Analysis of the Third Transmembrane Domain of the Human Type 1 Angiotensin II Receptor by Cysteine Scanning Mutagenesis*

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile103–Tyr127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, 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 TMD3 reporter cysteines engineered in a constitutively active AT1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.

The octapeptide hormone angiotensin II (AngII) 1 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 (1). Virtually all the known physiological effects of AngII are produced through the activation of the AT 1 receptor, which belongs to the G proteincoupled receptor (GPCR) superfamily (2,3). GPCRs all possess seven transmembrane domains (TMD), which provide structural support for signal transduction. The AT 1 receptor interacts with the G protein G q/11 , which activates a phospholipase C, which in turn generates inositol 1,4,5-trisphosphate and diacylglycerol from the cleavage of phosphatidylinositol 4,5bisphosphate (4,5). Inositol 1,4,5-trisphosphate causes the release of Ca 2ϩ from an intracellular store, whereas diacylglycerol activates protein kinase C.
Like other GPCRs, the AT 1 receptor undergoes spontaneous isomerization between its inactive state (favored in the absence of agonist) and its active state (induced or stabilized by the agonist) (6). The rotation or translation of TMD helices caused by rigid body movement occurs during the activation of GPCRs (7)(8)(9). It has been proposed that TMD3, TMD5, TMD6, and TMD7 may participate in the activation process of the AT 1 receptor by providing a network of interactions through the AngII-binding pocket (10). 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 (11), it was expected that the binding site of the AT 1 receptor would involve the seven, mostly hydrophobic TMDs and would be accessible to charged watersoluble ligands, such as 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 AT 1 receptor (12)(13)(14). 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 AT 1 receptor in the inactive conformation. The agonist AngII would disrupt this interaction and promote the active conformational state (15). 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 AT 1 receptor (16 -18).
The substituted-cysteine accessibility method (SCAM) (19 -21) 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 when 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 the wild type and therefore that the substituted cysteine lies in a similar orientation 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 such as 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 TMD7 that form the surface of the binding site pocket in the wild-type AT 1 receptor and in the constitutively active N111G-AT 1 receptor (22). Here we report the application of SCAM to probe TMD3 in the wild-type and constitutively active receptors.

EXPERIMENTAL PROCEDURES
Materials-Bovine serum albumin, bacitracin, and soybean trypsin inhibitor were from Sigma. The sulfhydryl-specific alkylating reagent MTSEA (CH 3 SO 2 -SCH 2 CH 2 NH 3 ϩ ) was purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). The cDNA clone for the human AT 1 receptor subcloned in the mammalian expression vector pcDNA3 was kindly provided by Dr. Sylvain Meloche (Université de Montréal). Lipofectamine TM 2000 and culture medium were obtained from Invitrogen. 125 I-[Sar 1 ,Ile 8 ]AngII (specific radioactivity ϳ1500 Ci/ mmol) was prepared with Iodo-GEN® (Perbio Science, Erembodegem, Belgium) according to the method of Fraker and Speck (23) and as reported previously (24).
Numbering of Residues in TMD3-Residues in TMD3 of the human AT 1 receptor were given two numbering schemes. First, residues were numbered according to their positions in the human AT 1 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 (25). By definition, the most conserved residue was assigned the position index "50," e.g. in TMD3, Arg 126 is the most conserved residue and was designated Arg 126(3.50) , whereas the upstream residue was designated Asp 125 (3.49) and the downstream residue was designated Tyr 127 (3.51) . This indexing simplified the identification of aligned residues in different GPCRs.
Oligodeoxynucleotide Site-directed Mutagenesis-Site-directed mutagenesis was performed on the wild-type AT 1 receptor with the overlap PCR method (Expand high fidelity PCR system; Roche Diagnostics). Briefly, forward and reverse oligonucleotides were constructed to introduce cysteine mutations between Ile 103 (3.27) and Tyr 127(3.51) . PCR products were subcloned into the HindIII-XbaI sites of the mammalian expression vector pcDNA3.1. Site-directed mutations were then confirmed by manual and automated DNA sequencing.
Cell Culture and Transfections-COS-7 cells were grown in Dulbecco's modified Eagle's medium 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 ϫ 10 6 cells/dish. When the cells reached ϳ90% confluency, they were transfected with 4 g of plasmid DNA and 15 l of Lipofectamine TM 2000. After 24 h, transfected cells were trypsinized, distributed into 12-well plates, and grown for an additional 24 h in complete Dulbecco's modified Eagle's medium containing 100 IU/ml penicillin and 100 g/ml streptomycin before the MTSEA treatment and binding assay.
Binding Experiments-COS-7 cells were grown for 36 h after transfection in 100-mm culture dishes, washed once with 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 MgCl 2 ), centrifuged at 2500 ϫ g for 15 min at 4°C, and resuspended in binding buffer (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM MgCl 2 , 0.1% bovine serum albumin, 0.01% bacitracin, 0.01% soybean trypsin inhibitor). Saturation binding experiments were done by incubating broken cells (20 -40 g of protein) for 1 h at room temperature with increasing concentrations of 125 I-[Sar 1 ,Ile 8 ]AngII in a final volume of 500 l. Nonspecific binding was determined in the presence of 1 M unlabeled [Sar 1 ,Ile 8 ]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 ␥ counting.
Treatment with MTSEA-The MTSEA treatment was performed according to the procedure of Javitch et al. (19), 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 (Dulbecco's modified Eagle's medium, 25 mM HEPES, 0.1% bovine serum albumin, pH 7.4) containing 0.05 nM 125 I [Sar 1 ,Ile 8 ]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 ␥ counting. The percentage of fractional binding inhibition was calculated as (1 Ϫ (specific binding after the MTSEA treatment/specific binding without the treatment)) ϫ 100. Molecular Modeling-All calculations were performed on a Silicon Graphics Octane2 work station. The theoretical structure of the hAT 1 receptor was generated by homology modeling based on the crystal structure of bovine rhodopsin (Protein Data Bank code 1F88). A pairwise sequence alignment between the two primary structures was performed using the Homology program of Insight II (Accelrys Inc., San Diego, CA). Once the structurally conserved regions (TMDs) were identified, the coordinates were transferred to the sequence of the hAT 1 receptor. The structures of the loops of hAT 1 were modeled using the loop generation program of Insight II. The potential energy of the model structure of hAT 1 was minimized using the molecular modeling package of Insight II/Discover with a consistent valence force field (26). Two  1

mutant receptors
Cells transfected with the appropriate receptor were assayed as described under "Experimental Procedures." Binding affinities (K d ) and maximal binding capacities (B max ) are expressed as the means Ϯ S.D. of values obtained in n independent experiments performed in duplicate. Mutants S105C, V108C, and S109C did not demonstrate any detectable binding activity.
disulfide bridges, Cys 18 -Cys 274 and Cys 101 -Cys 180 , were used as distance restraints. A distance-dependent dielectric constant of 4 was used with a simple harmonic potential for bond length energy. No cross-term energies were included, and the peptide bonds were forced to planarity. Data Analysis-Results are presented as means Ϯ S.D. Binding data (B max and K d ) were analyzed with the Kell program (Biosoft, Ferguson, MO), which uses a weighted nonlinear curve-fitting routine.

Binding Properties of Mutant Receptors Bearing Cysteines in
TMD3-To identify the residues in TMD3 that face the binding site pocket of the AT 1 receptor, we mutated 24 consecutive residues between Ile 103 (3.27) and Tyr 127(3.51) 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 substitutions, pharmacological parameters describing the equilibrium binding of the radiolabeled competitive ligand 125 I-[Sar 1 ,Ile 8 ]AngII such as K d and B max were determined (Table I). All the mutant receptors exhibited high binding affinity for 125 I-[Sar 1 ,Ile 8 ]AngII (similar to that of the wild-type AT 1 receptor) except for Y113C (3.37) , which had a moderate 6-fold decrease in binding affinity. Mutants S105C (3.29) , V108C (3.32) , and S109C (3.33) did not demonstrate any detectable binding activity and were not used for the SCAM analysis. B max values for all detectable receptors ranged from 210 fmol/mg to 1.3 pmol/mg.
Effect of Extracellularly Added MTSEA on the Binding Properties of Mutant Receptors-To verify whether the reporter cysteines introduced into the TMD3 of the AT 1 receptor were oriented toward the binding pocket, mutant receptors were treated with concentrations of MTSEA varying between 0.5 and 6 mM. We verified whether the wild-type AT 1 receptor, which contains 10 endogenous cysteines (Fig. 1), was sensitive to the MTSEA treatment. Fig. 2 shows that the various con-centrations of MTSEA had very little effect (no more than a 20% reduction) on the binding properties of the wild-type AT 1 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 A104C (3.28) and L112C (3.36) , whereas it had only a minor effect on the binding properties of the other mutant receptors. At higher MTSEA concentrations, the binding properties of mutant N111C (3.35) were also slightly affected (Fig. 3B). Overall, the most reactive cysteines were those substituted for Ala 104 (3.28) and Leu 112 (3.36) , whereas the cysteine substituted for Asn 111(3.35) was less reactive.
Altered Accessibility to TMD3 Reporter Cysteines in the Constitutively Active N111G-AT 1 Receptor-We made use of the constitutively active N111G-AT 1 receptor to assess and map the potentially altered accessibility of MTSEA to the engineered cysteines. We first determined the pharmacological properties of the 23 cysteine-substituted mutant receptors. Within the N111G-AT 1 receptor background, 20 cysteine-substituted mutants conserved a high binding affinity for the competitive ligand 125 I-[Sar 1 ,Ile 8 ]AngII (Table II), whereas one mutant (Y113C (3.37) -N111G-AT 1 receptor) displayed a moderate 5-fold decrease in binding affinity. The S105C (3.29) -N111G-AT 1 and V108C (3.32) -N111G-AT 1 receptors did not have any detectable binding activity and were not used for the SCAM analysis. Interestingly, the S109C (3.33) mutation that caused a drastic loss of binding affinity in the wild-type receptor background did not cause a significant loss of binding affinity (1.9 nM) when engineered in the N111G-AT 1 receptor background. B max values for all detectable receptors ranged from 311 fmol/mg to 1.8 pmol/mg (Table II). Fig. 4 (as well as Fig. 2) show that, like the wild-type recep- tor, the N111G-AT 1 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-AT 1 receptor mutants were also treated with increasing concentrations of MTSEA, and their binding properties were assessed with 125 I-[Sar 1 ,Ile 8 ]AngII. Fig. 4 also shows that the MTSEA treatment caused a large decrease in binding to the V116C (3.40) -N111G-AT 1 mutant, whereas it caused a large increase in binding to the Y113C (3.37) -N111G-AT 1 mutant. Fig. 5 summarizes the effect of the MTSEA treatment on the different cysteine-substituted N111G-AT 1 receptor mutants. As observed in the wild-type background, mutants A104C (3.28) -N111G-AT 1 (binding inhibition of 63%) and L112C (3.36) -N111G-AT 1 (binding inhibition of 54%) were very sensitive to 0.5 mM MTSEA. Interestingly, two other mutants (Y113C (3.37) -N111G-AT 1 and V116C (3.40) -N111G-AT 1 ) were also very sensitive to MTSEA (Fig. 5A), whereas a third mu- 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 AT 1 receptor. The white bars indicate mutant receptors for which binding activities were not appreciably reduced when 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 Ϯ S.D. of data from at least three independent experiments. tant, S109C (3.33) -N111G-AT 1 , had a low sensitivity to MTSEA treatment (Fig. 5B).

Protection against MTSEA Reaction by a Pretreatment with [Sar 1 ,Ile 8 ]
AngII-To confirm that reporter cysteines accessible to MTSEA are located within the binding pocket, receptor mutants were saturated with the competitive ligand [Sar 1 ,Ile 8 ]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 [Sar 1 ,Ile 8 ]AngII protected mutant receptors A104C (3.28) , L112C (3.36) , N111G-A104C (3.28) , N111G-S109C (3.33) , N111G-L112C (3.36) , and N111G-V116C (3.40) from the inhibitory effect of MTSEA, with protection levels ranging from 58 to 91%. Unexpectedly, pretreatment of the N111G-Y113C (3.37) receptor mutant with [Sar 1 ,Ile 8 ]AngII potentiated the effect of MTSEA on the binding properties of this mutant by a further 35% (Fig. 6). DISCUSSION The rationale of this study, which relied on SCAM analysis, was to gain an insight into the orientation of TMD3 of the AT 1 receptor by identifying the residues accessible to MTSEA within the binding site pocket. Mapping these residues in the ground state receptor and the constitutively active N111G background allowed us to measure relative changes in the position of certain residues, thus providing valuable information with which to infer a structural change underlying AT 1 receptor activation.
As reported previously, the insensitivity of the wild-type receptor to MTSEA suggests either that endogenous cysteines are not alkylated by MTSEA or that their alkylation does not affect the binding of the ligand (22). Our methodological approach of adding the MTSEA reagent to whole adherent cells expressing the AT 1 receptor essentially exposed only the extracellular ligand-accessible side of the receptor to MTSEA. Interestingly, most of the MTSEA-accessible residues that we identified with the SCAM approach lie in the middle (N111C (3.35) , L112C (3.36) ) to the top portion of TMD3 (A104C (3.28) ) (Fig. 7A). These results imply that the residues involved in the interaction with the ligand are mostly located within this interface. Thus, residue Ala 104 (3.28) would delineate the top of the binding pocket, whereas residues Asn 111 (3.35) and Leu 112 (3.36) would delineate the bottom of the water-accessible binding pocket of the receptor. By a mechanism that could be steric, electrostatic, or indirect, the alkylation of these residues with MTSEA would hamper the binding of the ligand. It is very likely that these two subsets of residues do not interact with the same portion of the ligand. Furthermore, it is assumed that water-accessible residues are in the binding site pocket if a competitive ligand protects them from the effect of MTSEA. The competitive ligand [Sar 1 ,Ile 8 ]AngII protected all the residues tested, thus supporting the notion that these specific residues within TMD3 are located in the binding pocket.  Based on the x-ray high resolution crystal structure of bovine rhodopsin and the pattern of the residues accessible to MTSEA, a ground state AT 1 receptor model was obtained (Fig. 7, A, C,  and D). The model predicts that MTSEA-sensitive Ala 104 (3.28) faces TMD7, Asn 111(3.35) is close to TMD2, whereas Leu 112(3.36) faces TMD6 (Fig. 7, C and D). Each one of these residues lies on the same ␣-helix face and is accessible within the hydrophilic binding pocket. Furthermore, the model predicts that endoge-nous Cys 121(3.45) is located on the opposite lipid-exposed face of the helix. This prediction is consistent with our results showing a low sensitivity of the wild-type receptor to MTSEA (Fig. 2). Our finding that residues Ala 104 (3.28) and Leu 112 (3.36) are located in the binding pocket of the AT 1 receptor is in accordance with the current models proposed for bovine rhodopsin and the dopamine D2 receptor. Indeed, residues Glu 113(3.28) and Gly 121(3.36) are thought to be contact points with retinal in the 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-AT 1 receptor. The white bars indicate mutant receptors for which binding activities were not appreciably reduced when compared with that of the N111G-AT 1 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 Ϯ S.D. of data from at least three independent experiments. crystal structure of bovine rhodopsin (11), whereas the SCAM approach was used to show that residues Phe 110 (3.28) and Cys 118 (3.36) are located in the binding pocket of dopamine D2 receptor (27). In light of these results, this orientation of TMD3 in the ligand-binding pocket appears to be a common feature of class A GPCRs.
To further investigate the mechanism by which the AT 1 receptor undergoes structural changes during the transition from its inactive to its active state, we took advantage of the constitutively active N111G-AT 1 receptor. It is believed that the isomerization of conformers toward the active state, which involves transmembrane movement, is stabilized by the binding of an agonist and would be mimicked in part by the constitutively active receptor (6,28). Thus, within the structural background of the N111G-AT 1 receptor, we verified the accessibility of TMD3 residues to MTSEA, and we compared the pattern obtained with that of the wild-type receptor. We found that Cys-substituted residues Ala 104 (3.28) and Leu 112(3.36) maintained their sensitivity to MTSEA in the N111G-AT 1 receptor background (Fig. 5). Interestingly, in the N111G-AT 1 receptor background (Fig. 7B), two additional Cys-substituted residues (Ala 109 (3.33) and Val 116(3.40) ) were found to be sensitive to the MTSEA treatment. In the protection assay, the competitive ligand [Sar 1 ,Ile 8 ]AngII offered effective protection to all the sensitive mutants (A104C (3.28) , A109C (3.33) , L1112C (3.36) , and V116C (3.40) ) against the alkylating effect of MTSEA, suggesting that these residues are located in the binding pocket. The increased number of sensitive Cys-substituted residues in the N111G-AT 1 receptor background (Fig. 7B) suggests that the accessibility of residues in TMD3 and their spatial proximity within the binding pocket were altered due to the single substitution of Asn 111 for Gly in TMD3. The molecular model- FIG. 7. Molecular modeling of the AT 1 receptor by homology with bovine rhodopsin. A ribbon representation of the AT 1 receptor generated by homology modeling with bovine rhodopsin is shown. The TMD3s of the MTSEA-sensitive wild-type AT 1 receptor (ground state) (A) and N111G-AT 1 receptor (activated state) (B) are shown from residues Asn 98 (3.22) to Tyr 127 (3.51) . Colored residues represent residues, the substitution of which by a cysteine rendered the receptor sensitive (red) or not (white) to MTSEA treatment. The gray portion at the top of the ribbon represents a stretch of residues that were not evaluated by SCAM analysis. The light blue residue corresponds to Tyr 113 . C and D, extracellular view (C) and side view (D) of the ground state AT 1 receptor. The color code for the helices (also identified with a roman numeral) is as follows: TMD1 (dark blue), TMD2 (green), TMD3 (white), TMD4 (purple), TMD5 (yellow), TMD6 (light blue), and TMD7 (red). Residues identified by SCAM as accessible in the ligand-binding pocket of ground state receptor are identified with an arrow (Asn 104 , Asn 111 , Leu 112 ).
ing of these results points to a significant structural change during the process of activation of the receptor. Therefore, in this model of receptor activation, the appearance in the binding pocket of 2 new residues from the same side of the ␣-helix would imply that transmembrane movement takes the form of a slight counterclockwise rotation (about 45°) of TMD3 (Fig. 8).
In the activated state of the receptor, the model predicts that residue Asn 111(3.35) (a residue that was found sensitive to MT-SEA in the ground state receptor) is now facing the interior of the protein, in the vicinity of TMD2 but out of the binding pocket.
Previous studies using the SCAM approach have proposed a rigid body motion of transmembrane helices as a mechanism by which the AT 1 receptor is activated. Miura et al. (2) provided evidence that the movement of TMD2 plays a role in regulating the activated state of the AT 1 receptor. Likewise, Boucard et al. (22) suggested that a lateral movement (translation) of TMD7 occurs as a consequence of AT 1 receptor activation. It was also reported that light activation of rhodopsin leads to rigid body movements of TMD3 and TMD6 (29,30) and that inhibition of these rigid body movements abolishes G protein activation. A rotation of TMD3 has recently been demonstrated to be a consequence of the activation of the melanocortin 4 receptor (31), another rhodopsin-like receptor.
A peculiar observation made when the N111G-AT 1 receptor was tested with the SCAM approach was the increase of binding (189%) of the N111G-Y113C (3.37) mutant following the MT-SEA treatment. Obviously, the alkylation of this mutant with MTSEA did not impose a constraint on the binding of the ligand but rather facilitated its interaction or increased its binding strength. Positive charges added by MTSEA at this position might promote ligand binding, possibly by stabilizing the histidine residue in position 6 or the C-terminal of the ligand. Even more peculiar is the observation that mutant N111G-Y113C (3.37) showed a significant increase in binding after alkylation with MTSEA in the presence of a saturating concentration of the competitive ligand [Sar 1 ,Ile 8 ]AngII. In accordance with what we suggested above, the competitive ligand might stabilize a receptor conformation that would facilitate alkylation of N111G-Y113C (3.37) by MTSEA and favor a high receptor binding efficiency. Further studies will be necessary to clarify these issues. Nonetheless, residue Tyr 113(3.37) does not meet the criteria to be considered as being part of the binding pocket of the constitutively active N111G-AT 1 receptor.
The highly conserved DRY sequence at the C-terminal end of TMD3 is generally considered to be a major structural determinant of GPCR function (32,33). Previous studies on the ␤2 adrenergic receptor and the 5-hydroxytryptamine 2A receptor have shown that an ionic interaction between the arginine of the DRY motif in TMD3 and a residue close to the cytosolic extremity of TMD6 stabilizes the inactive conformation of these receptors (34,35). Furthermore, disruption of this ionic interaction leads to a constitutively active 5-hydroxytryptamine 2A receptor (35). Our results with the constitutively active N111G-AT 1 receptor point to an altered accessibility of certain residues of TMD3. This change is likely due to a rotation of TMD3 that modifies the environment of the DRY motif. In the ground state model, Arg 126(3.50) lies close to the cytosolic end of TMD6, whereas in the activated state model, it is oriented toward the center of the binding pocket. This new orientation of Arg 126(3.50) is compatible with a destabilization of the inactive conformation and therefore might contribute to generating the active state. Such a pattern of helical movement related to the activation of the AT 1 receptor might be a general mechanism associated with the agonist-induced activation of GPCRs In conclusion, our data comparing the ground state versus an activated state of the AT 1 receptor strongly point toward a minor counterclockwise rotation of TMD3 within the binding pocket. This rotation exposes residues Ala 104 , Ala 109 , Asn 111 , Leu 112 , and Val 116 of TMD3 to a water-accessible crevice. This movement of TMD3 upon activation of the AT 1 receptor is reminiscent of the movement recently observed with rhodopsin (36), the AT 1 receptor (22,37), and the MC4 receptor (31) and might therefore be a structural feature common to numerous rhodopsin-like GPCRs.  (11) is shown. The water-accessible crevice forming the binding pocket is suggested to be located between TMDs 1, 2, 3, 5, 6, and 7. In the ground state, constraining intramolecular interactions help maintain the receptor in a basal state in which functional coupling with the G protein is kept to a minimum. During the process of activation (following agonist binding), intramolecular interactions are broken, and the receptor goes through a series of conformational rearrangements involving the different TMDs. We recently provided data suggesting that TMD7 can be excluded from the water-filled crevice by a translational movement (22). The present study provides data suggesting that TMD3 rotates slightly on its axis during activation of the AT 1 receptor.