مراجعة PREreview لMolecular Determinants of G Protein Subtype-Specific Coupling to the Angiotensin II Type 1 Receptor

Overall Assessment and Comments

A single G protein-coupled receptor (GPCR) can activate multiple distinct G proteins, leading to varying physiological responses. Therefore, understanding the molecular basis of G protein multi-coupling is central to receptor pharmacology. This is a common theme for many GPCRs and Angiotensin II type 1 receptor (AT1R) presents a good test case to investigate G protein subtype-specificity since it’s classically Gαq-coupled but also has activity at Gαi as well.

In this manuscript, Acheampong et al. presents a comprehensive exploration of the molecular determinants of why AT1R activation leads to differences in G protein subtype specificity. The authors employ structural insights, mutagenesis, cellular biosensors, and radioligand binding assays to show that AT1R activates Gαq more efficaciously than Gαi2 and demonstrate that the subtype-specific differences originate at the level of allosteric coupling between ligand and transducer binding. They provide clear evidence that residues in the C-terminal alpha helix of the α subunit in heterotrimeric G protein (α5 helix) drive the differential coupling of Gαq and Gαi2 and revealed 4 key residues that are sufficient to account for the difference in allostery. The importance of these residues is further demonstrated by residue substitutions that are able to rescue coupling effect of mini-Gi to similar levels of mini-Gq. They also demonstrate that residue Q17 on Gq, which had the greatest effect, acts through an interaction with AT1R ICL3 residue K232.

Taken together, the findings are well presented and provide key mechanistic insights into AT1R-G protein subtype activation. The data support the authors’ conclusion, and I have no major concerns. This work has a broader impact on our understanding of transducer selectivity across GPCRs and holds clear therapeutic implications for the rational design of subtype-selective drugs.

Major points

No major comments

Minor points

1. Figure 3 shows that Gαi2 and Gαq-specific residues in the α5 helix drive differential allosteric coupling in the cellular context. The manuscript would benefit in scope if the authors could also perform the experiments in figure 3 with the endogenous agonist, AngII, or discuss what trends would be expected if AngII was used for the rescue experiments. Even though TRV055 is an AngII analog, its effect on AT1R coupling to Gq and Gi may differ from that of AngII. The comparison would contribute to the molecular insights in allosteric transducer effect and broaden the implications of the 4 key residue analysis.

1. The manuscript would also benefit from a clearer rationale for why TRV055 was omitted from the experiments in Figure 4. The radioligand mutational scans (Figures 2 and 5) use TRV055 and this is because TRV055 maximizes the mini-Gq shift and hence the sensitivity of the assay. However, the SPASM experiments in Figure 4 use only AngII. This makes the two datasets hard to compare, and some discrepancies may reflect the ligand and not the assay. For example, mutant V26F exhibited no significant effect in the AngII-stimulated SPASM assay (Fig. 4f) yet yielded a significant effect when TRV055 was utilized in the radioligand competition assays.

1. It would be very informative to learn whether substitution of the 4 identified key residues into mini-Gq would successfully reduce signaling down to levels comparable to mini-Gi. This was done for the mini-Gi, but not the mini-Gq. Could the authors add a brief discussion to strengthen the narrative if this data is not available.

I reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.