The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation

Abstract

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

FIGURE 1.

Schematic representation of the human AT₁ receptor. The numbers indicate the positions of cysteines and other residues in the receptor. The gray closed circles represent cysteine residues that are thought to be linked via disulfide bridges, and the black closed circles represent cysteine residues whose side chains do not form a disulfide bridge. Mutated TMD5 residues are located between Thr-190 and Leu-217 inclusively. Potential N-glycosylation sites (Asn-4, Asn-176, and Asn-188) are indicated. Asn-111 in TMD3 is also shown in gray.

FIGURE 2.

MTSEA treatment of the wild-type AT₁ receptor and sensitive reporter cysteine-bearing mutant receptors. Intact COS-7 cells transiently expressing wild-type (WT), L197C, N200C, I201C, G203C, or F204C AT₁ receptors were incubated for 3 min at room temperature with increasing concentrations of freshly prepared MTSEA (0.5 to 6 mm). The intact cells were then incubated for 90 min at room temperature with 0.05 nm ¹²⁵I-[Sar¹, Ile⁸]AngII, and their binding properties were evaluated as indicated under “Experimental Procedures.” Each curve represents the mean ± S.D. of data obtained from at least three independent experiments.

FIGURE 3.

Effects of MTSEA on mutant AT₁ receptors bearing a reporter cysteine in TMD5. Intact COS-7 cells transiently expressing wild-type (WT) or mutant AT₁ receptors were incubated for 3 min at room temperature with freshly prepared 0.5 mm MTSEA (A) or 2 mm MTSEA (B). The intact cells were then incubated for 90 min at room temperature with 0.05 nm ¹²⁵I-[Sar¹, Ile⁸]AngII. The percentage of binding inhibition was calculated as indicated under “Experimental Procedures.” The vertical line represents an arbitrary threshold used to identify cysteine-sensitive mutants and was set at a value corresponding to binding inhibition 20% greater than the value for the wild-type AT₁ receptor. The white bars indicate mutant receptors for which binding activities were not appreciably reduced compared with the wild-type receptor after treatment with MTSEA. The gray and black bars indicate mutant receptors for which binding activities were slightly reduced (gray) or strongly reduced (black) after treatment with MTSEA. Each bar represents the mean ± S.D. of data from at least three independent experiments.

FIGURE 4.

MTSEA treatment of the N111G-AT₁ receptor and sensitive reporter cysteine-bearing mutant N111G-AT₁ receptors. Intact COS-7 cells transiently expressing the N111G-AT₁, L197C-N111G-AT₁, N200C-N111G-AT₁, I201C-N111G-AT₁, G203C-N111G-AT₁, or F204C-N111G-AT₁ receptors were incubated for 3 min at room temperature with increasing concentrations of freshly prepared MTSEA (0.5 to 6 mm). The intact cells were then incubated for 90 min at room temperature with 0.05 nm ¹²⁵I-[Sar¹, Ile⁸]AngII, and their binding properties were evaluated as indicated under “Experimental Procedures.” Each curve represents the mean ± S.D. of data from at least three independent experiments.

FIGURE 5.

Effect of MTSEA on N111G-AT₁ mutant receptors bearing a reporter cysteine in TMD5. Intact COS-7 cells transiently expressing the mutant N111G-AT₁ receptors were incubated for 3 min at room temperature with freshly prepared 0.5 mm MTSEA (A) or 2 mm MTSEA (B). The intact cells were then incubated for 90 min at room temperature with 0.05 nm ¹²⁵I-[Sar¹, Ile⁸]AngII. The percentage of binding inhibition was calculated as indicated under “Experimental Procedures.” The vertical line represents an arbitrary threshold used to identify cysteine-sensitive mutants. It was set at a value corresponding to binding inhibition 20% greater than the value for the N111G-AT₁ receptor. The white bars indicate mutant receptors for which binding activities were not appreciably reduced compared with that of the N111G-AT₁ receptor after treatment with MTSEA. The gray and black bars indicate mutant receptors for which binding activities were slightly reduced (gray) or strongly reduced (black) after treatment with MTSEA. Each bar represents the mean ± S.D. of data from at least three independent experiments.

FIGURE 6.

[Sar¹, Ile⁸]AngII protection of MTSEA-sensitive mutant receptors. Intact COS-7 cells transiently expressing MTSEA-sensitive mutant AT₁ receptors were preincubated for 1 h at 16 °C in the absence or presence of 100 nm [Sar¹, Ile⁸]AngII. The cells were then incubated for 3 min at 16 °C in the continued absence or presence of [Sar¹, Ile⁸]AngII with optimal MTSEA concentrations to achieve maximal binding inhibition of each receptor. The MTSEA concentrations were as follows: 0.5 mm for L197C-AT₁, N200C-AT₁, G203C-AT₁, F204C-AT₁, L197C-N111G-AT₁, N200C-N111G-AT₁, I201C-N111G-AT₁, and F204C-N111G-AT₁; and 2 mm for I201C-AT₁ and G203C-N111G-AT₁. The cells were then washed with ice-cold PBS and incubated for 3 h at 16 °C with 0.05 nm ¹²⁵I-[Sar¹, Ile⁸]AngII. Protection was calculated as described under “Experimental Procedures.” Each bar represents the mean ± S.D. of data from at least three independent experiments.

FIGURE 7.

Basal levels of inositol phosphates in cells expressing the wild-type (WT) and mutant AT₁ receptors. Transfected COS-7 cells were preloaded for 16–24 h with 10 μCi/ml [myo-³H]inositol. Inositol phosphates (sum of inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate) accumulated within a period of 30 min were determined as described under “Experimental Procedures.” These results represent the mean ± S.D. values from at least three independent experiments (done in triplicate) where inositol phosphate production was normalized for incorporation of [myo-³H]inositol into phospholipids and for receptor expression level (determined by saturation binding assays).

FIGURE 8.

Helical wheel representation of TMD5 reporter cysteines and their pattern of reactivity to MTSEA. The positions in TMD5 of MTSEA-alkylated cysteines affecting ligand binding are shown in a helical wheel representation as viewed from the extracellular side for receptors with no additional mutation in TMD5 (A) and for receptors in which Asn-111 has been mutated to Gly in addition to the reporter cysteine in TMD5 (B). The gray and black circles indicate mutant receptors for which binding activities were slightly reduced (gray) or significantly reduced (black) after the treatment with MTSEA. The white circles indicate those mutant receptors that were insensitive to MTSEA treatment or positions that resulted in little or no detectable binding when substituted for cysteine.