Fusion with Promiscuous Gα16 Subunit Reveals Signaling Bias at Muscarinic Receptors

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous alpha-subunit of G(16) protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with G alpha(16) limits access of other competitive G alpha subunits to the receptor, and thus enables us to study activation of G alpha(16) mediated pathway more specifically. Our data demonstrated agonist-specific activation of G(16) pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards G alpha(16) pathway at the M-2 receptor and at the same time impaired G alpha(16) signaling of iperoxo at M-5 receptors. Our data have shown that fusion proteins of muscarinic receptors with alpha-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.

Automated summary

This study focused on a complex evaluation of agonist bias at G-protein coupled receptors, showing that fusion of muscarinic receptors with G alpha(16) limits access of other G alpha subunits to the receptor, enabling more specific study of G alpha(16) pathway activation. Data demonstrated agonist-specific activation of G(16) pathway among individual subtypes of muscarinic receptors, highlighting the potential for fusion proteins as a suitable tool for studying agonist bias, especially at non-preferential pathways.