Portal de Periódicos da CAPES

Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay

2005; Elsevier BV; Volume: 280; Issue: 29 Linguagem: Inglês

10.1074/jbc.m413653200

1083-351X

Martin Clément, Stéphane Martin, Marie‐Eve Beaulieu, Caroline Chamberland, Pierre Lavigne, Richard Leduc, Gaétan Guillemette, Emanuel Escher,

Monoclonal and Polyclonal Antibodies Research

The peptide hormone angiotensin II (AngII) binds to the AT1 (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. A series of 44 transmembrane domain III, VI, and VII X → Met mutants photolabeled either with 125I-[Sar1,p′-benzoyl-l-Phe8]AngII or with 125I-[Sar1,p″-methoxy-p′-benzoyl-l-Phe8]AngII were purified and digested with cyanogen bromide. Several mutants produced digestion patterns different from that observed with wild type human AT1, indicating that they had a new receptor contact with position 8 of AngII. The following residues form this binding pocket: L112M and Y113M in transmembrane domain (TMD) III; F249M, W253M, H256M, and T260M in TMD VI; and F293M, N294M, N295M, C296M, and L297M in TMD VII. Homology modeling and incorporation of these contacts allowed us to develop an evidence-based molecular model of interactions with human AT1 that is very similar to the rhodopsin-retinal interaction. The peptide hormone angiotensin II (AngII) binds to the AT1 (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket. A series of 44 transmembrane domain III, VI, and VII X → Met mutants photolabeled either with 125I-[Sar1,p′-benzoyl-l-Phe8]AngII or with 125I-[Sar1,p″-methoxy-p′-benzoyl-l-Phe8]AngII were purified and digested with cyanogen bromide. Several mutants produced digestion patterns different from that observed with wild type human AT1, indicating that they had a new receptor contact with position 8 of AngII. The following residues form this binding pocket: L112M and Y113M in transmembrane domain (TMD) III; F249M, W253M, H256M, and T260M in TMD VI; and F293M, N294M, N295M, C296M, and L297M in TMD VII. Homology modeling and incorporation of these contacts allowed us to develop an evidence-based molecular model of interactions with human AT1 that is very similar to the rhodopsin-retinal interaction. The octapeptide hormone angiotensin II (AngII) 1The abbreviations used are: AngII, angiotensin II; hAT1, human angiotensin type 1 (receptor); Bpa, p-benzoyl-l-phenylalanine; ECL, extracellular loop; GPCR, G-protein coupled receptors; MPA, methionine proximity assay; SCAM, substituted cysteine accessibility method; TMD, transmembrane domain; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; wt, wild type. (Fig. 1A) is the active component of the renin-angiotensin system. Virtually all known physiological effects of AngII are produced through the activation of the hAT1 receptor, which belongs to the class A rhodopsin-like family of the heptahelical G protein-coupled receptor (GPCR) superfamily (1Miura S. Saku K. Seikagaku. 2003; 75: 382-386PubMed Google Scholar, 2Miura S. Zhang J. Boros J. Karnik S.S. J. Biol. Chem. 2003; 278: 3720-3725Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Elucidating the stereochemistry of the ligand-receptor interaction is vital for understanding the mechanism of ligand binding, GPCR activation, and, eventually, rational drug design. In the past, much effort was devoted to identifying the domains or individual residues of a given receptor that may interact with its ligand. Most experiments to address ligand-receptor interactions were performed with series of receptor mutants to identify specific residues critical to ligand binding (3Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (134) Google Scholar, 4Marie 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, 5Karnik S.S. Husain A. Graham R.M. Clin. Exp. Pharmacol. Physiol. Suppl. 1996; 3: S58-S66Crossref PubMed Google Scholar). It is, however, speculative to deduce precise structures of ligand-receptor interactions through mutagenesis studies alone. More direct approaches have therefore been used to study ligand-receptor interactions. Among these is photoaffinity labeling, which allows covalent incorporation of the ligand within its binding site, presumably at the contact area of the photolabel in the receptor. This ligand-receptor contact can be identified by specific enzymatic or chemical digestion of the labeled receptor (6Servant G. Dudley D.T. Escher E. Guillemette G. Biochem. J. 1996; 313: 297-304Crossref PubMed Scopus (38) Google Scholar) or by mass spectrometry (7Sachon E. Bolbach G. Lavielle S. Karoyan P. Sagan S. FEBS Lett. 2003; 544: 45-49Crossref PubMed Scopus (13) Google Scholar). The binding pockets within the transmembrane domains of several bioamine receptors have been identified using this kind of approach. The adenosine A1 receptor (8Kennedy A.P. Mangum K.C. Linden J. Wells J.N. Mol. Pharmacol. 1996; 50: 789-798PubMed Google Scholar) and the β2 adrenergic receptor (9Dohlman H.G. Song J. Apanovitch D.M. DiBello P.R. Gillen K.M. Semin. Cell Dev. Biol. 1998; 9: 135-141Crossref PubMed Scopus (42) Google Scholar, 10Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (1008) Google Scholar) are typical examples. Peptidergic receptors such as hAT1 and hAT2 (11Servant G. Laporte S.A. Leduc R. Escher E. Guillemette G. J. Biol. Chem. 1997; 272: 8653-8659Abstract Full Text Full Text PDF PubMed Scopus (43) 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), neurokinin receptors (13Bremer A.A. Leeman S.E. Boyd N.D. J. Biol. Chem. 2001; 276: 22857-22861Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and several other receptors from the secretin GPCR family B (14Dong M. Wang Y. Pinon D.I. Hadac E.M. Miller L.J. J. Biol. Chem. 1999; 274: 903-909Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) have been also studied using this approach. We previously identified ligand-contact points within the second extracellular loop (ECL) and the seventh transmembrane domain (TMD) of the hAT1 receptor (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, 15Boucard A.A. Wilkes B.C. Laporte S.A. Escher E. Guillemette G. Leduc R. Biochemistry. 2000; 39: 9662-9670Crossref PubMed Scopus (65) Google Scholar, 16Perodin J. Deraet M. Auger-Messier M. Boucard A.A. Rihakova L. Beaulieu M.E. Lavigne P. Parent J.L. Guillemette G. Leduc R. Escher E. Biochemistry. 2002; 41: 14348-14356Crossref PubMed Scopus (52) Google Scholar). Although photoaffinity labeling has been widely used to study peptidergic GPCR binding pockets, generally only a single contact point between a given ligand and its cognate receptor has been identified. The resulting information does not, however, induce sufficient restrictions to generate credible GPCR structures in the ligand-bound state using homology modeling. Labeling studies using benzophenone residues have identified many ligand-receptor contact points with a surprisingly high ratio of methionine contacts (17Li Y.M. Marnerakis M. Stimson E.R. Maggio J.E. J. Biol. Chem. 1995; 270: 1213-1220Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Kage R. Leeman S.E. Krause J.E. Costello C.E. Boyd N.D. J. Biol. Chem. 1996; 271: 25797-25800Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 19Bisello A. Adams A.E. Mierke D.F. Pellegrini M. Rosenblatt M. Suva L.J. Chorev M. J. Biol. Chem. 1998; 273: 22498-22505Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Despite the fact that methionine represents a small proportion of the proteinogenic amino acids in most receptors, p-benzoyl-l-phenylalanine (Bpa)-containing peptide labels (Fig. 1A) have been shown to incorporate into Met residues at a disproportionate frequency (13Bremer A.A. Leeman S.E. Boyd N.D. J. Biol. Chem. 2001; 276: 22857-22861Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Previous photochemical studies of the benzophenone radical have indicated that it exhibits strong selectivity for thioether groups (20Bobrowski K. Marciniak B. Hug G.L. J. Am. Chem. Soc. 1992; 104: 10279-10288Crossref Scopus (101) Google Scholar, 21Marciniak B. Bobrowski K. Hug G.L. J. Phys. Chem. 1993; 97: 11937-11943Crossref Scopus (97) Google Scholar). This selectivity would explain the high ratio of methionine insertion into target proteins through benzophenone photoaffinity labeling. This property can be exploited to introduce Met residues into target structures as "bait" with the goal of identifying other receptor residues that are in close proximity to the ligand label. The immediate molecular environment of this ligand residue can thus be determined. We used this strategy to investigate the binding environment of the C-terminal residue of AngII within the hAT1 receptor. We used two benzophenone-containing labeling peptides, [Sar1,Bpa8]AngII, a well characterized neutral antagonist on the hAT1 receptor (22Bosse R. Servant G. Zhou L.M. Boulay G. Guillemette G. Escher E. Regul. Pept. 1993; 44: 215-223Crossref PubMed Scopus (28) Google Scholar), and [Sar1, p′-MeO-Bpa8]AngII, which is also an antagonist. For the receptor, a systematic Met mutagenesis strategy was applied to TMD III, TMD VI, and TMD VII to identify other receptor contacts and thus define the binding environment of the C-terminal residue of AngII. Materials—Bovine serum albumin, bacitracin, soybean trypsin inhibitor, and CNBr were from Sigma-Aldrich. Acetonitrile was from Fisher Scientific. Culture media were from Invitrogen. FuGENE 6 transfection reagent and the protease inhibitor mixture were purchased from Roche Diagnostics. X-ray films (Kodak Biomax® MS) with intensifying screens from Fischer Scientific were used to visualize CNBr digestion fragments. Photolabeling yields were determined using Quantity One® quantitation software from Bio-Rad. Numbering of Residues—The residues of the human AT1 receptor were given two numbering schemes. First, residues were numbered based on their positions in the human AT1 receptor sequence. Second, residues were numbered based on their position relative to the most conserved residue in their respective TMDs within class A GPCRs (23Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2578) Google Scholar). By definition, the most conserved residue was assigned the position index 50, with incremental numbering of downstream residues and decremental numbering of upstream residues, respectively. This indexing simplifies the identification of aligned residues in different GPCRs. Oligodeoxynucleotide Site-directed Mutagenesis—Site-directed mutagenesis was performed on the wt-hAT1 receptor using the overlap PCR method described elsewhere (24Boucard A.A. Sauve S.S. Guillemette G. Escher E. Leduc R. Biochem. J. 2003; 370: 829-838Crossref PubMed Scopus (45) Google Scholar). Mutant receptors were subcloned into HindIII-XbaI sites of the mammalian expression vector pcDNA3.1. Site-directed mutations were confirmed by manual and automated DNA sequencing. Synthesis and Radioiodination of Photoligands—[Sar1,Bpa8]AngII was prepared according to Bosse et al. (22Bosse R. Servant G. Zhou L.M. Boulay G. Guillemette G. Escher E. Regul. Pept. 1993; 44: 215-223Crossref PubMed Scopus (28) Google Scholar). [Sar1,p′-MeO-Bpa8]AngII: p′-methoxy-p-methyl benzophenone was prepared according to Horner (25Horner M. Chem. Ber. 1952; 85: 520-530Crossref Scopus (4) Google Scholar). Photobromination, resin alkylation, and peptide syntheses were carried out as described previously (22Bosse R. Servant G. Zhou L.M. Boulay G. Guillemette G. Escher E. Regul. Pept. 1993; 44: 215-223Crossref PubMed Scopus (28) Google Scholar). The peptides were purified by reversed phase chromatography, which also permitted the separation of diastereomer peptides. Peptide purity as assessed by high performance liquid chromatography was at least 95%. The correct stereochemistry was assigned through comparison by high performance liquid chromatography with [Sar1,l-Bpa8]AngII and [Sar1,d-Bpa8]AngII made with Boc-l-Bpa and Boc-d-Bpa. Purified peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Tofspec2, Micromass). All 125I-AngII peptides (∼1500 Ci/mmol) were prepared using Iodogen® (Perbio Science, Erembodegem, Belgium) as described by Fraker and Speck (26Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3784) Google Scholar), except that an acetic acid buffer (pH 5.4) was used. The radiolabeled peptides were purified by high performance liquid chromatography on a C-18 column (Waters) with a 20-40% acetonitrile gradient in 0.05% aqueous trifluoroacetic acid. The specific radioactivity of the radiolabeled peptides was determined by self-displacement and saturation binding analysis. Cell Cultures and Transfection of COS-7 Cells—COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine, 10% (v/v) fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. The cells were incubated at 37 °C in a 5% CO2 atmosphere. Cells were transfected at ∼70% confluency with FuGENE 6 transfection reagent as per the manufacturer's instructions. Thirty-six hours after the initiation of transfection, the cells were washed once with phosphate-buffered saline (137 mm NaCl, 0.9 mm MgCl2, 3.5 mm KCl, 0.9 mm CaCl2, 8.7 mm Na2HPO4, and 3.5 mm NaHPO4) and immediately stored at -80 °C until used. Binding Studies and Photoaffinity Labeling—Frozen transfected COS-7 cells were thawed for 1 min at 37 °C. The broken cells were then gently scraped, resuspended in 10 ml of washing buffer (25 mm Tris-HCl (pH 7.4), 100 mm NaCl, and 5 mm MgCl2), and centrifuged (500 × g for 10 min at 4 °C). The pellet was dispersed in binding buffer (25 mm Tris-HCl (pH 7.4), 100 mm NaCl, 5 mm MgCl2, and 0.1% (w/v) bovine serum albumin). For binding studies, the broken cell suspension (50-80 μg of protein) was incubated for 60 min at room temperature in the presence of 0.1 nm 125I-[Sar1,Ile8]AngII (1,500 Ci/mmol) with increasing concentrations of test peptide (15 concentration points in duplicate from 10-12 to 10-5 m with half-log increases. Bound radioactivity was separated from free ligand by filtration at 4 °C through GF/C filters pre-soaked in binding buffer. Receptor-bound radioactivity was evaluated by γ-counting. Results are presented as means ± S.D. Binding data were analyzed with the Kell program (Biosoft, Ferguson, MO), which uses a weighted nonlinear curve-fitting routine. Maximal binding capacities were determined by approximation using the formula (B/T·IC50) (27Swillens S. Trends Pharmacol. Sci. 1992; 13: 430-434Abstract Full Text PDF PubMed Scopus (46) Google Scholar) from the displacement studies. For photolabeling studies, the broken cell suspension (1 mg of protein) was incubated for 90 min at room temperature in the presence of 3 nm 125I-[Sar1, Bpa8]AngII or 125I-[Sar1,p′-MeO-Bpa8]AngII. After centrifugation at 500 × g, the pelleted broken cells were washed once and resuspended in 0.5 ml of ice-cold washing buffer and then irradiated for 60 min on ice under filtered (Raymaster black light filters, catalog number 5873, Gates and Co. Inc., Franklin Square, NY.) UV light (365 nm) (100 watt mercury vapor lamp, serial number JC-Par-38, Westing-house). After centrifugation (2,500 × g for 10 min at 4 °C), the pellet was solubilized for 30 min at 4 °C in modified radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 8), 150 mm NaCl, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, and 1% (v/v) Nonidet P-40 supplemented with a protease inhibitor mixture (Complete EDTA-free) (Roche Diagnostics). The cell lysate was centrifuged (15,000 × g for 25 min at 4 °C) to remove insoluble material, and the supernatant was kept at -20 °C until used. Partial Purification of the Labeled Complex—The solubilized photolabeled receptor complexes were diluted in an equal volume of 2× Laemmli buffer (120 mm Tris-HCl (pH 6.8), 20% (v/v) glycerol, 4% (w/v) SDS, 200 mm dithiothreitol, and 0.05% (w/v) bromphenol blue) and incubated for 60 min at 37 °C. SDS-PAGE was performed as described previously (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (213983) Google Scholar) using a 7.5% polyacrylamide preparative gel. The gel was then cut into slices, and the radioactive content was measured using a γ-counter. The labeled receptor was passively eluted from the gel slices into fresh electrophoresis buffer (25 mm Trizma Tris base (pH 8.3), 250 mm glycine, and 0.1% (w/v) SDS) for 3-4 days at 4 °C with gentle agitation as described by Blanton and Cohen (29Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (212) Google Scholar). The eluate (∼40 ml) was concentrated to a final volume of 0.100-0.250 ml using an Amicon-10 filter (Millipore) and stored at -20 °C. CNBr Hydrolysis—The partially purified photolabeled receptor (3,500-10,000 cpm) was diluted in a 3:5 mixture of 30% trifluoroacetic acid and CNBr dissolved in 100% acetonitrile to obtain a final concentration of 50 mg/ml. Samples were incubated at room temperature in the dark for 16-18 h. One milliliter of water was added to terminate the reaction. The samples were lyophilized and resuspended in Laemmli buffer (1× final), loaded at 2,500-3,500 cpm on 16.5% SDS-polyacrylamide Tris-Tricine gels (Bio-Rad), and revealed by autoradiography on x-ray films (Kodak Biomax® MS). 14C-Labeled low molecular protein standards (Invitrogen) were used to determine the apparent molecular masses. Running conditions and fixation procedures were performed according to the manufacturer's instructions. Inositol Phosphate Production—COS-7 cells were seeded in six-well plates, transfected, and labeled for 24 h in serum-free, inositol-free Dulbecco's modified Eagle's medium containing 10 μCi/ml myo-[3H]-inositol (Amersham Biosciences). Cells were washed twice with phosphate-buffered saline containing 0.1% (w/v) dextrose and then incubated in stimulation buffer (Dulbecco's modified Eagle's medium containing 25 mm Hepes, 10 mm LiCl, and 0.1% bovine serum albumin, pH 7.4) for 30 min at 37 °C. Inositol phosphate production was induced with 100 nm AngII for 10 min at 37 °C in stimulation buffer. Incubations were terminated by the addition of ice-cold perchloric acid (5% (v/v) final concentration). 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 the inositol phosphates was applied to an AG1-X8 resin column (Bio-Rad). The 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. Molecular Modeling—All calculations were performed on a Silicon Graphics Octane2 work station (Silicon Graphics Inc. Mountain View, CA). Molecular modeling of the hAT1 receptor and the complex [Sar1,Bpa8]AngII-hAT1 receptor was done with the INSIGHTII suite of programs (Homology, Discover, and Biopolymer; Accelrys, San Diego, CA). The molecular model of hAT1 (Swiss-Prot accession number P30556) was based on the bovine rhodopsin structure (Protein Data Bank code 1L9H) (30Okada T. Fujiyoshi Y. Silow M. Navarro J. Landau E.M. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5982-5987Crossref PubMed Scopus (664) Google Scholar). The sequence alignment (supplemental data, available in the on-line version of this article) between the hAT1 receptor and the bovine rhodopsin used to identify and assign the structurally conserved regions had all the strictly conserved residues of family A members aligned: Asn-55-Asn-46(1.50), Asp-83-Asp-74(2.50), Arg-135-Arg-126(3.50), Trp-161-Trp-153(4.50), Pro-215-Pro-207(5.50), Pro-267-Pro-255(6.50), and Pro-303-Pro-299(7.50). The coordinates of the assigned structurally conserved regions were then transferred to the sequence of hAT1, followed by ECL2 and ECL3 generation with the data base in HOMOLOGY. The conserved disulfide bond between ECL1 and ECL2 was then added to hAT1, and the potential energy was minimized sequentially using Discover with a consistent valence force field (31Dauber-Osguthorpe P. Roberts V.A. Osguthorpe D.J. Wolff J. Genest M. Hagler A.T. Proteins. 1988; 4: 31-47Crossref PubMed Scopus (1992) Google Scholar). In the first step of the minimization, all of the heavy atoms were fixed and all atoms, except those of the TMD backbones, were free to move. In the final step, all of the atoms were unrestrained. To allow ligand [Sar1,Bpa8]AngII to be incorporated into the receptor, a cavity has to be generated. This was accomplished by a slight rotation (7°) of the φ angle of Asn-231. The Bpa molecule was modeled with INSIGHTII BUILDER and was placed between TMD III, TMD VI, and TMD VII as suggested by the photolabeling results. A first minimization of the complex between hAT1 and Bpa (the coulombic terms were turned off) was performed using restraints (2 Å < d < 7Å) between the Cβ atoms of the photolabeled Met residues and the ketone oxygen of Bpa. The backbone atoms of hAT1 were held in their position during this step. An extended Ang-(1-7) peptide (Sar-Arg-Val-Tyr-Val-His-Pro) was then appended to the N-terminal of Bpa. A subsequent minimization step was performed until the maximum derivative was <0.1 kcal/mol. The complex was further refined by adding a second disulfide bond between Cys-18 and Cys-274 (3Yamano Y. Ohyama K. Chaki S. Guo D.F. Inagami T. Biochem. Biophys. Res. Commun. 1992; 187: 1426-1431Crossref PubMed Scopus (134) Google Scholar) and by relaxing the ECL and performing a final overall minimization step. Site-directed Mutagenesis and Photoaffinity Labeling of hAT1—To identify the receptor residues that participate in the ligand binding pocket of the C-terminal amino acid of AngII, 44 X → Met mutations were induced in TMD III, TMD VI, and TMD VII of wt-hAT1 (Fig. 2). Each mutant receptor was transiently expressed in COS-7 cells. Membranes containing wt-hAT1 or two selected mutants, F293M(7.44) and N294M(7.45), were photolabeled with 3 nm 125I-[Sar1,Bpa8]AngII or 125I-[Sar1,p′-MeO-Bpa8]AngII. They produced a broad band migrating diffusely between 75 and 180 kDa on SDS-polyacrylamide gels (Fig. 1B, lanes 1-3). Labeling was completely prevented when the experiments were carried out in the presence of 10 μm AngII (Fig. 1B, lanes 4-6) (22Bosse R. Servant G. Zhou L.M. Boulay G. Guillemette G. Escher E. Regul. Pept. 1993; 44: 215-223Crossref PubMed Scopus (28) Google Scholar), confirming the specificity of the labeling. Photolabeling of all ligand binding receptor mutants was performed in the same manner. Because both photoligands produced identical results, only those performed with 125I-[Sar1,Bpa8]AngII are shown in Figs. 3, 4, 5. 125I-[Sar1, p′-MeO-Bpa8]AngII and 125I-[Sar1,Bpa8]AngII produced comparable labeling yields of 20-34% covalent incorporation into wt-hAT1 and all the mutants. Covalent incorporation yields were calculated from the ratio of the total radioactivity in the 75-180 kDa bands versus the total specific binding observed before photolysis.Fig. 4A, CNBr cleavage of photolabeled wt and TMD VI mutant hAT1 receptors. SDS-PAGE was carried as described in Fig. 3A. Lane 1, wt-hAT1; lane 2, F249M(6.44)-hAT1; lane 3, W253M(6.48)-hAT1; lane 4, H256M(6.51)-hAT1; lane 5, T260M(6.55)-hAT1; lane 6, F261M(6.56)-hAT1; lane 7, L262M(6.57)-hAT1; lane 8, V264M(6.59)-hAT1; lane 9, L265M(6.60)-hAT1; and lane 10, 125I-[Sar1,Bpa8]AngII. B, schematic degradation patterns of photolabeled hAT1 and TMD VI mutants. Radioactively labeled fragments are indicated as thick gray bars and the photolabeling peptides as thinner gray bars. The calculated molecular masses of the proteins are indicated in kDa and include the mass of the labeled peptide. For mutants with simultaneous TMD VI and TMD VII labeling, the necessary fragmentation patterns are presented in detail. The respective Met positions are indicated by boldfaced numbers. The results are representative of experiments performed at least in triplicate. Single letter amino acid abbreviations are used with position numbers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5A, CNBr cleavage of photolabeled wt and TMD III mutant hAT1 receptors. SDS-PAGE was carried as described in Fig. 3A. Lane 1, Wt-hAT1; lane 2, I290M(7.41)-hAT1; lane 3, A291M(7.42)-hAT1; lane 4, F293M(7.44)-hAT1; lane 5, N294M(7.45)-hAT1; lane 6, N295M(7.46)-hAT1; lane 7, C296M(7.47)-hAT1; lane 8, L297M(7.48)-hAT1; lane 9, L300M(7.51)-hAT1; and lane 10, 125I-[Sar1, Bpa8]AngII. B, schematic degradation patterns of photolabeled hAT1 and TMD VII mutants. Radioactively labeled fragments are indicated as thick gray bars and the photolabeling peptides as thinner gray bars. The calculated molecular masses of the proteins are indicated in kDa and include the mass of the labeled peptide. The respective Met positions are indicated by boldfaced numbers. The results are representative of experiments performed at least in triplicate. Single letter amino acid abbreviations are used with position numbers.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Digestion of the Labeled Receptor Mutants—To identify the covalently modified regions, the labeled receptors were treated with CNBr and analyzed by SDS-PAGE. Autoradiography revealed a typical 7.2-kDa fragment from wt-hAT1 consisting of the C-terminal sequence (285-334 plus ligand) (Figs. 3,4, 5), which comprises parts of TMD VII, the C-terminal tail, and the photoligand (Figs. 3,4, 5). The typical 10-kDa fragment from incomplete digestion at position Met-334 was also present (285-359 plus ligand) (Figs. 3,4, 5). Both fragments were identified in previous studies (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, 16Perodin J. Deraet M. Auger-Messier M. Boucard A.A. Rihakova L. Beaulieu M.E. Lavigne P. Parent J.L. Guillemette G. Leduc R. Escher E. Biochemistry. 2002; 41: 14348-14356Crossref PubMed Scopus (52) Google Scholar). The following X → Met hAT1 mutants had exactly the same labeling pattern as wt-hAT1, indicating that the introduced Met residue did not participate in the labeling process: A106M(3.30), S107M(3.31), V108M(3.32), S109M(3.33), F110M(3.34), N111M(3.35), A114M(3.38), S115M(3.39), V116M(3.40), F117M(3.41), L118M(3.42) and L119M(3.43) in TM-DIII; and I245M(6.40), V246M(6.41), L247M(6.42), F248M(6.43), F250M(6.45), F251M(6.46), S252M(6.47), I254M(6.49), Q257M6.52), I258M(6.53), F259M(6.54), F261M(6.56), L262M(6.57), V264M(6.59), and L265M(6.60) in TMD VI (selected results are shown in Figs. 3A and 4A). Two TMD III mutants (L112M(3.36) and Y113M(3.37)) displayed new fragments besides those associated with TMD VII, namely two new bands each (Fig. 3, lanes 2 and 3), one at 6.4 kDa (91-134 plus ligand) and the other below the 3.4-kDa marker, suggesting ligand release. TMD VI mutant F249M(6.44) had a new band at 5.2 kDa (244-284 plus ligand) (Fig, 4, lane 2). Mutant W253M(6.48) also had a band at 5.2 kDa (244-284 plus ligand) and weak bands at 7.2 and 10 kDa (Fig, 4, lane 3). Mutant H256M(6.51) had the TMD VII-associated bands together with a band below 3.4 kDa, suggesting ligand release (Fig. 4, lane 4), whereas mutant T260M(6.55) had a faint band that migrated below the 3.4-kDa band in addition to the TMD VII-associated fragments (Fig. 4, lane 5). The TMD VII Met mutants had different profiles because of the non-Met labeling of residues Phe-293(7.44) and Asn-294(7.45) in wt-hAT1 (16Perodin J. Deraet M. Auger-Messier M. Boucard A.A. Rihakova L. Beaulieu M.E. Lavigne P. Parent J.L. Guillemette G. Leduc R. Escher E. Biochemistry. 2002; 41: 14348-14356Crossref PubMed Scopus (52) Google Scholar). Met labeling thus had to be identified through ligand release as shown for residues Phe-293(7.44) and Asn-294(7.45) (16Perodin J. Deraet M. Auger-Messier M. Boucard A.A. Rihakova L. Beaulieu M.E. Lavigne P. Parent J.L. Guillemette G. Leduc R. Escher E. Biochemistry. 2002; 41: 14348-14356Crossref PubMed Scopus (52) Google Scholar). Mutants I290M(7.41) and A291M(7.42) produced patterns comparable with that of wt-hAT1, namely 291-334 plus ligand and 292-334 plus ligand instead of 285-334 plus ligand (Fig.