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. 2013 Jan 4;288(1):540-51.
doi: 10.1074/jbc.M112.392514. Epub 2012 Nov 8.

Long range effect of mutations on specific conformational changes in the extracellular loop 2 of angiotensin II type 1 receptor

Hamiyet Unal  1 , Rajaganapathi Jagannathan, Anushree Bhatnagar, Kalyan Tirupula, Russell Desnoyer, Sadashiva S Karnik
Affiliations

Long range effect of mutations on specific conformational changes in the extracellular loop 2 of angiotensin II type 1 receptor

Hamiyet Unal et al. J Biol Chem. .

Abstract

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific "lid" conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar(1),Gln(2),Ile(8)] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.

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Figures

FIGURE 1.
FIGURE 1.
Model of rat AT1R. A, secondary structure model of rat AT1R. ECL2 residues substituted with reporter cysteines are highlighted in dark blue. Cysteine residues involved in formation of disulfide bonds are shown in yellow. Nonessential cysteines are replaced with alanine as described previously (11). The gain of function substitution residue Asn111 on TMIII is highlighted in green. The loss of function substitution residue Asp281 on ECL3 is shown in a red circle. The interactions of Asn111 and Asp281 with AngII previously mapped by site-directed mutagenesis are shown with solid lines. The residues involved in both AngII and losartan binding are boxed. B, model of rat AT1R showing position of residues Asn111 and Asp281 relative to Cys180 located in the middle of ECL2 in the AT1R. The backbone Cα trace of AT1R model is shown in gray, and the ECL2 region is highlighted in magenta. The Cα-Cα distances of Asn111 (green) and Asp281 (red) from Cys180 (yellow) are shown as dashed lines. The distance between Cys180 and Asp281 in AngII- and losartan-bound states did not change substantially (not shown). The native residues replaced by Cys reporters are represented as blue sticks. PyMOL (version 0.99rc6) was used to visualize the protein structures and generate images.
FIGURE 2.
FIGURE 2.
Expression of ECL2 single-cysteine mutants of N111G-CYS−AT1R and D281A-CYS−AT1R. Expression of HA-CYS−AT1R and HA-tagged single-cysteine mutants of N111G-CYS−AT1R and D281A-CYS−AT1R in transiently transfected COS1 cells was analyzed. Untransfected (UT) cells served as negative controls. Actin expression levels are shown as loading controls. IB, immunoblot.
FIGURE 3.
FIGURE 3.
MTSEA-biotin accessibility of representative mutants. A, structure of MTSEA-biotin. The region of the molecule that is modified with reporter Cys is shown in red. B, immunoprecipitated receptors were probed with anti-HA (left panel) or streptavidin-HRP (right panel) to estimate receptor pulldown and biotinylation levels, respectively. The same blot is used for probing with HA and streptavidin-HRP. The blots for representative mutants N111G-N174C and N111G-N176C are shown under three experimental conditions: in the absence of ligand (top panel), in the presence of 1 μm AngII (middle panel), and in the presence of 10 μm losartan (bottom panel). The 42-kDa monomeric receptor band was used for determination of MTSEA-biotin accessibility. The HA signal intensity and streptavidin-HRP signal intensity of each sample are compared with the N111G-CYS−AT1R in the same gel as indicated by the numbers below the bands. The corresponding plots show the MTSEA-biotin relative accessibility, which is the ratio of relative streptavidin-HRP signal to relative HA signal for a particular sample. Relative MTSEA-biotin accessibility of each mutant is compared with the N111G-CYS−AT1R in the same gel. The insets show schematic representations of reporter cysteines that point up when inaccessible (shown as SH in green) and point down when accessible, when reacted with MTSEA-biotin (shown as S-SR in red). IB, immunoblot.
FIGURE 4.
FIGURE 4.
MTSEA-biotin accessibility maps of ECL2 single-cysteine mutants in the empty states. The MTSEA-biotin relative accessibility of mutants are expressed as the means ± S.E., n = 3. The red line shown on the graph designates the significance cut off that determines the accessibility of mutants. Mutants with significantly higher accessibility compared with control are indicated with red asterisks. A–C, MTSEA-biotin accessibility maps of ECL2 mutants in HA-CYS−AT1R (A), N111G-CYS−AT1R (B), and D281A-CYS−AT1R (C) are shown in the absence of ligand. Note that the scales in A and B are different from that in C. D–F, the molecular dynamics of ECL2 frames that best fit accessibility data are shown in CYS−AT1R (D), N111G-CYS−AT1R (E), and D281A-CYS−AT1R (F). TM helices are shown in gray. The ECL2 backbone is shown in magenta. The side chains replaced by Cys reporter residues are shown in red when accessible and green when inaccessible. The disulfide-bonded cysteines Cys101 (TMIII) and Cys180 (ECL2) are shaded in yellow.
FIGURE 5.
FIGURE 5.
Concentration-dependent change in MTSEA-biotin accessibility. A, AngII and induced calcium mobilization in CYS−AT1R and N111G-CYS−AT1R transfected COS1 cells is shown upon stimulation with 0–1 μm AngII. B and C, the accessibility of N176C is shown at different concentrations of AngII in CYS−AT1R (B) and N111G-CYS−AT1R (C). D, dose-response inhibition of CYS−AT1R and N111G-CYS−AT1R with 0–10 μm candesartan is shown. E and F, the accessibility of N176C is shown at different concentrations of candesartan in CYS−AT1R (E) and N111G-CYS−AT1R (F). The data indicate the higher variability in the accessibility measurements when the concentration of ligands is closer to ≈Kd in both CYS−AT1R and N111G-CYS−AT1R and indicate the rationale behind using saturation concentrations.
FIGURE 6.
FIGURE 6.
MTSEA-biotin accessibility maps of ECL2 single-cysteine mutants in the N111G-AT1R in the presence of ligands. A, AngII and [Sar1,Ile4,Ile8]AngII induced calcium mobilization in CYS−AT1R and N111G-CYS−AT1R. CYS−AT1R and N111G-CYS−AT1R transfected COS1 cells are stimulated with 0–1 μm AngII or [Sar1,Ile4,Ile8]AngII. B and C, MTSEA-biotin accessibility maps of ECL2 mutants in N111G-CYS−AT1R in the presence of AngII (B) and [Sar1,Ile4,Ile8]AngII (C) are shown. B–E, the gray shading shows the accessibility pattern of ECL2 mutants in the absence of ligand. D, molecular dynamics simulation frames of ECL2 that fit accessibility data in the presence of AngII (orange spheres) are shown. The peptide and ECL2 backbones are shown in gray. The ECL2 backbone that corresponds to the best of all frames is shown in magenta. The side chains replaced by Cys reporter residues are shown in red when accessible and green when inaccessible. E, dose response inhibition of N111G-CYS−AT1R with 0–10 μm losartan or candesartan is shown. F and G, MTSEA-biotin accessibility maps of ECL2 mutants in N111G-CYS−AT1R in the presence of losartan (F) and candesartan (G) are shown. H, molecular dynamic simulation frames of ECL2 in the presence of losartan (cyan spheres) are shown.
FIGURE 7.
FIGURE 7.
A–C, MTSEA-biotin accessibility maps of ECL2 single-cysteine mutants in the D281A-CYS−AT1R in the presence of AngII (A), [Sar1,Gln2,Ile8]AngII (B), and losartan (C) are shown. D–F, molecular dynamics simulations of ECL2 in the presence of AngII (D, orange spheres), [Sar1,Gln2,Ile8]AngII (E), and losartan (F, cyan spheres) are shown. The peptide backbone is shown in gray. The ECL2 backbone that corresponds to the best of all frames is shown in magenta. The side chains replaced by Cys reporter residues are shown in red when accessible and green when inaccessible.
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