Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

Stéphane A. Laporte, Antony A. Boucard, Guy Servant, Gaétan Guillemette, Richard Leduc and Emanuel Escher

Department of Pharmacology Medical School Université de Sherbrooke Sherbrooke, Québec J1H 5N4 Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII, whereas the N-terminal amino acid of [Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The octapeptide hormone angiotensin II (AngII) exerts its numerous physiological effects on cardiovascular, endocrine, and neuronal systems by interacting with specific receptors (1). Two pharmacologically distinct types of receptors have been identified for this vasoactive peptide: AngII type 1 (AT1) and AngII type 2 (AT2) (2). The AT1 receptor mediates all the known physiological actions of AngII, including regulation of blood pressure and of water and electrolyte balance (3). The functional roles of the AT2 receptor are not well defined, but recent studies suggest that it could act as a physiological antagonist of AT1-mediated pressor effect and that it could regulate central nervous system functions related to locomotion and exploratory behavior (4, 5). The AT1 and AT2 receptors have been cloned from several species and are members of the G protein-coupled receptor family (GPCRs) characterized by their topography consisting of seven {alpha}-helical transmembrane domains. The two receptors exhibit high affinities for AngII even though they display a low degree of sequence identity at the amino acid level (34%).

The elucidation of primary structures of numerous GPCRs has prompted investigators to identify domains in receptors directly involved in ligand binding. One approach to the identification of the sites interacting between a ligand and its receptor is to covalently link a selective radioactive ligand to its cognate receptor and, by chemical or enzymatic fragmentation of the complex, to identify photoligand-binding domains of the receptor. This technique has been invaluable in mapping the ligand-binding pocket of the ß-adrenergic receptors (6, 7), the GnRH receptor (8), adenosine receptor (9), NK-1 receptor (10), and more recently, by our group, the AT2 receptor (11). This approach complements some of the limitations of site-directed mutagenesis, deletion analysis, or chimeric receptors in that it is a more direct attempt to identify ligand-binding domains. We previously reported the use of photoreactive AngII analogs for covalent labeling of AT receptors (12). In the present study, two photoreactive analogs were prepared by replacing the position 1 aspartyl or the position 8 phenylalanine of AngII with p-benzoyl-L-phenylalanine (Bpa) (13). These two photoreactive AngII analogs were used to covalently label the human AT1 (hAT1) receptor expressed in COS-7 cells. Targeted enzymatic and chemical fragmentation of the photoaffinity labeled receptor identified two distinct receptor domains that are specifically labeled by the two photoreactive AngII analogs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Specificity of Photoreactive Analogs in Binding Experiments and Photoaffinity Labeling
Figure 1Go shows the primary structures of AngII and photoreactive AngII analogs used in this study. Asp1 and Phe8 were respectively replaced by Bpa to give [Bpa1]AngII and [Sar1,Bpa8]AngII. By competitive binding assays, we first determined that [Sar1,Bpa8]AngII and [Bpa1]AngII exhibit respective IC50 values of 0.8 ± 0.2 nM (n = 3) and 17.7 ± 1.3 nM (n = 3) for the hAT1 receptor expressed in COS-7 cells. The functional properties of the two analogs were then analyzed by measurement of the ligand-induced inositol-1,4,5-trisphosphate production in COS-7 cells expressing the hAT1 receptor. Incubation of the cells with a high concentration of [Bpa1]AngII (1 µM) caused an increase of inositol 1,4,5-trisphosphate comparable to that obtained with AngII, whereas the same treatment with [Bpa8]AngII had no effect on inositol phosphate production (results not shown), thus suggesting the agonist and the antagonist nature of [Bpa1]AngII and [Sar1,Bpa8]AngII, respectively. It had been previously shown that [Sar1,Bpa8]AngII is a competitive antagonist on the rabbit aorta preparation (13). In photoaffinity labeling experiments, 125I-[Sar1,Bpa8]AngII (Fig. 2AGo) and 125I-[Bpa1]AngII (Fig. 2BGo) specifically labeled the hAT1 receptor that migrated as a glycoprotein of 110 kDa, as previously described (14). The labeling of the hAT1 receptor by the two photoreactive analogs was completely abolished by L158,809 (1 µM), an AT1 receptor-selective ligand and by AngII (1 µM) (Fig. 2Go), thereby confirming the specificity and the selectivity of the labeling. Although both photoreactive analogs successfully labeled the hAT1 receptor, determination of covalent incorporation yields (calculated from the ratio of total radioactivity found in isolated bands to total specific binding observed before photolysis) revealed that 125I-[Sar1,Bpa8]AngII, with an approximately 30% yield of covalent incorporation, was far more effective than 125I-[Bpa1]AngII (~1% yield of covalent incorporation). This difference is well illustrated in Fig. 2Go where the intensity of the band labeled with 125I-[Sar1,Bpa8]AngII is clearly higher (2 h autoradiography) than the one labeled with 125I-[Bpa1]AngII (20 h autoradiography).



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Figure 1. Amino Acid Sequence of the Photoreactive AngII Analogs

Residues represented in bold characters correspond to amino acid modifications. The absorption of a photon at approximately 350 nm by the Bpa moiety results in the formation of a diradicaloid state in which the electrophilic electron-deficient oxygen interacts with C-H bonds to produce a benzpinacol-type compound.

 


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Figure 2. Specific Photoaffinity Labeling of the Human AT1 Receptor Expressed in COS-7 Cells

Cells expressing hAT1 receptor were photolabeled with 125I-[Bpa8]AngII (A) or 125I-[Bpa1]AngII (B) in the absence or presence of 1 µM L-158,809 or 1 µM AngII. Samples (40 µg of protein/lane) were run on 8% acrylamide separating gel followed by autoradiography for 2 h (A) or 20 h (B). 14C-labeled protein standards of the indicated molecular masses (kDa) were run in parallel.

 
Mapping the 125I-[Sar1,Bpa8]AngII Contact Point
125I-[Sar1,Bpa8]AngII-photolabeled hAT1 receptor was partially purified, as indicated in Materials and Methods, and digested with glycopeptidase-F (PNGase-F). Treatment of the photolabeled complex with PNGase-F shifted the mobility of 125I-[Sar1,Bpa8]AngII-hAT1 receptor complex to an apparent molecular mass (Mr) of 34 kDa, corroborating the glycosylated nature of hAT1 receptor (Fig. 3Go). Digestion of the photolabeled receptor with Endoproteinase Glu-C (Staphylococcus aureus V8 protease) in ammonium carbonate buffer [a condition under which it specifically cleaves peptide bonds on the C-terminal of glutamic acid (15)] produced two fragments of 15.8 kDa and 17.8 kDa (Fig. 3Go). Treatment of the 15.8-kDa and 17.8-kDa fragments with PNGase-F had no effect on the mobility of the two V8 protease digestion products, indicating that they were not glycosylated (results not shown). The best candidate for the 15.8-kDa fragment (estimated Mr of the peptide + photolabel) is the peptide located between glutamate 227 and glutamate 357 (Fig. 4Go). The 17.8-kDa fragment would correspond to the incomplete digestion of peptide 186–357.



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Figure 3. Enzymatic Digestions of 125I-[Sar1,Bpa8]AngII-Labeled AT1 Receptor

Partially purified photolabeled hAT1 receptor (50,000–100,000 cpm) was incubated in the absence (control) or in the presence of PNGase-F (80 U/ml) for 24 h at room temperature. Photolabeled wild-type receptor was incubated with 800 µg/ml of V8 protease for 4 days at room temperature. Photolabeled A221E mutant hAT1 receptor was incubated with 800 µg/ml of V8 protease for 24 h at room temperature. Samples were then run on a 16.5% acrylamide Tris-Tricine separating gel followed by autoradiography. 14C-labeled protein standards of the indicated molecular masses (kDa) were run in parallel.

 


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Figure 4. Two-Dimensional Representation of the Primary Structure of the hAT1 Receptor and Its Potential Sites of Enzymatic Cleavage and Chemical Hydrolysis

Arrows indicate recognition sites for V8 protease; glutamic acid residues 173, 185, 227, and 357, as well as the A221E mutant bold circles indicate recognition sites for endo Lys-C; solid circles indicate sites of hydrolysis by CNBr; C indicates cysteine residues. Putative N-glycosylation sites on asparagines 4, 176, and 188 are also shown.

 
To confirm the location of the binding domain for 125I-[Sar1,Bpa8]AngII, we used a previously described mutant of the hAT1 receptor in which a glutamate residue was introduced at position 221 (16). We suspected that by introducing another glutamate residue near the Glu227, we could eliminate the partial digestion product of 17.8 kDa, since the mutant would become more susceptible to cleavage by V8 protease at position 221. The hAT1A221E mutant receptor displays activation and affinity profiles highly similar to those of the wild-type hAT1 receptor (16). Photolabeling of hAT1A221E with 125I-[Sar1,Bpa8]AngII resulted in incorporation yields identical to those obtained with the hAT1 receptor. Figure 3Go shows that V8 protease treatment of the partially purified receptor mutant released a major digestion product of 15.8 kDa comparable to fragment 228–357 generated by the same proteolytic cleavage of the wild-type receptor. This experiment clearly identifies the 222–357 fragment as the label-bearing segment of the receptor protein (see Fig. 4Go). Interestingly, the enzymatic cleavage of the hAT1A221E mutant receptor with V8 protease was almost complete after a 24-h incubation, whereas the digestion of the wild-type receptor was still incomplete even after a 4-day incubation.

To further delineate the binding domain for 125I-[Sar1,Bpa8]AngII, photolabeled and undigested hAT1 receptor was submitted to hydrolysis with CNBr, which cleaves specifically at the carboxy-terminal side of methionine residue (17). Figure 5AGo shows that, under these conditions, a major digestion product of 7.5 kDa was obtained. Considering the previous results obtained with V8 protease digestion and the location of the methionine residues in this sequence (243, 284, and 334), this apparent molecular mass identifies the fragment as the 285–334 polypeptide of the seventh transmembrane domain/cytoplasmic tail of the hAT1 receptor. To support this assumption and to narrow down the size of the label-bearing receptor segment, the partially purified 7.5-kDa CNBr fragment was submitted to Endo Lys-C proteolysis, which specifically cleaves on the carboxylic side of lysine residue (18). Figure 5BGo shows that digestion of the 7.5-kDa CNBr fragment with Endo Lys-C produced a 3.6 kDa fragment that could correspond to either of the three peptides located between proline 285 and lysines 307, 308, or 310 of the hAT1 receptor (Fig. 4Go). The 7.5-kDa CNBr fragment was also submitted to chemical hydrolysis with 2-nitro-5-thiocyanobenzoic acid (TNB-CN), which cleaves at the amino-terminal side of cysteine residue (19). Figure 5BGo indicates that the 7.5-kDa CNBr fragment contained at least one cysteine residue since it was converted to a 3.1-kDa fragment, upon cleavage with TNB-CN. Indeed, the 125I-[Sar1,Bpa8]AngII binding domain contains two cysteine residues at positions 289 and 296 (Fig. 4Go). Complete cleavage at these residues should produce either the labeled peptides 285–288 or 289–295. Both fragments are expected to migrate with an estimated Mr of 1.7 and 2.2 kDa, respectively. However, we could only detect a 3.1-kDa fragment probably corresponding to the partial hydrolysis of the peptide at position 296, which would generate the labeled 285–295 fragment. The possibility that the labeled fragment corresponds to peptide 296–334 must be excluded because it would be expected to migrate with an approximative Mr of 6.4 kDa. Taken together, these results indicate that 125I-[Sar1,Bpa8]AngII labels the fragment encompassing residues 285–295 within the seventh transmembrane domain of hAT1 receptor.



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Figure 5. Chemical and Enzymatic Fragmentation of 125I-[Sar1,Bpa8]AngII-Labeled AT1 Receptor

A, Partially purified photolabeled hAT1 receptor (65,000 cpm) was incubated in the absence (control) or the presence of CNBr (100 mg/ml) for 24 h at room temperature in the dark. Samples were then run on a 16.5% acrylamide Tris-Tricine separating gel followed by autoradiography. The 7.5-kDa fragment was recovered by passive elution from gel slices and in panel B was incubated in the absence (control) or the presence of endo Lys-C (40 µg/ml) or 25 mM TNB-CN as described in Materials and Methods. Samples were run on a 16.5% acrylamide Tris-Tricine separating gel followed by autoradiography. 14C-labeled protein standards of the indicated molecular masses (kDa) were run in parallel.

 
Mapping the 125I-[Bpa1]AngII Contact Point
125I-[Bpa1]AngII-photolabeled hAT1 receptor was partially purified, as indicated in Materials and Methods, and digested with Endo Lys-C, which produced a broad band of approximately 31 kDa (Fig. 6AGo). The high Mr and the broadness of the band suggested a polypeptide that was glycosylated. Treatment of the endo Lys-C digestion product with PNGase-F generated a 7.5-kDa fragment that migrated as a sharp band, indicating that the polypeptide was completely deglycosylated (Fig. 6Go). Similar results were obtained with a simplified protocol in which the photolabeled hAT1 receptor was simultaneously digested with Endo Lys-C and PNGase-F (results not shown). Since potential sites for the N-glycosylation of the hAT1 receptor are located in the amino-terminal ectodomain and in the second extracellular loop, the cleavage by Endo Lys-C must have occurred at the two lysine residues located at positions 146 and 199 (Fig. 4Go). Cleavage at these residues would yield a fragment of 7.5 kDa (including the photolabeled analog of 1.3 kDa). Further narrowing of the contact point in this fragment was not achieved on the native hAT1 receptor due to the very low labeling yield obtained with 125I-[Bpa1] AngII. To narrow down the contact point of 125I-[Bpa1]AngII, a mutant hAT1I165M receptor was constructed and expressed in COS-7 cells. This mutant receptor showed a high affinity (0.9 nM) for AngII and was expressed at a density (1.5 pmol/mg) comparable to that of native AT1 receptor. Digestion of the photolabeled mutant receptor with CNBr yielded a glycosylated fragment, which after deglycosylation migrated with a Mr of 10.1 kDa, corresponding to the only possible fragment 166–243 (see Fig. 4Go). Taken together, these results suggest that 125I-[Bpa1]-AngII labels the fragment encompassing residues 166–199, which is essentially the second extracellular loop of AT1 receptor.



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Figure 6. Endo Lys-C Digestion of 125I-[Bpa1]AngII-Labeled AT1 Receptor

Partially purified photolabeled hAT1 receptor (150,000 cpm) was initially incubated with Endo Lys-C (80 µg/µl) for 18 h at 37 C. An aliquot was then subjected to deglycosylation by incubating with PNGase-F (80 U/ml) for 24 h at room temperature. Samples were run on a 10% acrylamide separating gel followed by autoradiography. 14C-labeled protein standards of the indicated molecular masses (kDa) were run in parallel.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Numerous structure-activity studies performed on AngII have demonstrated that the aromatic group of Phe8 at the C terminus of the peptide carries the informationneeded for the biological response (20). In the present work, we have used a photoaffinity labeling approach with the aim to identify contact points between the AT1 receptor and its peptide ligands. Our results indicate that the C-terminal end of [Sar1,Bpa8]AngII interacts with the inner portion of the seventh transmembrane domain. Using a similar approach, Desarnaud et al. (21) had suggested that the C-terminal portion of Ang II interacts with the third transmembrane domain of AT1 receptor. This apparent discrepancy is probably due to the photochemical properties and pathways that are fundamentally different for the two labeling moieties. Desarnaud’s study used the 4'-azidophenyl moiety, whereas in the present study we used the much more efficient 4'-benzoyl-phenyl moiety. Although structurally the reactive part of both molecules is at the same position corresponding to the para-hydrogen of the phenyl nucleus in the endogenous ligand AngII, they follow very distinct pathways. It was previously believed that the azido-phenyl moiety produces an irreversible aryl nitrene upon light activation and that this nitrene inserts into a carbon-hydrogen bond in the close vicinity. Recent studies, however, have shown that this type of arylnitrene is extremely short-lived and immediately rearranges into a ketenimine (22). This latter moiety has a much longer lifetime and is not able to undergo a C-H insertion but is susceptible to nucleophile attack from solvent or from a polar protein sidechain. This ketenimine is therefore either hydrolyzed (thus not incorporated) or in the absence of water, it reacts with a Lys, Cys, Ser, or Asn sidechain in the immediate vicinity or, if none of these side chains is available, it reacts further away. The Bpa residue, however, produces a reversible benzophenone radical that either reacts in its vicinity in a nondiscriminative manner or hydrolyzes or rearranges. In the latter two cases the benzophenone is reconstituted and can be reactivated again. The C-H insertion reaction of the benzophenone radical is therefore only possible in its immediate vicinity, and the incorporation yield is inversely related to the distance. If high incorporation yields are observed, then a close and tight interaction between the photosensitive moiety and the receptor can be assumed. Low incorporation yields, on the other hand, may be due to longer distances, the presence of water, or a combination of both.

These different labeling mechanisms could explain the different results obtained between the present study and that reported by Desarnaud et al. Interestingly, this raises the possibility that the third transmembrane domain is in close proximity to the seventh transmembrane domain of AT1 receptor, since the secondary photoproduct of the arylnitrene, the cyclic ketenimine, has to search for a nucleophilic group. This suggestion is strongly supported by a site-directed mutagenesis study that proposed that the spatial proximity of Asn111 in the third transmembrane domain and Asn295 in the seventh transmembrane domain of the AT1 receptor could be responsible, in part, for maintaining the receptor in a conformational constraint that would be relaxed upon agonist binding (23). The proximity of the third and the seventh transmembrane helices has also been suggested in three-dimensional models of other GPCRs (24, 25, 26).

Binding domains on the AT1 receptor have also been extensively studied using mutagenesis approaches coupled with molecular modeling (for review, see Refs. 27, 28). Activation of the AT1 receptor appears to require interaction of the Phe8 side chain of AngII with His256 in the sixth transmembrane domain, an interaction that is stabilized by docking of the {alpha}-COOH group of Phe8 to Lys199 in the fifth transmembrane domain of AT1 receptor (29, 30). It was also suggested that the aromatic Trp253 in the sixth transmembrane domain stabilizes the ionic bridge formed between Lys199 in the fifth transmembrane of AT1 receptor and the carboxylate anion of Phe8 of AngII (31). The C-terminal residue of AngII would therefore be confined within a binding pocket circumscribed by transmembrane domains III, V, VI, and VII. Again, this is in agreement with the crucial role played by the Phe8 side chain of AngII in activating the AT1 receptor (20) and the involvement of intrahelical amino acids in GPCR activation upon agonist binding (32). In a related study, we have previously shown that [Sar1,Bpa8]AngII incorporates in the third transmembrane domain of AT2 receptor (11) (Fig. 7Go). On the basis of these results, it is tempting to speculate that the binding pockets of AT1 receptor and AT2 receptor are different. However, as mentioned for AT1 receptor, the third and the seventh transmembrane domains of AT2 receptor may be in close proximity, and hence local conformation brought about by the different primary structure of AT2 receptor would contribute to the formation of a distinct binding pocket.



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Figure 7. Two-Dimensional Representation of AngII-Binding Domains of the Human AT1 and Rat AT2 Receptors

The ligand-binding domains are represented by solid circles and numbered 1 for those domains interacting with the Bpa moiety of [Bpa1]AngII and 8 for those domains interacting the Bpa moiety of [Sar1,Bpa8]AngII. The amino-terminal end of AngII would make contact with the hAT1 receptor within residues 147–199, whereas it would make contact with the extracellular amino-terminal tail of the rAT2 receptor (11 ). The carboxy-terminal end of AngII would make contact with the seventh transmembrane domain of the hAT1 receptor (residues 285–295), whereas it would make contact with the third transmembrane domain of the rAT2 receptor (residues 129–138) (11 ).

 
The results of the present study also indicate that the N-terminal portion of [Bpa1]AngII interacts with a segment of the second extracellular loop of AT1 receptor. These results are in agreement with a previous study demonstrating how charged residues of the second extracellular loop are major docking points for the N-terminal amino acid of AngII. Using a site-directed mutagenesis approach, Feng et al. (30) suggested that the N-terminal Asp of AngII interacts with His183 found in the second extracellular loop of AT1 receptor. Further studies on receptor mutants with additional cleavage sites will be necessary to narrow down the site of contact between AT1 receptor and the N terminus of AngII.

The AngII molecule has an estimated length of 30 Å (probably less in solution), which is about the size of a transmembrane domain (~40 Å). To account for the interaction of our AngII analogs with the contact sites identified in this study, the second extracellular loop must be in proximity to the seventh transmembrane domain of AT1 receptor. In a previous study on the AT2 receptor, we also showed that the N terminus of the photoligand interacts with an extracellular domain (the N-terminal ectodomain), whereas the C terminus of the photoligand interacts with the third transmembrane domain of AT2 receptor (Fig. 7Go) (11). A similar pattern of peptide binding was also found when the binding domains of NK-1 receptor were examined with its cognate ligand Substance P. Using a photoaffinity-labeling approach, it was proposed that the C terminus of Substance P inserts into a binding pocket formed between transmembranes II and VI, whereas the N-terminus of the peptide interacts with an extracellular loop of the receptor (10).

It has been determined that the binding domain of heptahelical receptors for several small physiological ligands, like bioamines, are formed by residues located within the transmembrane helices (32). On the other hand, glycoprotein hormone receptors provide only extracellular regions as docking sites for their much larger ligands. Indeed, although extracellular domains of receptors have been implicated for the binding of glycoprotein hormones like TSH and FSH, recent studies have shown that small ligands, such as glutamate and calcium ion, can also interact with these regions. These ligands appear to bind to extracellular sites located in an elongated N terminus of their respective receptors (33). As suggested in the present study, the principle that governs small peptide hormone binding probably encompasses a mixture of both these above mentioned situations. The size of peptides conceivably allows them to find their way to the lower portion of the transmembrane domains. The fact that [Sar1, Bpa8]Ang was characterized as an antagonist in functional studies should not invalidate our findings with respect to the agonistic behavior of AngII. It has been suggested by site-directed mutagenesis that nonpeptide antagonists do not dock to the same parts of the AT1 receptor. Contrary to nonpeptide ligands, structure-activity relationship studies by us and others (20, 35), using a variety of AngII peptide analogs, indicate that AngII peptide antagonists and agonists have highly similar interaction profiles on the AT1 receptor.

Furthermore, in favor of a similar positioning of [Sar1]AngII (Kd of 0.9 mM) and of [Sar1, Bpa8]AngII within the AT1 receptor are studies on the agonist-to-antagonist transition observed with analogs having aromatic substitutions in position 8. Increasing the bulk of the aromatic nucleus gradually decreases efficacy until, on rabbit aorta strips and on IP3 production, agonistic behavior can no longer be observed but strong binding is evident (37). It is therefore highly unlikely that the Phe8->Bpa8 transition substantially alters or disturbs the peptide orientation in the AT1 binding pocket.

In conclusion, we have identified two peptide-binding regions in the AT1 receptor with the use of specific AngII photoreactive analogs. This approach also allowed the determination of the ligand molecule’s orientation in its binding pocket. Our results would support a model in which the C terminus of AngII interacts with the seventh transmembrane domain of AT1 receptor and the N-terminus of AngII interacts with the second extracellular loop of AT1 receptor. In future experiments, it will be important to identify which amino acid, within the identified fragments, interacts with the photoreactive analog. In addition, systematic mapping of the contact points between the AT1 receptor and AngII analogs containing the Bpa moiety at all possible positions could be revealing. At present, we believe that models of the three-dimensional structure of the AT1 receptor-binding pocket should accommodate the results of the present study.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Infection/Transfections
COS-7 cells were grown in DMEM containing 2 mM L-glutamine and 10% (vol/vol) FBS. Cells were seeded in 10-cm2 plates and infected at a density of 1.5–2 x 106 cells with a Vaccinia virus recombinant expressing the T7 RNA polymerase (VV:TF7–3) as previously described (38). Briefly, cells were infected for 1 h at 37 C with VV:TF7–3 (multiplicity of infection = 2), washed once with serum-free DMEM, and transfected with 4 µg of pcDNA3 into which was inserted the DNA encoding human AT1 (kindly provided by Dr. S. Meloche, Université de Montréal) and 30 µl of Lipofectamine (GIBCO/BRL, Gaithersburg, MD) in 8 ml of serum-free DMEM, according to the manufacturer’s instructions. The cells were incubated for 5 h at 37 C, and the media were replaced with complete DMEM containing 100 IU/ml penicillin and 100 µg/ml streptomycin. Infected/transfected cells were grown for 18–24 h before binding assays or photoaffinity labeling. The hAT1 I165M mutant was constructed as already reported elsewhere (16) and transfected as described above.

Binding Assays and Photoaffinity Labeling
[Bpa1]AngII and [Sar1,Bpa8]AngII were synthesized in our laboratories as previously described (13). 125I-AngII, 125I-[Bpa1]AngII, and 125I-[Sar1,Bpa8]AngII (specific radioactivity, ~1000 Ci/mmol) were prepared with iodogen as described by Fraker and Speck (39) but in an acetic acid buffer (pH 5.4). The labeled peptides were purified by HPLC on a C-18 column (10 µm) (Allteck Associates Inc., Deerfield, IL; No 29004) with a 20–40% acetonitrile gradient. The specific radioactivity of the labeled hormones was determined by self-displacement and saturation binding analysis.

Infected-transfected COS-7 cells were gently scraped and centrifuged at 200 x g for 10 min at 4 C. The pellet was washed once with ice-cold binding medium without BSA (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1 mg/ml soybean trypsin inhibitor, 0.1 mg/ml bacitracin) and resuspended in binding medium containing 2 mg/ml BSA. In binding experiments, COS-7 cells were incubated in binding medium containing 0.2 nM of [125I]AngII and selected concentrations of [Sar1,Bpa8]AngII or [Bpa1]AngII. Incubations were performed at room temperature for 60 min. Nonspecific binding was measured in the presence of 1 µM of unlabeled AngII. Bound radioactivity was separated from free ligand by filtration through GF/C filters presoaked in binding buffer. In photoaffinity-labeling experiments, cells were incubated with 5 nM of 125I-[Sar1,Bpa8]AngII or 45 nM of 125I-[Bpa1]AngII in the presence or absence of 1 µM L158,809 (an AT1-selective, nonpeptide analog, Merck & Co., Inc., Wilmington, DE) or 1 µM AngII. After a 60-min incubation period at room temperature, cells were washed once with ice-cold binding medium (without BSA) and irradiated for 60 min at 0 C under filtered (Raymaster black light filters number 5873, George W. Gates & Co. Inc., Franklin Square, NY) UV light (365 nm) (2 x 100 W mercury vapor lamp JC-Par-38, Westinghouse Electric Corp., Pittsburgh, PA). Cells were centrifuged at 200 x g for 10 min at 4 C. The pellet was solubilized in a buffer containing 100 mM Na2HPO4, pH 8.5, 25 mM EDTA, 0.1 mg/ml soybean trypsin inhibitor, and 1% (vol/vol) Nonidet P-40 and then submitted to one cycle of freezing and thawing. Cell lysates were centrifuged at 13,000 x g for 10 min at 4 C to remove insoluble material, and supernatants were kept at -80 C until further analysis.

Partial Purification of the Labeled Complex
The solubilized photolabeled receptors were diluted with an equal volume of 2x loading buffer [120 mM Tris-HCl, pH 6.8, 20% (vol/vol) glycerol, 4% (wt/vol) SDS, 200 mM dithiothreitol, and 0.05% (wt/vol) bromophenol blue] and incubated for 1 h at 37 C. SDS-PAGE was performed as described by Laemmli (40) using 8% gels. The gel was then dried and exposed to x-ray film (Kodak BIOMAX MS; Eastman Kodak, Rochester, NY) with an intensifying screen. After autoradiography, radioactive bands were excised from dried gels and rehydrated with appropriate digestion buffer. The gel slices were macerated and eluted with 2 ml of buffer for 3–4 days at 4 C under gentle agitation. The eluted proteins were filtered (Acrodisc 0.45 µm; Gelman Sciences, Inc., Ann Arbor, MI), and the gel slices were washed with 10 ml of digestion buffer. The whole eluate (12–15 ml) was concentrated approximately 150 times using Centriprep-10 and Centricon-10 cartridges (Amicon, Inc., Beverly, MA), and the partially purified proteins were aliquoted and kept at -80 C. This passive elution protocol is similar to that described by Blanton and Cohen (41).

Proteolytic, Chemical, and Endoglycosidase Digestions
For endoglycosidase digestions, partially purified photolabeled receptors were resuspended in digestion buffers containing 0.2% (vol/vol) Nonidet P-40. PNGase-F (33–100 U/ml) was added and samples were incubated overnight at room temperature.

For proteolytic digestions, partially purified photolabeled receptors (10,000–300,000 cpm) were resuspended in 25 µl of digestion buffer containing 100 mM NH4HCO3, pH 8.0, and 0.1% (wt/vol) SDS. Samples were incubated for 3–4 days at room temperature with indicated amounts of V8 protease. Partially purified photolabeled receptors were also digested for 18–24 h at 37 C with indicated amounts of endo Lys-C in 25 µl of digestion buffer containing 25 mM Tris-HCl, pH 8.5, 1 mM EDTA, and 0.1% (wt/vol) SDS. All digestions were terminated by adding an equal volume of 2x loading buffer and boiling the samples for 3 min. When subsequent digestions were needed, products of the first digestion were identified by SDS-PAGE followed by autoradiography and recovered from nonfixed gels by passive elution for 12–18 h at 37 C with 500 µl of digestion buffer. Extracted proteins were lyophilized and submitted to chemical or enzymatic digestion as described.

For CNBr hydrolysis, partially purified photolabeled receptors (60,000–450,000 cpm) were resuspended in 50 µl of 70% (vol/vol) trifluoroacetic acid, and CNBr (50 µl) was added to a final concentration of 100 mg/ml. Samples were incubated at room temperature, in the dark, for 24–36 h. Reactions were terminated by adding 500 µl of water. Samples were lyophilized, resuspended in denaturing buffer, and analyzed by SDS-PAGE. TNB-CN hydrolysis was performed using a modified procedure (19). Briefly, the isolated CNBr fragment was resuspended in digestion buffer containing 100 mM Tris-HCl, pH 8.5, 5 mM DTT, and 0.1% (wt/vol) SDS and incubated at 37 C for 30 min. A 5-fold excess of TNB-CN (25 mM) over thiol present was added, and the pH was readjusted, if necessary, to 8.5 with NaOH. The mixture was then allowed to stand at room temperature for 60 min. An equal volume of 200 mM sodium borate buffer, pH 10.5, was added and the reaction was incubated at 70 C for 2 h. Hydrolysis was terminated by adding an equal volume of 2x loading buffer.

Analysis of Products of Proteolysis and Chemical Cleavage
The products of proteolysis and deglycosylation were analyzed by SDS-PAGE using 16.5% acrylamide Tris-Tricine gels (Bio-Rad, Richmond, CA) followed by autoradiography on x-ray films (Kodak BIOMAX MS). 14C-labeled low molecular protein standards (GIBCO/BRL) were used to determine apparent molecular masses. Running conditions and fixation and coloration of gels were performed according to the manufacturer’s instructions.


    FOOTNOTES
 
Address requests for reprints to: Emanuel Escher, Ph.D., Département de pharmacologie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, CANADA. E-mail: e.escher{at}courrier.usherb.ca

This work was supported by grants from the Heart and Stroke Foundation of Canada (HSFC) and the Medical Research Council of Canada (MRCC). S.A.L. and J.P. are recipients of studentships from HSFC. G.S. is the recipient of a studentship from MRCC-Ciba-Geigy. E.E. is the recipient of the J.C. Edwards Chair in Cardiovascular Research. G.G. is the recipient of an MRCC Scientist Award. R.L. is a scholar from the Fonds de la Recherche en Santé du Québec.

This work is part of the Ph.D. thesis of S.A.L.

Received for publication May 18, 1998. Revision received December 17, 1998. Accepted for publication January 14, 1999.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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