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The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation

2009; Elsevier BV; Volume: 284; Issue: 18 Linguagem: Inglês

10.1074/jbc.m808113200

1083-351X

Ivana Domazet, Brian J. Holleran, Stéphane Martin, Pierre Lavigne, Richard Leduc, Emanuel Escher, Gaétan Guillemette,

Computational Drug Discovery Methods

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. The octapeptide hormone angiotensin II (AngII) 5The abbreviations used are: AngII, angiotensin II; AT1, angiotensin II type-1; GPCR, G protein-coupled receptor; TMD, transmembrane domain; SCAM, substituted cysteine accessibility method; MTSEA, methanethiosulfonateethylammonium; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. is the active component of the renin-angiotensin system. It exerts a wide variety of physiological effects, including vascular contraction, aldosterone secretion, neuronal activation, and cardiovascular cell growth and proliferation (1de Gasparo M. Catt K.J. Inagami T. Wright J.W. Unger T. Pharmacol. Rev. 2000; 52: 415-472PubMed Google Scholar). Virtually all of the known physiological effects of AngII are produced through the activation of the AT1 receptor, which belongs to the G protein-coupled receptor (GPCR) superfamily (2Burnier M. Circulation. 2001; 103: 904-912Crossref PubMed Scopus (425) Google Scholar, 3Miura S. Saku K. Karnik S.S. Hypertens. Res. 2003; 26: 937-943Crossref PubMed Scopus (99) Google Scholar). GPCRs possess seven transmembrane domains (TMD), which provide structural support for signal transduction. The AT1 receptor interacts with the G protein Gq/11, which activates a phospholipase C, which in turn generates inositol 1,4,5-trisphosphate and diacylglycerol from the cleavage of phosphatidylinositol 4,5-bisphosphate (4Balla T. Baukal A.J. Guillemette G. Morgan R.O. Catt K.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9323-9327Crossref PubMed Scopus (67) Google Scholar, 5Kojima I. Kojima K. Kreutter D. Rasmussen H. J. Biol. Chem. 1984; 259: 14448-14457Abstract Full Text PDF PubMed Google Scholar). Inositol 1,4,5-trisphosphate causes the release of Ca2+ from an intracellular store, whereas diacylglycerol activates protein kinase C. Like other GPCRs, the AT1 receptor undergoes spontaneous isomerization between its inactive state (favored in the absence of agonist) and its active state (induced or stabilized by the agonist) (6Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Movement of TMD helices through translational or rotational displacement is believed to be essential to achieve the active state (7Ghanouni P. Steenhuis J.J. Farrens D.L. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5997-6002Crossref PubMed Scopus (306) Google Scholar, 8Dunham T.D. Farrens D.L. J. Biol. Chem. 1999; 274: 1683-1690Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). It has been proposed that TMD3, TMD5, TMD6, and TMD7 may participate in the activation process of the AT1 receptor by providing a network of interactions through the AngII-binding pocket (9Inoue Y. Nakamura N. Inagami T. J. Hypertens. 1997; 15: 703-714Crossref PubMed Scopus (95) Google Scholar). The dynamics of this network are thought to be modified following agonist binding, thereby forcing the receptor to form new interactions between the TMDs. Based on homology with the high resolution structure of rhodopsin, the archetypal GPCR (10Palczewski K. Kumasaka T. Hori T. et al.Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar), it was expected that the binding site of the AT1 receptor would involve the seven mostly hydrophobic TMDs and would be accessible to charged water-soluble ligands, like AngII. For this receptor, the binding site would thus be contained within a water-accessible crevice, the binding pocket, extending from the extracellular surface of the receptor to the transmembrane portion. Using a photoaffinity labeling approach, we directly identified ligand-contact points within the second extracellular loop and the seventh TMD of the AT1 receptor (11Boucard A.A. Wilkes B.C. Laporte S.A. Escher E. Guillemette G. Leduc R. Biochemistry. 2000; 39: 9662-9670Crossref PubMed Scopus (65) Google Scholar, 12Laporte S.A. Boucard A.A. Servant G. Guillemette G. Leduc R. Escher E. Mol. Endocrinol. 1999; 13: 578-586Crossref PubMed Scopus (38) Google Scholar, 13Perodin J. Deraet M. Auger-Messier M. et al.Biochemistry. 2002; 41: 14348-14356Crossref PubMed Scopus (52) Google Scholar). Interestingly, numerous mutagenesis studies have provided the basis for a model in which an interaction between Asn-111 in TMD3 and Tyr-292 in TMD7 maintains the AT1 receptor in the inactive conformation. The agonist AngII would disrupt this interaction and promote the active conformational state (14Joseph M.P. Maigret B. Bonnafous J.C. Marie J. Scheraga H.A. J. Protein Chem. 1995; 14: 381-398Crossref PubMed Scopus (67) Google Scholar). In support of this model, it was further shown that substitution of Asn-111 for a residue of smaller size (Ala or Gly) confers constitutive activity on the AT1 receptor (15Balmforth A.J. Lee A.J. Warburton P. Donnelly D. Ball S.G. J. Biol. Chem. 1997; 272: 4245-4251Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Groblewski T. Maigret B. Larguier R. Lombard C. Bonnafous J.C. Marie J. J. Biol. Chem. 1997; 272: 1822-1826Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Feng Y.H. Miura S. Husain A. Karnik S.S. Biochemistry. 1998; 37: 15791-15798Crossref PubMed Scopus (85) Google Scholar). The substituted cysteine accessibility method (SCAM) (18Javitch J.A. Li X. Kaback J. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10355-10359Crossref PubMed Scopus (132) Google Scholar, 19Javitch J.A. Shi L. Liapakis G. Methods Enzymol. 2002; 343: 137-156Crossref PubMed Scopus (55) Google Scholar, 20Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (596) Google Scholar) is an ingenious approach for systematically identifying the residues in a TMD that contribute to the binding site pocket of a GPCR. Consecutive residues within TMDs are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substituted mutant is unchanged compared with wild-type receptor, it is assumed that the structure of the mutant receptor, especially around the binding site, is similar to that of wild type and therefore that the substituted cysteine lies in an orientation similar to that of the wild-type residue. In TMDs, the sulfhydryl of a cysteine oriented toward the binding site pocket should react faster with a positively charged sulfhydryl reagent like methanethiosulfonate-ethylammonium (MTSEA) than sulfhydryls facing the interior of the protein or the lipid bilayer. Two criteria are used for identifying engineered cysteines on the surface of the binding site pocket: (i) the reaction with MTSEA alters binding irreversibly, and (ii) the reaction is retarded by the presence of ligand. We previously used this approach to identify the residues in TMD3, TMD6, and TMD7 that form the surface of the binding site pocket in the wild-type AT1 receptor and in the constitutively active N111G-AT1 receptor (21Martin S.S. Boucard A.A. Clement M. Escher E. Leduc R. Guillemette G. J. Biol. Chem. 2004; 279: 51415-51423Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 22Martin S.S. Holleran B.J. Escher E. Guillemette G. Leduc R. Mol. Pharmacol. 2007; 72: 182-190Crossref PubMed Scopus (26) Google Scholar, 23Boucard A.A. Roy M. Beaulieu M.E. Lavigne P. Escher E. Guillemette G. Leduc R. J. Biol. Chem. 2003; 278: 36628-36636Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Here we report the application of SCAM to probe TMD2 in the wild-type and constitutively active receptors. Materials—Bovine serum albumin, bacitracin, and soybean trypsin inhibitor were from Sigma. The sulfhydryl-specific alkylating reagent MTSEA (CH3SO2-SCH2CH2NH3+) was purchased from Toronto Research Chemicals, Inc. (Toronto, Canada). The cDNA clone for the human AT1 receptor subcloned in the mammalian expression vector pcDNA3 was kindly provided by Dr. Sylvain Meloche (Université de Montréal). LipofectAmine2000® and culture media were obtained from Invitrogen. 125I-[Sar1,Ile8]AngII (specific radioactivity ∼1500 Ci/mmol) was prepared with IODO-GEN® (Perbio Science, Erembodegem, Belgium) according to the method of Fraker and Speck (24Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar) and as previously reported (25Guillemette G. Escher E. Biochemistry. 1983; 22: 5591-5596Crossref PubMed Scopus (54) Google Scholar). Numbering of Residues in TMD2—Residues in TMD2 of the human AT1 receptor were given two numbering schemes. First, residues were numbered according to their positions in the human AT1 receptor sequence. Second, residues were also indexed according to their position relative to the most conserved residue in the TMD in which it is located (26Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4347) Google Scholar). By definition, the most conserved residue was assigned the position index “50” (e.g. in TMD2, Asp-74 is the most conserved residue and was designated Asp-74(2.50), whereas the upstream residue was designated Leu-75(2.51) and the downstream residue was designated Ala-73(2.49)). This indexing simplified the identification of aligned residues in different GPCRs. Oligodeoxynucleotide Site-directed Mutagenesis—Site-directed mutagenesis was performed on the wild-type AT1 receptor with the overlap PCR method (Expand High Fidelity PCR System; Roche Applied Science). Briefly, forward and reverse oligonucleotides were constructed to introduce cysteine mutations between Leu-70(2.46) and Trp-94(2.70). PCR products were subcloned into the HindIII-XbaI sites of the mammalian expression vector pcDNA3.1. Site-directed mutations were then confirmed by automated DNA sequencing by aligning the AT1 sequence with multiAlin (26Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4347) Google Scholar). Cell Culture and Transfection—COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 2 mm l-glutamine and 10% (v/v) fetal bovine serum. The cells were seeded into 100-mm culture dishes at a density of 2 × 106 cells/dish. When the cells reached ∼90% confluence, they were transfected with 4 μg of plasmid DNA and 15 μl of lipofectAmine2000®. After 24 h, transfected cells were trypsinized, distributed into 12-well plates, and grown for an additional 24 h in complete DMEM containing 100 IU/ml penicillin and 100 μg/ml streptomycin before the MTSEA treatment and binding assay. Binding Experiments—COS-7 cells were grown for 36 h post-transfection in 100-mm culture dishes, washed once with phosphate-buffered saline (PBS), and subjected to one freeze-thaw cycle. Broken cells were then gently scraped into washing buffer (25 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm MgCl2), centrifuged at 2500 × g for 15 min at 4 °C, and resuspended in binding buffer (25 mm Tris-HCl, pH 7.4, 100 mm NaCl, 5 mm MgCl2, 0.1% bovine serum albumin, 0.01% bacitracin, 0.01% soybean trypsin inhibitor). Saturation binding experiments were done by incubating broken cells (20–40 μg of protein) for 1 h at room temperature with increasing concentrations of 125I-[Sar1,Ile8]AngII in a final volume of 500 μl. Nonspecific binding was determined in the presence of 1 μm unlabeled [Sar1,Ile8]AngII. Bound radioactivity was separated from free ligand by filtration through GF/C filters presoaked for at least 3 h in binding buffer. Receptor-bound radioactivity was evaluated by γ-counting. Treatment with MTSEA—MTSEA treatment was performed according to the procedure of Javitch et al. (18Javitch J.A. Li X. Kaback J. Karlin A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10355-10359Crossref PubMed Scopus (132) Google Scholar), with minor modifications. Two days after transfection, the cells, which were grown in 12-well plates, were washed with PBS and incubated for 3 min at room temperature with freshly prepared MTSEA at the desired concentrations (typically from 0.5 to 6 mm) in a final volume of 0.2 ml. The reaction was stopped by washing the cells with ice-cold PBS. Intact cells were then incubated in binding medium (DMEM, 25 mm HEPES, 0.1% bovine serum albumin, pH 7.4) containing 0.05 nm 125I-[Sar1,Ile8]AngII for 90 min at room temperature. After washing with ice-cold PBS, the cells were lysed with 0.1 n NaOH, and the radioactivity was evaluated by γ-counting. The percentage of sfractional binding inhibition was calculated as (1 – (specific binding after the MTSEA treatment/specific binding without the treatment)) × 100. Protection against MTSEA Reaction by [Sar1,Ile8]AngII—Transfected cells grown in 12-well plates were washed once with PBS and incubated in the presence or absence of 100 nm [Sar1,Ile8]AngII for 1 h at 16 °C (to avoid internalization of receptors). The cells were washed to remove excess ligand and then treated with MTSEA. The cells were washed three times with ice-cold PBS and once with an acidic buffer (150 mm NaCl, 50 mm acetic acid, pH 3.0) to dissociate bound ligand. They were then incubated for 3 h at 16 °C in binding medium (DMEM, 25 mm HEPES, 0.1% bovine serum albumin, pH 7.4) containing 0.05 nm 125I-[Sar1,Ile8]AngII. The percentage of protection was calculated as ((inhibition in the absence of [Sar1,Ile8]AngII)–(inhibition in the presence of [Sar1,Ile8]AngII)/(inhibition in the absence of [Sar1,Ile8]AngII)) × 100. Phospholipase C Assay—Inositol phosphate accumulation was determined as described previously (27Lanctot P.M. Leclerc P.C. Escher E. Leduc R. Guillemette G. Biochemistry. 1999; 38: 8621-8627Crossref PubMed Scopus (63) Google Scholar). In brief, COS-7 cells were seeded in 6-well plates, transfected, and labeled for 16 h in serum-free M199 containing 10 μCi/ml [myo-3H]inositol (MP Biomedicals, Solon, OH). Cells were washed twice with PBS plus 0.1% (w/v) dextrose and then incubated in stimulation buffer (DMEM containing 25 mm HEPES, 10 mm LiCl, and 0.1% bovine serum albumin, pH 7.4) for 30 min at 37 °C. Incubations were terminated by the addition of ice-cold perchloric acid (final concentration, 5% (v/v)). Water-soluble inositol phosphates were then extracted with an equal volume of a 1:1 (v/v) mixture of 1,1,2-trichlorotrifluoroethane and tri-N-octylamine. The samples were mixed vigorously and centrifuged at 2500 × g for 30 min. The upper phase containing inositol phosphates was applied to an AG1-X8 resin column (Bio-Rad). Inositol phosphates were eluted sequentially by the addition of an ammonium formate/formic acid solution of increasing ionic strength. Fractions containing inositol phosphates were collected and measured in a liquid scintillation counter. Data Analysis—Results are presented as means ± S.D. Specific binding data (Bmax and Kd) were analyzed with Prism version 4.0 for Windows (GraphPad Software, San Diego CA), using a one-site binding hyperbola nonlinear regression analysis. Binding Properties of Mutant Receptors Bearing Cysteines in TMD2—To identify the residues in TMD2 that face the binding site pocket of the AT1 receptor, we mutated 24 consecutive residues between Leu-70(2.47) and Trp-94(2.70) to cysteine, one at a time. Each mutant receptor was transiently expressed in COS-7 cells. To assess the conservation of the global conformation of these receptors after the substitution, pharmacological parameters describing the equilibrium binding of the radiolabeled competitive ligand 125I-[Sar1,Ile8]AngII, such as Kd and Bmax, were determined (Table 1). All mutant AT1 receptors exhibited high binding affinity for 125I-[Sar1,Ile8]AngII (similar to that of the wild-type AT1 receptor) except for mutants P82C(2.58), W84C(2.60), Y87C(2.63), Y92C(2.68), and W94C(2.70), which did not demonstrate any detectable binding activity and were not used for the SCAM analysis. Bmax values for all detectable receptors ranged from 490 to 4282 fmol/mg of protein.TABLE 1Binding Properties of [Sar1,Ile8]AngII to cysteine-substituted hAT1 mutant receptorsKdBmaxnnmfmol/mgWild type (Cys-76)0.6 ± 0.34282 ± 90311L70C0.7 ± 0.21428 ± 7964A71C0.9 ± 0.32729 ± 5233L72C0.4 ± 0.21923 ± 5494A73C0.4 ± 0.11814 ± 3093D74C1.2 ± 0.71141 ± 3204L75C0.9 ± 0.41405 ± 2323F77C3.1 ± 0.62256 ± 4783L78C0.4 ± 0.11412 ± 3003L79C0.5 ± 0.21833 ± 10203T80C0.3 ± 0.01918 ± 7113L81C4.0 ± 1.32415 ± 8723L83C1.8 ± 0.21288 ± 6913A85C0.9 ± 0.31074 ± 4054V86C0.6 ± 0.31709 ± 1403T88C3.2 ± 1.95633 ± 723A89C0.5 ± 0.11061 ± 4513M90C1.6 ± 1.0490 ± 203E91C2.1 ± 0.61622 ± 9503R93C0.8 ± 0.31152 ± 673 Open table in a new tab Effect of Extracellularly Added MTSEA on the Binding Properties of Mutant Receptors—To verify whether the reporter cysteines introduced into the TMD2 of the AT1 receptor were oriented toward the binding pocket, mutant receptors were treated with concentrations of MTSEA varying between 0.5 and 6 mm. We also verified whether the wild-type AT1 receptor, which contains 10 endogenous cysteines (Fig. 1), was sensitive to MTSEA treatment. Fig. 2 shows that various concentrations of MTSEA had very little effect (no more than a 25% reduction) on the binding properties of the wild-type AT1 receptor, indicating that the endogenous cysteines made a relatively small contribution to the binding site pocket. A 3-min treatment with 0.5 mm MTSEA (Fig. 3A) strongly inhibited the binding properties of mutants L81C(2.57)-AT1 (binding inhibition of 65%), A85C(2.61)-AT1 (binding inhibition of 71%), and T88C(2.64)-AT1 (binding inhibition of 75%), whereas it had only a minor effect on the binding properties of the other mutant receptors. At higher MTSEA concentrations (2 mm and above), the binding properties of mutant receptors D74C(2.50)-AT1 and A89C(2.65)-AT1 were also slightly affected (Fig. 3B). Overall, the most reactive cysteines were those substituted for L81C(2.57), A85C(2.61), and T88C(2.64), whereas the cysteine substituted for D74C(2.50) and A89C(2.65) were less reactive.FIGURE 2MTSEA treatment of the wild-type AT1 receptor and sensitive reporter cysteine-bearing mutant receptors. Intact COS-7 cells transiently expressing wild-type, D74C, L81C, A85C, T88C, or A89C AT1 receptors were incubated for 3 min at room temperature with increasing concentrations of freshly prepared MTSEA (0.5–6 mm). The intact cells were then incubated for 90 min at room temperature with 0.05 nm 125I-[Sar1,Ile8]AngII, and their binding properties were evaluated as indicated under “Experimental Procedures.” Each curve represents the means ± S.D. of data obtained from at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Effects of MTSEA on different mutant AT1 receptors bearing a reporter cysteine in TMD2. Intact COS-7 cells transiently expressing wild-type or mutant AT1 receptors were incubated for 3 min at room temperature with freshly prepared 0.5 mm MTSEA (A) or 2 mm MTSEA (B). The intact cells were then incubated for 90 min at room temperature with 0.05 nm 125I-[Sar1,Ile8]AngII. The percentage of binding inhibition was calculated as indicated under “Experimental Procedures.” The vertical line represents an arbitrary threshold used to identify cysteine-sensitive mutants and was set at a value corresponding to binding inhibition 20% greater than the value for the wild-type AT1 receptor. The white bars indicate mutant receptors for which binding activities were not appreciably reduced compared with the wild-type receptor after treatment with MTSEA. The gray and black bars indicate mutant receptors for which binding activities were slightly reduced (gray) or strongly reduced (black) after treatment with MTSEA. Each bar represents the means ± S.D. of data from at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Altered Accessibility to TMD2 Reporter Cysteines in the Constitutively Active N111G-AT1 Receptor—We made use of the constitutively active N111G-AT1 receptor to assess and map the potentially altered accessibility of MTSEA to the engineered cysteines. We first determined the pharmacological properties of the 24 cysteine-substituted mutant receptors. Within the N111G-AT1 receptor background, 18 cysteine-substituted mutants conserved a high binding affinity for the competitive ligand 125I-[Sar1,Ile8]AngII (Table 2). The mutant receptors P82C(2.58)-N111G-AT1, L83C(2.59)-N111G-AT1, W84C(2.60)-N111G-AT1, Y87C(2.63)-N111G-AT1, Y92C(2.68))-N111G-AT1, and W94C(2.70)-N111G-AT1 did not have any detectable binding activity and were not used for the SCAM analysis. Bmax values for all detectable receptors ranged from 570 to 3967 fmol/mg protein (Table 2).TABLE 2Binding properties of [Sar1,Ile8]AngII to cysteine-substituted hAT1 mutant receptors bearing the N111G mutationKdBmaxnnMfmol/mgN111G0.8 ± 0.23003 ± 88510L70C3.6 ± 1.63087 ± 2353A71C1.3 ± 0.22787 ± 7773L72C0.9 ± 0.62688 ± 8773A73C0.6 ± 0.13967 ± 15164D74C1.8 ± 0.11781 ± 7753L75C0.6 ± 0.02336 ± 6893F77C0.7 ± 0.03004 ± 2133L78C0.6 ± 0.11645 ± 7043L79C0.5 ± 0.01868 ± 2803T80C0.4 ± 0.03395 ± 4563L81C1.7 ± 0.21780 ± 2523A85C0.9 ± 0.1622 ± 4293V86C2.6 ± 0.6570 ± 2023T88C0.7 ± 0.1819 ± 6083A89C1.0 ± 0.41019 ± 8543M90C2.9 ± 1.0980 ± 4923E91C2.1 ± 0.61622 ± 9503R93C1.2 ± 0.51175 ± 753 Open table in a new tab Fig. 4 (see also Fig. 2) shows that, like the wild-type receptor, the N111G-AT1 receptor was relatively insensitive to a 3-min treatment with MTSEA concentrations ranging from 0.5 to 2 mm, again indicating the relatively low contribution of the endogenous cysteines to the binding site pocket. Cysteine-substituted N111G-AT1 receptor mutants were also treated with increasing concentrations of MTSEA, and their binding properties were assessed with 125I-[Sar1,Ile8]AngII. Fig. 5 summarizes the effect of the MTSEA treatment on the different cysteine-substituted N111G-AT1 receptor mutants. As observed in the wild-type background, 0.5 mm MTSEA decreased the binding activity of the A85C(2.61)-N111G-AT1 and T88C(2.64)-N111G-AT1 by 78 and 82%, respectively. It is noteworthy that the V86C(2.62)-N111G-AT1 mutant, which was insensitive to MTSEA in the wild-type background, had reduced binding activity by 47% in the N111G-AT1 background after a treatment with 0.5 mm MTSEA. On the other hand, L81C(2.57)-N111G-AT1 which exhibited a significant reduction in binding activity in the wild-type background, became less sensitive to MTSEA in the N111G-AT1 background (reduction of 39% in binding activity after a treatment with 2 mm MTSEA) and that D74C(2.50)-N111G-AT1 became completely insensitive to treatment with MTSEA.FIGURE 5Effect of MTSEA on different mutant N111G-AT1 receptors bearing a reporter cysteine in TMD2. Intact COS-7 cells transiently expressing the mutant N111G-AT1 receptors were incubated for 3 min at room temperature with freshly prepared 0.5 mm MTSEA (A) or 2 mm MTSEA (B). The intact cells were then incubated for 90 min at room temperature with 0.05 nm 125I-[Sar1, Ile8]AngII. The percentage of binding inhibition was calculated as indicated under “Experimental Procedures.” The vertical line represents an arbitrary threshold used to identify cysteine-sensitive mutants. It was set at a value corresponding to binding inhibition 20% greater than the value for the N111G-AT1 receptor. The white bars indicate mutant receptors for which binding activities were not appreciably reduced compared with that of the N111G-AT1 receptor after treatment with MTSEA. The gray and black bars indicate mutant receptors for which binding activities were slightly reduced (gray) or strongly reduced (black) after treatment with MTSEA. Each bar represents the means ± S.D. of data from at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Protection against MTSEA Reaction by a Pretreatment with [Sar1,Ile8]AngII—To confirm that reporter cysteines accessible to MTSEA are located within the binding pocket, receptor mutants were saturated with the competitive ligand [Sar1,Ile8]AngII prior to the MTSEA treatment. The cells were then washed with an acid buffer to dissociate the bound ligand. The receptors were then assayed for binding with the radiolabeled competitive ligand. Fig. 6 shows how a preincubation with the competitive ligand [Sar1,Ile8]AngII protected mutant receptors D74C(2.50)-AT1, L81C(2.57)-AT1, A85C(2.61)-AT1, T88C(2.64)-AT1, A89C(2.65)-AT1, L81C(2.57)-N111G-AT1, A85C(2.61)-N111G-AT1, V86C(2.62)-N111G-AT1, T88C(2.64)-N111G-AT1, and A89C(2.65)-N111G-AT1 from the inhibitory effect of MTSEA, with protection levels ranging from 59 to 91%. Functional Properties of Wild-type and Mutant AT1 Receptors—The functional properties of wild-type and mutant AT1 receptors were evaluated by measuring phospholipase C activity in transiently transfected COS-7 cells. Fig. 7 shows the relative amounts of inositol phosphates accumulated under basal conditions. Cells expressing cysteine mutant receptors in the wild-type background produced inositol phosphates at levels that were not significantly different from that produced by cells expressing the wild-type AT1 receptor. The basal level of inositol phosphates found in cells expressing cysteine mutant receptors in the N111G background was at least 3-fold higher than that found in cells expressing the wild-type AT1 receptor, thereby confirming the constitutive activity of all receptors bearing both cysteine and N111G mutations. These results show that cysteine mutations within TMD2 do not compromise the functional integrity of either the ground state AT1 receptor or the constitutively active N111G-AT1 receptor. The rationale of this