A Domain for G Protein Coupling in Carboxyl-terminal Tail of Rat Angiotensin II Receptor Type 1A
1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês
10.1074/jbc.272.38.23631
ISSN1083-351X
AutoresTomoaki Sano, Kenji Ohyama, Yoshiaki Yamano, Yoshiko Nakagomi, Shinpei Nakazawa, Mitsuhiro Kikyo, Heigoro Shirai, Jonathan S. Blank, John H. Exton, Tadashi Inagami,
Tópico(s)Hormonal Regulation and Hypertension
ResumoTo delineate domains essential for Gq protein coupling in the C-terminal region (C-tail) of rat angiotensin II (Ang II) receptor type 1A (AT1A), we modified the putative cytosolic regions of the receptor by truncation or alanine substitution and determined resultant changes in the guanosine 5′-3-O-(thio)triphosphate (GTPγS) effect on Ang II binding and inositol trisphosphate production by the agonist. Independently, we studied the effect of synthetic C-tail peptides (P-5) and its alanine substitution analogs on [35S]GTPγS binding to Gq. Effects of GTPγS on Ang II binding (shift to a low affinity form) and inositol trisphosphate production in the deletional mutant receptor 1–317 AT1A was similar to wild type AT1A, whereas in shorter C-terminal deletion mutants 1–309, 1–311, 1–312, 1–313 AT1A, and substitutional mutants Y312A, F313A, and L314A these activities were markedly reduced. The binding of [35S]GTPγS to Gq was promoted by the synthetic C-terminal peptide P-5 but not when mutated at Tyr312, Phe313, or Leu314. Results indicate that Tyr312, Phe313, and Leu314 in cytosolic carboxyl-terminal region of rat AT1A are essential for coupling and activation of Gq. To delineate domains essential for Gq protein coupling in the C-terminal region (C-tail) of rat angiotensin II (Ang II) receptor type 1A (AT1A), we modified the putative cytosolic regions of the receptor by truncation or alanine substitution and determined resultant changes in the guanosine 5′-3-O-(thio)triphosphate (GTPγS) effect on Ang II binding and inositol trisphosphate production by the agonist. Independently, we studied the effect of synthetic C-tail peptides (P-5) and its alanine substitution analogs on [35S]GTPγS binding to Gq. Effects of GTPγS on Ang II binding (shift to a low affinity form) and inositol trisphosphate production in the deletional mutant receptor 1–317 AT1A was similar to wild type AT1A, whereas in shorter C-terminal deletion mutants 1–309, 1–311, 1–312, 1–313 AT1A, and substitutional mutants Y312A, F313A, and L314A these activities were markedly reduced. The binding of [35S]GTPγS to Gq was promoted by the synthetic C-terminal peptide P-5 but not when mutated at Tyr312, Phe313, or Leu314. Results indicate that Tyr312, Phe313, and Leu314 in cytosolic carboxyl-terminal region of rat AT1A are essential for coupling and activation of Gq. Angiotensin II (Ang II) 1The abbreviations used are: Ang II, angiotensin II; AT1A, angiotensin type 1A receptor; AT2, angiotensin type 2 receptor; ICL2, second intracellular loop; ICL3, third intracellular loop; PLC, phospholipase C; InsP3, inositol-1,4,5-trisphosphate; Mut, mutant; del, deletion; GTPγS, guanosine 5′-3-O-(thio)triphosphate; G protein, guanyl nucleotide-binding protein; CHO-K1, Chinese hamster ovary cells. 1The abbreviations used are: Ang II, angiotensin II; AT1A, angiotensin type 1A receptor; AT2, angiotensin type 2 receptor; ICL2, second intracellular loop; ICL3, third intracellular loop; PLC, phospholipase C; InsP3, inositol-1,4,5-trisphosphate; Mut, mutant; del, deletion; GTPγS, guanosine 5′-3-O-(thio)triphosphate; G protein, guanyl nucleotide-binding protein; CHO-K1, Chinese hamster ovary cells.receptor type 1A (AT1A) is a seven-transmembrane, G protein-coupled receptor (1Sasaki K. Yamano Y. Bardhan S. Iwai N. Murray J.J. Hasegawa M. Matsuda Y. Inagami T. Nature. 1991; 351: 230-232Crossref PubMed Scopus (772) Google Scholar, 2Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1164) Google Scholar). Ang II activates Gq, Gi, and Go proteins through the AT1A receptor (3Inagami T. Iwai N. Sasaki K. Yamano Y. Bardhan S. Chaki S. Guo D.F. Furuta H. J. Hypertens. 1992; 10: 713-716Crossref PubMed Scopus (58) Google Scholar, 4Crawford K.W. Frey E.A. Cote T.E. Mol. Pharmacol. 1992; 41: 154-162PubMed Google Scholar, 5Mitchell, J., Murphy, E. A., and Northup, J. K. (1991)Endocrine Society Abstract , 199.Google Scholar). The GTP·Gqα complex stimulates phospholipase C (PLC) resulting in inositol trisphosphate (InsP3) generation (6Griendling K.K. Rittenhouse S.E. Brock T.A. Ekstein L.S. Gimbrone Jr., M.A. Alexander R.W. J. Biol. Chem. 1986; 261: 5901-5906Abstract Full Text PDF PubMed Google Scholar, 7Hescheler J. Rosenthal W. Hinsch K.D. Wulfern M. Trautwein W. Schultz G. EMBO J. 1988; 7: 619-624Crossref PubMed Scopus (100) Google Scholar). 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 (8Franke R.R. Konig B. Sakmar T.P. Khorana H.G. Hofmann K.P. Science. 1990; 250: 123-125Crossref PubMed Scopus (301) Google Scholar, 9Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 10O'Dowd B.F. Hnatowich M. Regan J.W. Leader W.M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 15985-15992Abstract Full Text PDF PubMed Google Scholar). 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) (11Chazenbalk G.D. Nagayama Y. Russo D. Wadsworth H.L. Rapoport B. J. Biol. Chem. 1990; 265: 20970-20975Abstract Full Text PDF PubMed Google Scholar). 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 (12Namba T. Sugimoto Y. Negishi M. Irie A. Ushikubi F. Kakizuka A. Ito S. Ichikawa A. Narumiya S. Nature. 1993; 365: 166-170Crossref PubMed Scopus (513) Google Scholar). Studies by Wang et al. (13Wang C. Jayadev S. Escobedo J.A. J. Biol. Chem. 1995; 270: 16677-16682Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) using chimeras of human AT1 and AT2 suggested that the N-terminal portion of ICL3 was important for Gq 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 AT1Areceptor in COS7 cells showed that the last 50 amino acid residues (beyond Phe309) were also important for Gqcoupling (14Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar). Thomas et al. (15Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) reported that truncation of the last 45 amino acid residues of the rat AT1A 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 AT1A, 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 Gq activation with the aim of defining a region in the C-tail essential for the coupling to Gq in rat AT1A. The entire coding region of rat kidney AT1A was cloned into EcoRI site of a plasmid pUC19 (16Iwai N. Yamano Y. Chaki S. Konishi F. Bardhan S. Tibbett C. Sasaki K. Hasegawa M. Matsuda Y. Inagami T. Biochem. Biophys. Res. Commun. 1991; 177: 299-304Crossref PubMed Scopus (213) Google Scholar). 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 (17Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4880) Google Scholar). Sites of truncation and substitution are shown in Fig.1. The mutated DNA sequences were confirmed by Sanger's dideoxynucleotide sequencing method (18Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52250) Google Scholar). The mutated AT1A cDNA was excised with enzymesBamHI and XhoI and introduced into the expression vector pCDNA1. Forty μg of plasmid constructs containing the wild type or mutated rat AT1A 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 of125I-Ang II was selected. AT1A-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 AT1A 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. InsP3was 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 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 InsP3assay kit (NEN Life Science Products). 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 (19Chaki S. Inagami T. Biochem. Biophys. Res. Commun. 1992; 182: 388-394Crossref PubMed Scopus (97) Google Scholar). 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 mmMgCl2, 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 nm125I-Ang II at 25 °C for 60 min in the presence of varying concentrations of GTPγS. For studying the time course of ligand binding, membranes were incubated with 0.1 nm125I-Ang II for 60 min at 37 °C to attain binding equilibrium and unlabeled Ang II (1 μm) or GTPγS (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 Gq proteins from bovine liver were purified to homogeneity as published (20Taylor S.J. Smith J.A. Exton J.H. J. Biol. Chem. 1990; 265: 17150-17156Abstract Full Text PDF PubMed Google Scholar). [35S]GTPγS binding to 10 nm purified heterotrimeric Gq 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]GTPγS in the absence of phospholipids as described by Okamoto et al. (9Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar). Briefly, Gq 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]GTPγS to Gq was measured in the presence of 1 μm GTPγS and 25 mmMg2+ at room temperature by the method of Northup et al. (21Northup J.K. Smigel M.D. Gilman A.G. J. Biol. Chem. 1982; 257: 11416-11423Abstract Full Text PDF PubMed Google Scholar) as a positive control. The results of experiments with the synthetic peptide study was examined by unpaired Student'st test. p values less than 0.05 were considered significant. As shown in TableI the dissociation constants (K d) and B max values of the wild type AT1A 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 IBinding affinity of [Sar1-Ile8]Ang II binding for rat AT1A wild type receptor and mutant receptorsK dB maxnmfmol/mg proteinWild type1.6 ± 0.118.8 ± 1.4Mut 310-del2.4 ± 0.216.0 ± 1.1Mut 312-del2.4 ± 0.217.8 ± 1.6Mut 313-del2.0 ± 0.315.2 ± 1.8Mut 314-del1.8 ± 0.215.6 ± 1.6Mut 318-del2.5 ± 0.317.8 ± 1.8Mut Y312A2.4 ± 0.416.6 ± 1.8Mut F313A2.6 ± 0.415.5 ± 1.9Mut L314A2.8 ± 0.314.8 ± 1.2Mut K310,311Q2.7 ± 0.318.3 ± 1.4Data represent results of three identical series of binding isotherms followed by Scatchard analysis. Results are presented as means ± S.D. Open table in a new tab Data represent results of three identical series of binding isotherms followed by Scatchard analysis. Results are presented as means ± S.D. As shown in Fig.2, the binding of 125I-Ang II to wild type AT1A, Mut 318-del, and Mut K310,311Q receptors were dose-dependently decreased by GTPγS, whereas the effect of GTPγS (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. 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 AT1A 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). GTPγS (10 μm) markedly shortened the half-life time of the spontaneous dissociation in the wild type AT1A, 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 AT1A and Mut 318-del were shortened in the presence of both Ang II and GTPγS (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). Binding of Ang II to AT1A activates a PLC via Gq resulting in stimulation of InsP3 formation. Thus, increased InsP3 formation by Ang II can be considered to indicate effective coupling to Gq of the mutants. In unmutated AT1A, 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). Gq was incubated for 10 min in the presence of [35S]GTPγS with peptides representing domains in the cytoplasmic segments of native AT1A (P-1 to P-5) or mutated peptides of P-5. As shown in Fig.5, Peptides P-3 and P-5 activated Gq as well as positive control. The Gq-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]GTPγS was significantly lower in Mut Y, Mut F (p < 0.01) and Mut L (p < 0.05) than in P-5. The cytoplasmic C-terminal (C-tail) region was shown to play an essential role in agonist-induced receptor internalization (15Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). 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 Gq activation. Different experimental approaches produce results leading to different and sometimes contradicting conclusions. Wang et al. (13Wang C. Jayadev S. Escobedo J.A. J. Biol. Chem. 1995; 270: 16677-16682Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) using chimeric human AT1 with a grafted AT2C-tail that shows Gq activation concluded that the major determinant of Gq 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 Gq-coupled inositol 1,4,5-trisphosphate formation (14Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar). 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 Gq coupling in Mut 310-del and complete recovery of the Gq activation in Mut 318-del narrowed the Gq coupling domain to residues 310–317 (Figs. 1 and 4) (14Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar). The observation of a robust activity with Mut 315-del by Thomas et al. (15Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) 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 Gq coupling was also determined by loss of the well known GTPγS-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 Gq, and binding to [35S]GTPγS was examined. Again, alanine substitution of Tyr312-Phe313-Leu314 singly or three together significantly reduced GTPγS 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 AT1A 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 Gqα activation and GTPγS 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 (22Schreiber R.E. Prossnitz E.R. Ye R.D. Cochrane C.G. Bokoch G.M. J. Biol. Chem. 1994; 269: 326-331Abstract Full Text PDF PubMed Google Scholar), 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 α-helical structure of the N-terminal region of the C-tail has been proposed by Probst et al. (23Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (676) Google Scholar). The β-adrenergic receptor uses the C-terminal region of ICL3 and the N-terminal region of C-tail for Gs activation (10O'Dowd B.F. Hnatowich M. Regan J.W. Leader W.M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1988; 263: 15985-15992Abstract Full Text PDF PubMed Google Scholar). 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 Gq just as well as the C-tail peptide (P-5) (Fig. 5) supports the observation of Hunyadyet al. (24Hunyady L. Bor M. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1995; 270: 16602-16609Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) that the deletion of a sequence (215–226) in this domain abolished Gq activation. It also supports the observation of Wang et al. (13Wang C. Jayadev S. Escobedo J.A. J. Biol. Chem. 1995; 270: 16677-16682Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) that in chimeras of AT1 and AT2, ICL3 plays dominant roles in Gq coupling. Shirai et al. (25Shirai H. Takahashi K. Katada T. Inagami T. Hypertension. 1995; 25: 726-730Crossref PubMed Google Scholar) showed the same ICL3 domain activates Gi1, Gi2, and Go by using the synthetic peptide P-3. These results indicate that AT1A may use and require the tripeptide sequence of C-tail in collaboration with ICL3 in Gqactivation. 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 Gi, Go, and Gq. 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 Gq coupling (14Ohyama K. Yamano Y. Chaki S. Kondo T. Inagami T. Biochem. Biophys. Res. Commun. 1992; 189: 677-683Crossref PubMed Scopus (128) Google Scholar). 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 (26Marie J. Maigret B. Joseph M.-P. Larguier R. Nouet S. Lombard C. Bonnafous J.-C. J. Biol. Chem. 1994; 269: 20815-20818Abstract Full Text PDF PubMed Google Scholar, 27Strosberg A.D. Eur. J. Biochem. 1991; 196: 1-10Crossref PubMed Scopus (201) Google Scholar). The conserved sequence NPLFY at the bottom of transmembrane domain 7 was shown to contribute to both agonist binding and signal transduction (24Hunyady L. Bor M. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1995; 270: 16602-16609Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). 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 Gi activator domain of insulin growth factor II receptor (9Okamoto T. Murayama Y. Hayashi Y. Inagaki M. Ogata E. Nishimoto I. Cell. 1991; 67: 723-730Abstract Full Text PDF PubMed Scopus (228) Google Scholar). However, in AT1 mutation to Lys-Lys-Phe-Gln310-Gln311 did not have any effect on Gq coupling. This observation helped our work in narrowing the Gq activating domain to Tyr312-Phe313-Leu314. In summary, the present study presents evidence that a Gqcoupling site in the type 1A angiotensin receptor AT1Ashould 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 Gq protein. We are grateful to Japanese Cancer Research Resources Bank (JCRB)-Cell for the gift of CHO-K1 cell.
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