Fusion with Promiscuous Gα(16) 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 α-subunit of G(16) protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα(16) limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα(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 Gα(16) pathway at the M(2) receptor and at the same time impaired Gα(16) signaling of iperoxo at M(5) receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways.