Polar Residues in the Transmembrane Domains of the Type 1 Angiotensin II Receptor Are Required for Binding and Coupling
1996; Elsevier BV; Volume: 271; Issue: 3 Linguagem: Inglês
10.1074/jbc.271.3.1507
ISSN1083-351X
AutoresCatherine Monnot, Claire Bihoreau, Sophie Conchon, Kathleen M. Curnow, Pierre Corvol, Éric Clauser,
Tópico(s)Computational Drug Discovery Methods
ResumoType 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism.Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation. Type 1 angiotensin receptors (AT1) are G-protein coupled receptors, mediating the physiological actions of the vasoactive peptide angiotensin II. In this study, the roles of 7 amino acids of the rat AT1A receptor in ligand binding and signaling were investigated by performing functional assays of individual receptor mutants expressed in COS and Chinese hamster ovary cells. Substitutions of polar residues in the third transmembrane domain with Ala indicate that Ser105, Ser107, and Ser109 are not essential for maintenance of the angiotensin II binding site. Replacement of Asn111 or Ser115 does not alter the binding affinity for peptidic analogs, but modifies the ability of the receptor to interact with AT1 (DuP753)- or AT2 (CGP42112A)-specific ligands. These 2 residues are probably involved in determining the binding specificity for these analogs. The absence of G-protein coupling to the Ser115 mutant suggests that this residue, in addition to previously identified residues, Asp74 and Tyr292, participates in the receptor activation mechanism. Finally, Lys102 (third helix) and Lys199 (fifth helix) mutants do not bind angiotensin II or different analogs. Co-expression of these two deficient receptors permitted the restoration of a normal binding site. This effect was not due to homologous recombination of the cDNAs but to protein trans-complementation. INTRODUCTIONThe physiological actions of the vasoactive octapeptide hormone angiotensin II (AngII) in the cardiovascular, endocrine, and neuronal systems are mediated by membrane-bond receptors. Two pharmacologically distinct AngII receptors have been identified: AT1 and AT2(1.Chiu A.T. Herblin W.F. McCall D.E. Ardecky R.J. Carini D.J. Duncia J.V. Pease L.J. Wong P.C. Wexler R.R. Johnson A.L. Timmermans P.B.M.W.M. Biochem. Biophys. Res. Commun. 1989; 165: 196-203Crossref PubMed Scopus (836) Google Scholar, 2.Whitebread S. Mele M. Kamber B. De Gasparo M. Biochem. Biophys. Res. Commun. 1989; 163: 284-291Crossref PubMed Scopus (758) Google Scholar). AT1 receptors bind biphenylimidazole antagonists such as DuP753 with high affinity and pentapeptide analogs such as CGP42112A with low affinity, whereas AT2 receptors have the reverse affinities for these compounds. Cloning of both receptor types has revealed that they belong to the seven transmembrane domain receptor family(3.Murphy T.J. Alexander R.W. Griendling K.K. Runge M.S. Bernstein K.E. Nature. 1991; 351: 233-236Crossref PubMed Scopus (1166) Google Scholar, 4.Sasaki K. Yamano Y. Bardhan S. Iwai N. Murray J.J. Hasegawa M. Matsuda Y. Inagami T. Nature. 1991; 351: 230-233Crossref PubMed Scopus (773) Google Scholar, 5.Kambayashi Y. Bardhan S. Takahashi K. Tsuzuki S. Inui H. Hamakubo T. Inagami T. J. Biol. Chem. 1993; 268: 24543-24546Abstract Full Text PDF PubMed Google Scholar, 6.Mukoyama M. Nakajima M. Horiuchi M. Sasamura H. Pratt R.E. Dzau V.J. J. Biol. Chem. 1993; 268: 24539-24542Abstract Full Text PDF PubMed Google Scholar). Two closely related AT1 isoforms (AT1A and AT1B) have been identified in rat and mouse species(7.Iwai N. Inagami T. FEBS Lett. 1992; 298: 257-260Crossref PubMed Scopus (381) Google Scholar, 8.Sasamura H. Hein L. Krieger J.E. Pratt R.E. Kobilka B.K. Dzau V.J. Biochem. Biophys. Res. Commun. 1992; 185: 253-259Crossref PubMed Scopus (279) Google Scholar). AT1 receptors have been shown to be coupled to G-proteins and to activate phospholipase C (PLC)(9.Bottari S.P. de Gasparo M. Steckeling U.M. Levens N.R. Frontiers Neuroendocrinol. 1993; 14: 123-171Crossref PubMed Scopus (338) Google Scholar, 10.Timmermans P.B.M.W.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar). This results in inositol trisphosphate (IP3) generation, which then causes an increase in intracellular calcium concentrations, and diacylglycerol formation, which leads to protein kinase C activation(11.Farese R.V. Larson R.E. Davis J.S. Endocrinology. 1984; 114: 302-304Crossref PubMed Scopus (63) Google Scholar, 12.Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3297) Google Scholar).The molecular location of the ligand binding domain of the G-protein-coupled receptors has been intensively investigated using genetic, biochemical, and biophysical approaches. The binding site for small molecules such as the bioamine neurotransmitters involves polar residues of the transmembrane domains (TM)(13.Strader C.D. Ming Fong T. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). In contrast, the binding site for large hormones such as the pituitary glycoproteins is located in the large amino-terminal extracellular domain of the corresponding receptors(13.Strader C.D. Ming Fong T. Tota M.R. Underwood D. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (991) Google Scholar). The ligand binding domains of peptide receptors, such as AT1, have been more recently investigated. Peptide binding by AT1 receptors is dependent on the presence of four extracellular cysteines (14.Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar) as well as on several additional residues located in the extracellular domains(15.Hjorth S.A. Schambye H.T. Greenlee W.J. Schwartz T.W. J. Biol. Chem. 1994; 269: 30953-30959Abstract Full Text PDF PubMed Google Scholar). Whereas the binding of non-peptidic ligands is unaffected by these extracellular mutations, numerous polar residues of the hydrophobic transmembrane segments are determinant in binding of non-peptidic antagonists(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 17.Ji H. Leung M. Zhang Y. Catt K.J. Sandberg K. J. Biol. Chem. 1994; 269: 16533-16536Abstract Full Text PDF PubMed Google Scholar, 18.Marie 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, 19.Schambye H.T. Hjorth S.A. Bergsma D.J. Sathe G. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7046-7050Crossref PubMed Scopus (116) Google Scholar). Two of these residues, Asp74 and Tyr292, also play an essential role in the coupling of AT1A to PLC(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 18.Marie 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).Biochemical analysis (20.Desarnaud F. Marie J. Lombard C. Larguier R. Seyer R. Lorca T. Jard S. Bonnafous J.C. Biochem. J. 1993; 289: 289-297Crossref PubMed Scopus (27) Google Scholar) and molecular modeling studies (21.Underwood D.J. Strader C.D. Rivero R. Patchett A.A. Greenlee W. Prendergast K. Chem. Biol. 1994; 1: 211-221Abstract Full Text PDF PubMed Scopus (65) Google Scholar) 2P. Broto, unpublished results. indicate a major role for the third and fifth transmembrane segments (TM-III and TM-V) in AngII binding. To define the functional roles of polar residues in these transmembrane domains, substitutions of different polar residues into Ala (K102A, S105A, S107A, S109A, N111A, S115A, and K199A) were therefore created in the rat AT1A receptor (Fig. 1). The pharmacological profiles of the mutated receptors for peptidic and non-peptidic agonists and antagonists, as well as their signaling properties were analyzed.Some of the mutants were defective for the binding of AngII and its analogs. Since the mutations were located in different domains of the AT1A receptor, we investigated whether the recently described mechanism of intermolecular complementation could be observed for this peptide hormone receptor. This phenomenon was described for different mutants of the α2C-adrenergic receptors as well of the M2 and M3 muscarinic receptors when they were co-expressed, suggesting that molecular association was occurring between complementary transmembrane domains of two different defective receptors(22.Kobilka B.K. Kobilka T.S. Daniel K. Regan J.W. Caron M.G. Lefkowitz R.J. Science. 1988; 240: 1310-1316Crossref PubMed Scopus (604) Google Scholar, 23.Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (296) Google Scholar, 24.Maggio R. Vogel Z. Wess J. FEBS Lett. 1993; 319: 195-200Crossref PubMed Scopus (118) Google Scholar). The ligand binding and signaling properties of four binding defective AT1A mutants were therefore analyzed after their co-transfections in COS cells, in different paired combinations.EXPERIMENTAL PROCEDURESSite-directed, Insertion, and Deletion MutagenesisExpression plasmids coding for the mutated receptors were constructed following two types of strategies, using either the wild type rat AT1A cDNA fragment (2.2 kilobase pairs) inserted in M13mp19, or a described synthetic cDNA sequence (1.1 kilobase pairs) into which multiple restriction sites had been introduced(25.Conchon S. Monnot C. Sirieix M.E. Bihoreau C. Corvol P. Clauser E. Biochem. Biophys. Res. Commun. 1994; 199: 1347-1354Crossref PubMed Scopus (8) Google Scholar).K102A, S107A, K199A, and Δ(168-188) were generated in the M13mp19 construct. Four oligonucleotides were synthesized on a PCR-Mate (Applied Biosystems): 5′-CAC CTA TGT GCC ATC GCT TCG-3′ for replacement of Lys102; 5′-GCT TCG GCC GCC GTG AGC TTC-3′ for replacement of Ser107; 5′-GGC CTT ACC GCC AAT ATT CTG-3′ for replacement of Lys199; 5′-G CCA GCT GTC ATC CAC CGA TGC TCG ACG CTC CCC ATA GGG CTG-3′ deleting the sequence coding for the second extracellular loop. An uracil-containing M13mp19-AT1A DNA was used as template in an in vitro mutagenesis reaction using the synthetic oligonucleotides (Muta-Gene D kit, Bio-Rad). Transformation into a strain with a functional uracil N-glycosylase allows selection against the paternal unmutated strand.The other mutations were performed by deletion of a restriction fragment and replacement with an appropriate linker. The Δ(3-25) mutant was obtained by deleting the 360-base pair HindIII-BspHI fragment corresponding to the amino-terminal extracellular region of AT1A and replacing with double-stranded linker corresponding to sense oligonucleotide 5′-AG CTT ACC ATG GCC TGC TAC ATA TTT GTC-3′.The S105A, S109A, N111A, and S115A mutants were constructed using four double-stranded linkers corresponding to sense oligonucleotides: 5′-CG GCC GCT TCC GTG AGC TTC AAC CTC TA-3′, replacing Ser105; 5′-CG AGC GCC TCC GTG GCC TTC AAC CTC TA-3′, both replacing Ser109 and changing a Eco47III restriction site to a BglI site for screening purpose; 5′-CG TCC GCT TCC GTG AGC TTC GCC CTC TA-3′, replacing Asn111 as well as suppressing a Eco47III restriction site; 5′-C GCG GCC GTG TTC CTT CTC AC-3′, replacing Ser115. These linkers were inserted, respectively, in the NruI-MluI (for Ser105, Ser109, and Asn111) and MluI-PmlI (for Ser115) single restriction sites of the synthetic cDNA.The other AT1A mutants were constructed using the same strategy. These constructions were performed in order to tag the amino (Ins[Nter]) and/or carboxyl terminus (Ins[Cter]) of the AT1A coding sequence and were used here as a control to verify the absence of homologous recombination between two co-transfected AT1A cDNAs. The two inserted sequences, corresponding to the sense oligonucleotides (FLAG sequence, SIS, Eastman Kodak Co.): 5′-AG CTT ACC ATG GAC TAC AAA GAC GAT GCC GAT AAG GCC CTT AAC TCT TC-3′ (5′ sequence) and 5′-C GAA GTG GAG GAC GAT GAC GAT AAA GAC TAC AAA GAC GAT GAC GAT AAA TGA CGG ACC GT-3′ (3′ sequence) were, respectively, inserted in the HindIII-EagI and BstBI-XbaI restriction sites of the synthetic cDNA. To produce the construction Ins[Nter-Cter] containing the double insertion, the 560-base pair EcoRI-XbaI fragment of Ins[Cter] was inserted into the expression vector containing HindIII-EcoRI fragment of Ins[Nter].The mutated sequences were verified by dideoxy sequencing using Sequenase version 2 (United States Biochemical Corp.). D74N was constructed as described previously(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar). All the mutated AT1A cDNAs were subcloned into the expression vector pECE(26.Ellis L. Clauser E. Morgan D. Edery M. Roth R.A. Rutter W.J. Cell. 1986; 45: 721-732Abstract Full Text PDF PubMed Scopus (695) Google Scholar).Expression in COS-7 and CHO CellsThe COS-7 cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum plus 0.5 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Boehringer Mannheim). Two days after plating (3 × 106 cells/75 cm3), cells were transfected or co-transfected with 10 μg of each plasmid DNA, unless otherwise indicated, by the DEAE-dextran-chloroquine method(27.Thibonnier M. Auzan C. Madhun Z. Wilkins P. Berti-Mattera L. Clauser E. J. Biol. Chem. 1994; 269: 3304-3310Abstract Full Text PDF PubMed Google Scholar). Binding studies or inositol phosphate measurements were done 48 h after transfection.CHO K1 cells were maintained in Ham's F-12 medium (Boehringer Mannheim) supplemented with 10% fetal calf serum plus 0.5 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The CHO.AT1A clone has been previously described(16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 28.Teutsch B. Bihoreau C. Monnot C. Bernstein K.E. Murphy T.J. Alexander R.W. Corvol P. Clauser E. Biochem. Biophys. Res. Commun. 1992; 187: 1381-1388Crossref PubMed Scopus (40) Google Scholar). To establish the CHO.S107A, CHO.N111A and CHO.S115A clones, CHO cells were co-transfected with 10 μg of the corresponding plasmid and 2 μg of the selection marker pSV2neo using the calcium phosphate precipitation method(29.Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar). Transfected cells were selected by their resistance to 750 μg/ml G418 (Life Technologies, Inc.) and cloned by limiting dilution.Binding Experiments[Sar1]AngII was labeled by the chloramine-T method. Monoiodinated [Sar1,Tyr(125I)4]AngII (2000 Ci/mmol; 1 Ci = 37 GBq), hereafter called 125I-[Sar1]AngII, was purified by high performance liquid chromatography. [3H]AngII and [3H]DuP753 were purchased from DuPont NEN. Saturation and competitive binding assays were performed as described(30.Conchon S. Monnot C. Teutsch B. Corvol P. Clauser E. FEBS Lett. 1994; 349: 365-370Crossref PubMed Scopus (93) Google Scholar). Competition binding experiments were carried out using 0.8-1 nM125I-[Sar1]AngII and increasing concentrations (10-11 to 10-4M) of the various ligands. Each experiment was carried out in duplicate. Binding data were analyzed with a non-linear least-squares curve fitting procedure, Ebda-Ligand (Elsevier-Biosoft, Cambridge, United Kingdom)(31.Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7760) Google Scholar).Determination of Inositol Phosphate Production[3H]Inositol phosphate (IP) production in response to increasing concentrations of AngII was as described previously(32.Torrens Y. Daguet de Montety M. El Etr M. Beaujouan J. Glowinski J. J. Neurochem. 1989; 52: 1913-1918Crossref PubMed Scopus (92) Google Scholar). Cells were subcultured in 12-well plates and labeled with 2 μCi/ml [3H]myoinositol for 24 h and then incubated with AngII at 37°C for 30 min in presence of 10 mM LiCl. After purification on a Dowex anion exchange resin (AG® 1-X8 resin, Bio-Rad), the total radiolabeled IP fraction was measured.RT-PCR Analysis for Detection of Homologous RecombinationTotal RNA was prepared from COS-7 cells transfected with Ins[Nter], Ins[Cter], or Ins[Nter-Cter] plasmids using the guanidine thiocyanate method(33.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar). Total RNA (5 μg) was treated with RNase-free DNase (Boehringer Mannheim) to avoid plasmid DNA amplification. Complementary DNAs were synthesized with Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim) using 10 pmol of a primer, corresponding to the 3′ region of the carboxyl-terminal insertion sequence 5′-TTT ATC GTC ATC GTC TTT GT-3′. This particular primer allows only reverse transcription of mRNA containing the carboxyl-terminal insertion sequence to avoid any heterologous amplification between the Ins[Nter] and Ins[Cter] cDNAs. In these experiments, only Ins[Cter] and Ins[Nter-Cter] cDNAs can be subsequently amplified. Ten percent of the cDNA synthesized was treated with DNase-free RNase (Boehringer Mannheim) and then amplified using 1 unit of Taq DNA polymerase (Boehringer Mannheim) and 10 pmol of each primer in a 25-μl volume. Thirty cycles of the PCR were performed using 94°C, 30 s; 60°C, 30 s; 72°C, 1 min. The primers used for PCR amplification correspond to the following sequences: primer 1 (5′-AAA GAC GAT GCC GAT AAG GCC CT-3′); primer 2 (5′-A AGG ATC CAA GAT GAC TGC CCC A-3′); primer 3 (5′-A CTC CAC TTC GAA ACA AGA CGC A-3′) and primer 4 (5′-C GTC TTT GTA GTC TTT ATC GTC A-3′). Their locations are indicated in Fig. 4A. The primer pairs (1;3) and (2;4) are able to amplify the Ins[Nter] and Ins[Cter] plasmid DNAs, respectively, and the three primer pairs (1;3), (2;4), and (1;4) are able to amplify the Ins[Nter-Cter].Figure 4:Messenger RNA detection by RT-PCR. A, Schematic representation of the Ins[Nter-Cter] cDNA containing the two insertion sequences. The two rectangles correspond to the inserted heterologous sequences, and the continuous line corresponds to the AT1A cDNA. Locations of the PCR primers 1, 2, 3, and 4 are indicated by arrows. B, RT-PCR experiments were performed on mRNAs isolated from COS cells transfected with Ins[Nter], Ins[Cter] or Ins[Nter-Cter] or co-transfected with Ins[Nter] and Ins[Cter]. The PCR product amplified is a 1-kilobase pair band. Asterisk indicates absence of band due to specific reverse transcription of cDNAs containing the carboxyl-terminal insertion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)StatisticsStatistical analysis was performed with a paired Student's t test.RESULTS AND DISCUSSIONResidues or Domains Involved in AT1A Binding SitesTo determine the roles of several polar residues in TM-III and TM-V in the binding of both peptidic and non-peptidic ligands, a series of single point mutated rat AT1A receptors were generated (Fig. 1). These mutant receptors were transiently expressed in COS-7 cells and evaluated by binding assays.Two single point mutants K102A and K199A are unable to bind the peptidic agonists 125I-[Sar1]AngII (Table 1) and [3H]AngII, or the non-peptidic antagonist [3H]DuP753 (data not shown). These data suggest that these mutations result in a loss of the structural integrity necessary for peptide and non-peptide binding or that they are not expressed at the membrane. Latter experiments (see below) show that co-expression of these two receptor mutants results in normal ligand binding. It is therefore concluded that these mutants are expressed at the cell surface and that Lys102 and Lys199 are essential for the AT1A binding site. Two previous studies also describe the important role of Lys102 and Lys199, respectively(14.Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar, 15.Hjorth S.A. Schambye H.T. Greenlee W.J. Schwartz T.W. J. Biol. Chem. 1994; 269: 30953-30959Abstract Full Text PDF PubMed Google Scholar). In the case of Lys102, it was proposed that its substitution would provoke an overall alteration in receptor structure in view of its position at the neighboring disulfide bridge. Our co-expression study indicates that substitution of this residue does not cause a global alteration in receptor structure as protein complementation can occur to produce chimeric receptors, functional in binding AngII. Thus, it seems likely that Lys102 represents an overlapping point in binding site for peptidic (AngII and [Sar1]AngII) and non-peptidic ligands (DuP753). A similar argument can be made for Lys199. Since substitution of this residue with Gln causes a major decrease in affinity for AngII (14.Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (128) Google Scholar) and substitution with Ala provokes the complete abolition of AngII and DuP753 binding, we propose that Lys199 is required for ionic interaction with carboxyl-terminal COOH of AngII as well as with the acidic group of the tetrazole of DuP753.TABLE 1 Open table in a new tab Five single point mutants in the TM-III, S105A, S107A, S109A, N111A, and S115A, recognized 125I-[Sar1]AngII with Kd values similar to those of the wild-type receptor (Table 1). The pharmacology of these five mutants for different peptidic or non-peptidic ligands was then analyzed (Table 2). All of these mutants exhibited AngII and [Sar1,Ala8]AngII binding affinities that did not markedly differ from those of the wild-type receptor. However, some differences are observed in the binding affinity of the pseudopeptidic CGP42112A or non-peptidic DuP753 compounds, specific for AT2 and AT1 receptors, respectively, for the N111A and S115A mutants. N111A showed a significant increase (8-fold) in affinity for CGP42112A and a significant decrease (45-fold) in affinity for DuP753. S115A had an affinity for the DuP753 similar to the wild-type but a significantly increased affinity for CGP42112A (7-fold).TABLE 2 Open table in a new tab These results indicate that the polar residues Ser105, Ser107, and Ser109 are not essential for the binding of peptidic or non-peptidic ligand to the AT1A receptor. The absence of a major role for Ser107 in the binding of [Sar1,Ala8]AngII and DuP753 has been demonstrated previously(17.Ji H. Leung M. Zhang Y. Catt K.J. Sandberg K. J. Biol. Chem. 1994; 269: 16533-16536Abstract Full Text PDF PubMed Google Scholar). This Ser residue is conserved in mammalian AT1 receptors, but it is replaced by an alanine in the amphibian AngII receptor. Our data suggest that it is not responsible for the specific pharmacological profile observed for the amphibian receptor, which recognizes peptidic but not non-peptidic antagonists. Furthermore, our results demonstrate that this residue is not a contributor to the binding of either peptidic agonists, or of the specific AT1/AT2 non-peptidic ligands. In contrast, these studies stress the importance of the residues Asn111 and Ser115 in binding the specific AT1/AT2 ligands. These results could be related to two previous reports; the substitution of Asp74, present in TM-II, by an asparagine (16.Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar) and the substitution of the Tyr292, present in TM-VII, by a phenylalanine (18.Marie 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) provoke a lower affinity for DuP753 and a higher affinity for CGP42112A. Despite their locations on different transmembrane domains, these 4 residues are probably positioned within a small distance of each other in the plasma membrane, suggesting that Asp74, Asn111, Ser115, and Tyr292 could belong to a specific AT1/AT2 ligand binding site. Molecular modeling studies suggest that these residues lie in a plane that is three or four α-helical turns below the membrane surface and therefore buried deep in the lipid bilayer.Residues Involved in G-protein Coupling and Mechanisms of Receptor ActivationAgonist binding to the AT1 receptor leads to the activation of PLC, which hydrolyzes a membrane phospholipid (phosphoinositide diphosphate) and produces two second messengers, IP3 and diacylglycerol. Therefore, the efficiency of AT1 coupling and signaling can be estimated by measuring the increased production of either specific IP3 or total IP in response to increasing concentrations of agonist. It is generally accepted that the intensity of the maximal IP response to agonist (Emax) is dependent on the number of binding sites at the surface of the cells and that the half-maximal response is obtained with an agonist concentration (EC50) similar to the Ki of the agonist(34.Gershengorn M.C. Heinflink M. Nussenzveig D.R. Hinkle P.M. Falck-Pedersen E. J. Biol. Chem. 1994; 269: 6779-6783Abstract Full Text PDF PubMed Google Scholar). Consequently, the coupling efficiency of different AT1A mutants can be estimated and compared using the EC50 and the ratio Emax/Bmax. Using these parameters, coupling of S105A, S107A, S109A, N111A, and S115A mutant receptors was compared with that of the wild-type AT1A after transient expression in COS-7 cells. No detectable IP response was observed in non-transfected COS-7 cells (data not shown), whereas a dose-dependent stimulation of IP production was measured in cells expressing the wild-type, the S105A, S107A, S109A, and N111A mutants. The half-maximal response (EC50) of these different AT1A receptors was obtained with AngII concentrations varying between 0.26 and 0.44 nM (Table 3), which are in a similar range to the corresponding Ki values of AngII for these receptors (Table 2).TABLE 3 Open table in a new tab The maximal stimulation of IP production (Fig. 2) is similar to the wild-type receptor for S105A and S109A and corresponds to a 6-fold increase above the basal production of total IP. These results show a functional coupling for these two mutants. In contrast, the maximal stimulation of IP production by the S107A and N111A mutants (Fig. 2) was lower (2-3-fold increase above the basal production), but these receptors were expressed at lower levels in COS-7 cells (1.1-1.3 × 105 sites/cell) than the wild-type or other mutants (4.3 to 6.0 × 105 sites/cell). Similarly, after stable expression in CHO cells, the expression level was also reduced by 5- or 6-fold for the S107A and N111A mutants as compared to the wild-type receptor (Table 1). However, the ratio Emax/Bmax× 105 sites and the EC50 value were similar for the two mutants as compared to the wild-type receptor (Table 3). These results indicate that the S107A and N111A mutations do not alter the ability of the receptor to couple to G-protein, but probably interfere with the biosynthesis and/or cell surface expression of AT1A.Figure 2:AngII-induced stimulation of total inositol phosphate production in COS cells expressing wild-type or mutant AT1A receptors. Dose-response curves were performed for the wild-type (▪), S105A (•), S107A (▵), S109A (▴), N111A (□), and S115A (○) AT1A receptors. The results are expressed as the ratio of the [3H]IP fraction (counts/min) derived from cells after exposure to agonist versus those obtained from cells exposed to buffer alone. Data points represent the mean ± S.E. of three independent experiments carried out in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutation of Ser115 results in a dramatic reduction of the ability of the receptor to mediate AngII-induced IP formation, despite the fact that this receptor is expressed at similar levels to those of the wild-type receptor in CHO cells (Table 3). Therefore, this polar residue in TM-III plays a crucial role in agonist-induced activation of the AT1A receptor, responsible for G-protein coupling and signal transduction. Two other polar residues (Asp74 and Tyr292) deeply located in the TM-II and -VII, respectively, have also been shown to be essential for receptor activation and co
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