Activation of the angiotensin II type I receptor by a nonpeptide agonist

Figures

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  Fig. 1.. L-162,313 is an AT1R partial agonist. (A) Chemical structures of small-molecule ligands for AT1R. (B) Activation of G⍺q mediated inositol monophosphate in response to L-162,313 and AngII (IPone, CisBio). (C) G⍺q mediated calcium mobilization (FLIPR Calcium 6, Molecular Devices). (D) AT1R mediated dissociation of the Gq heterotrimer monitored via BRET (9). (E) L-162,313 and AngII desensitize calcium mobilization upon rechallenge with AngII. (F) Recruitment of Nluc-β-arrestin2 to AT1R-mVenus. (G) Endosomal translocation of Nluc-β-arrestin2 to FYVE-linked mVenus. Error bars represent the mean SE from at least three independent experiments. EV represents empty vector control to ensure signals are receptor dependent.

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  Fig. 2.. Nanobody AT118-R facilitates structure determination of L-162,313-bound AT1R. (A) Competition binding between the radiolabeled antagonist [3H]-olmesartan and unlabeled agonists L-162,313, AngII, or the antagonist losartan in cell membranes containing an AT1R-miniGs/q/iN fusion protein. The biphasic curve reports on ligand affinity to G⍺q bound (1st phase) and free (2nd phase) AT1R. Calculated Ki values and fraction of receptor in the high affinity G⍺q bound state are found in SI Appendix, Table S2. (B) Nanobody AT118-R allosterically increases the binding of L-162,313 to AT1R in competition radioligand binding assays in detergent micelles. (C) Nanobody AT118-R has no effect on AngII mediated inositol monophosphate production, but suppresses activation by L-162,313. Error bars represent the mean SE from at least three independent experiments. (D) Representative density of L-162,313 from cryo-EM map. (E) Global cryo-EM map of protein construct used for structure determination colored by component: AT118-R (gray), AT1R (light blue), BRIL (purple), anti-BRIL Fab (light pink), anti-Fab nanobody (magenta). (F) Model of the L-162,313-bound AT1R complex from local refinement maps. Image…

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  Fig. 3.. L-162,313 activation mechanism. (A) L-162,313 binds in the same orientation as small-molecule antagonists, making contacts with orthosteric site residues that interact with both AT1R antagonists and agonists. (B) L-162,313’s isobutyl substituent is in close proximity to L1123.36 in our inactive-state structure (blue). We hypothesize that a steric clash between the isobutyl and L1123.36 promotes its rotation toward its active-state position (pink). (C) Conformational changes initiated near the L-162,313 binding site are propagated through AT1R’s transmembrane core. (D) Movement of L1123.36 promotes TM3 rotation and conformational change in additional AT1R activation switches, including N1113.35, W2536.46, and Y2927.43. (E) Rotation of L1123.36 is coupled to the release of a hydrogen bond between N1113.35 and N2957.46, which stabilizes inactive-state AT1R.

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  Fig. 4.. Pharmacological and signaling effects of AT1R variants. (A) Changes in affinity represented as ΔLogKd values for [3H]-Olmesartan and ΔLogKi values for AngII, L-162,313, and L-162-propyl. (B) Change in EC50 value for Gαq dissociation. The L-162 propyl derivative shows minimal activation of WT AT1R and an EC50 value could not be fit (SI Appendix, Fig. S5). Variants that supported L-162-propyl mediated signaling are displayed with an EC50 value < −2. Variants that eliminated L-162,313 mediated activation are displayed with an EC50 value > 2. (C) Change in EC50 value for β-arrestin recruitment (SI Appendix, Fig. S6). Variants that eliminated L-162,313 or L-162-propyl mediated signaling are displayed with an EC50 value > 2. ΔLog values are calculated by subtracting the WT Log Affinity or EC50 value.

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  Fig. 5.. (A) Structures of synthesized L-162,313 derivatives containing various thiophene substituents. (B) L-162-neopentyl supports AT1R mediated dissociation of the Gq heterotrimer, whereas L-162-propyl and L-162-phenyl fail to robustly activate AT1R. (C) L-162-propyl and L-162-phenyl inhibit activation of AT1R by AngII. Error bars represent the mean SE from at least three independent experiments.