Ligand-specific conformation of extracellular loop-2 in the angiotensin II type 1 receptor

Abstract

The orientation of the second extracellular loop (ECL2) is divergent in G-protein coupled receptor (GPCR) structures determined. This discovery provoked the question, is the ECL2 conformation differentially regulated in the GPCRs that respond to diffusible ligands? We have determined the conformation of the ECL2 of the angiotensin II type 1 receptor by reporter-cysteine accessibility mapping in different receptor states (i.e. empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of an N-terminal epitope-tagged receptor surrogate lacking all non-essential cysteines and then measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a steptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility. Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a lid conformation similar to that of ECL2 in rhodopsin was induced upon binding both agonist and antagonist, but exposing different accessible segments delimited by the highly conserved disulfide bond. Our study reveals the ability of ECL2 to interact with diffusing ligands and to adopt a ligand-specific lid conformation, thus, slowing down dissociation of ligands when bound. Distinct conformations induced by the bound agonist and the antagonist around the conserved disulfide bond suggest an important role for this disulfide bond in producing different functional states of the receptor.

FIGURE 1.

Secondary structure model of a typical GPCR. The rat AT1a receptor shows the interactions of AT1R with Ang II previously mapped by site-directed mutagenesis (solid black lines) and cross-linking experiments (dashed lines) (, , , , –58). ECL2 residues are highlighted in light gray. Native free cysteines are shown in dark filled circles, which were replaced with Ala in the HA-CYS⁻AT1R.

FIGURE 2.

Comparison of HA-AT1R and HA-CYS⁻AT1R. A, Western blot analysis of HA-AT1R (lane 4) and HA-CYS⁻AT1R (lane 5) expression in COS1 cells. Cells which are untransfected (UT) (lane 1), transfected with pMT3 (lane 2) and CYS⁻AT1R without HA tag (lane 3) are shown as negative controls. Three differentially glycosylated monomeric bands of the receptor and higher molecular weight oligomeric forms of the receptor can be seen. B, localization of HA-AT1R (panel 2) and HA-CYS⁻AT1R (panel 3) on the plasma membrane of COS1 cells. Untransfected cells are shown as negative control (panel 1). Cells were labeled with DAPI (blue) for nucleus and with HA primary antibody and Alexa fluor 568 (red) secondary antibody for HA-tagged receptors. C, saturation curves of HA-AT1R and HA-CYS⁻AT1R determined using [¹²⁵I]-[Sar¹, Ile⁸]Ang II. The inset shows the corresponding Scatchard plot. D, cell surface density of HA-AT1R and HA-CYS⁻AT1R expressed as mean B_max values derived from Scatchard plots. Error bars indicate the S.E., n = 3. E, ERK1/2 phosphorylation upon treatment with 1 μm Ang II on untransfected cells (lanes 1 and 2), HA-AT1R (lanes 3 and 4)-, and HA-CYS⁻AT1R (lanes 7 and 8)-transfected cells. ERK1/2 phosphorylation is inhibited by losartan treatment prior to Ang II treatment (lanes 5 and 9) and together with Ang II treatment (lanes 6 and 10). Total ERK levels are shown as loading control. F, measurement of intracellular Ca²⁺ mobilization upon 1 μm Ang II treatment in HA-AT1R- and HA-CYS⁻AT1R-transfected COS1 cells. The time point of Ang II treatment is indicated by an arrow.

FIGURE 3.

Expression of ECL2 single cysteine mutants. Expression analysis of HA-CYS⁻AT1R (lane 2) and HA-tagged single cysteine mutants (lanes 3–22) in transiently transfected COS1 cells. Untransfected cells served as negative control (lane 1). Actin expression levels are shown as loading control.

FIGURE 4.

MTSEA-biotin accessibility of representative mutants. A, structure of MTSEA-biotin, the part of the molecule that modify with reporter Cys is shown in the box. B, schematic representation of experimental design for measuring MTSEA-biotin accessibility. C, immunoprecipitated receptors were probed for both HA (left) and streptavidin-HRP (right) to detect receptor expression and biotinylation levels, respectively. Please note that the same blot is used for probing with HA and streptavidin-HRP. The blots for representative mutants H183C and E185C are shown under three experimental conditions; in the absence of ligand (upper), in the presence of 1 μm Ang II (middle), and in the presence of 1 μm losartan (lower). The fully glycosylated monomeric receptor band at 41.9 kDa was used for determination of MTSEA-biotin accessibility. HA signal intensity and streptavidin-HRP signal intensity of each sample is compared with the HA-CYS⁻AT1R in the same gel as indicated by 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 particular sample. Relative MTSEA-biotin accessibility of each mutant is compared with the HA-CYS⁻AT1R in the same gel. The inset is the schematic representation of reporter cysteines which point up when inaccessible and point down when accessible, reacted with MTSEA-biotin (shown as -SR). D, MTSEA-biotin accessibility of HA-AT1R and HA-CYS⁻AT1R.

FIGURE 5.

MTSEA-biotin accessibility maps of ECL2 single cysteine mutants. The MTSEA-biotin relative accessibility of mutants are expressed as mean ± S.E., n = 3. The red line shown on the graph designates the significance cutoff (S.E. of HA-CYS⁻AT1R accessibility, n = 9) that determines the accessibility of mutants. Mutants with significantly higher accessibility compared with HA-CYS⁻AT1R are indicated with an red asterisk. The conserved Cys¹⁸⁰ residue is marked by gold star. A, MTSEA-biotin accessibility map of ECL2 mutants in the absence of ligand. The autoantibody binding epitope sequences are indicated in dark red. B, MTSEA-biotin accessibility map of ECL2 mutants in the presence of AngII. C, MTSEA-biotin accessibility map of ECL2 in the presence of losartan. D, molecular dynamics simulation of ECL2 in the absence of ligand. TMI, TMII, TMIII, TMVI, and TMV are shown in the model. The ECL2 residues and side chains are shown in a stick and surface model. The accessible residues are shown in red. The disulfide-bonded cysteines Cys¹⁰¹ (TMIII) and Cys¹⁸⁰ (ECL2) are shaded in yellow. E, molecular dynamics simulation of ECL2 in the presence of Ang II. Ang II is shown as a magenta stick. The accessible residues are shown in red. F, molecular dynamics simulation of ECL2 in the presence of losartan. Losartan is shown as blue stick. The accessible residues are shown in red.

FIGURE 6.

Predicted hydrogen-bonding network formed by inaccessible residues of ECL2. The ECL2 is highlighted in dark gray. A, intramolecular interactions of ECL2 with the TM helices (red) in the absence of ligand. B, intramolecular interactions of ECL2 with the TM helices (red) in the presence of Ang II. Ang II is shown in yellow spheres. Two intermolecular hydrogen bonding network between Ang II and ECL2 (blue) and between Ang II and TM helices (green) are also shown. C, intramolecular interactions of ECL2 with the TM helices (red) in the presence of losartan. Losartan is shown in cyan spheres. Two intermolecular hydrogen-bonding network between losartan and ECL2 (blue) and between losartan and TM helices (green) are also shown.