Unraveling allostery within the angiotensin II type 1 receptor for Gα _q and β-arrestin coupling

Abstract

G protein–coupled receptors engage both G proteins and β-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gα q and β-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gα q coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to β-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred β-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gα q coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gα q and β-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Biased Signaling in Mutated Variants of β2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations

“…It should be noted that residues in the intracellular regions (other than transducer binding) can also play a crucial role in allosteric communications, resulting in specific downstream signaling. For instance, a recent experimental and simulation study on AT1R revealed that several residues in TM4 favored β-arrestin signaling upon mutations, either due to enhanced β-arrestin recruitment or reduced G q protein coupling . Interestingly, in our simulations, TM4 exhibits a stronger allosteric coupling with the agonist in B2RY compared to the β-arrestin favoring receptor B2RTYY (Figure b), suggesting a similar role of TM4 residues in β 2 AR transducer selectivity.…”

“…For example, the G s -binding residue E62 ICL1 and the TM4 residue T146 4.38 show a substantial decrease in n SOP in B2RTYY relative to B2RWT (Figure and Table S3). Though the intracellular tip residue T146 4.38 of β 2 AR is not in direct contact with G s , an alanine substitution for this conserved threonine yielded β-arrestin specificity for AT1R, suggesting the significance of T 4.38 in biased signaling of class A GPCRs. Furthermore, the TM7 residue C327 7.54 , which exhibited notable variation in 19 F NMR spectra upon stimulating β 2 AR with β-arrestin biased agonist, along with its neighboring residue Y326 7.53 that interact with β-arrestin/GRK (Figure c,d), exhibits either comparable or enhanced allosteric signals from the agonist in B2RTYY, while a reduction is observed in B2RY, with respect to B2RWT (Figure and Table S3).…”

Biased Signaling in Mutated Variants ofβ2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations

“…For instance, a recent experimental and simulation study on AT1R revealed that several residues in TM4 favored β -arrestin signaling upon mutations, either due to enhanced β -arrestin recruitment or reduced G q protein coupling. 126 Interestingly, in our simulations, TM4 exhibits a stronger allosteric coupling with the agonist in B2RY compared to the β -arrestin favoring receptor B2RTYY (Figure 6b), suggesting a similar role of TM4 residues in β 2 AR transducer selectivity.…”

“…For example, the G s binding residue E62 ICL1 , and the TM4 residue T146 4.38 show a substantial decrease in n SOP in B2RTYY relative to B2RWT (Figure 7 and Table S3). Though the intracellular tip residue T146 4.38 of β 2 AR is not in direct contact with G s , an alanine substitution for this conserved threonine yielded β -arrestin specificity for AT1R, 126 suggesting the significance of T 4.38 in biased signaling of class A GPCRs. Furthermore, the TM7 residue C327 7.54 , which exhibited notable variation in 19 F NMR spectra upon stimulating β 2 AR with β -arrestin biased agonist, 43 along with its neighboring residue Y326 7.53 that interact with β -arrestin/GRK (Figures 6c and 6d), exhibits either comparable or enhanced allosteric signals from the agonist in B2RTYY, while a reduction is observed in B2RY, with respect to B2RWT (Figure 7 and Table S3).…”

Bias translation: The final frontier?

“…Currently new approaches to the active quest for biased molecules have been developed (i.e., DNA-encoded libraries) (Cai et al, 2023). At the receptor level, techniques employing molecular dynamic simulations (Suomivuori et al, 2020), receptor structure (Cao et al, 2023;Sengmany et al, 2020;Vuckovic et al, 2023) et al, 2010). In fact, the low intrinsic efficacy of the new reputedly biased opioid agonist TRV130 is postulated to be an important part of this molecule's therapeutic profile (Che et al, 2021).…”