changes and led to erroneous results that could be misinterpreted.

Tyr²⁹² in transmembrane domain 7 was reported to be essential for G protein coupling (26). The conserved sequence NPLFY at the bottom of transmembrane domain 7 was shown to contribute to both agonist binding and signal transduction (24). Thus, the junctional area of AT₁ between transmembrane domain 7 and C-tail seems to play an important role in receptor signaling. This area of AT₁ also contains the sequence KKFKK that was shown to be an unusual G_i activator domain of insulin growth factor II receptor (9). However, in AT₁ mutation to Lys-Lys-Phe-Gln³¹⁰-Gln³¹¹ did not have any effect on G_q coupling. This observation helped our work in narrowing the G_q activating domain to Tyr³¹²-Phe³¹³-Leu³¹⁴.

In summary, the present study presents evidence that a G_q coupling site in the type 1A angiotensin receptor AT_1A should reside between residues 312 and 318 in the C-terminal tail, and the specific sequence Tyr³¹²-Phe³¹³-Leu³¹⁴ is essential for coupling and activation of the G_q protein.