Molecular dissection of G protein preference using G_sα chimeras reveals novel ligand signaling of GPCRs

<jats:p>Although only 16 genes have been identified in mammals, several Gα subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Gα activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G<jats:sub>s</jats:sub>α and the carboxyl termini of the various Gα proteins determine their receptor coupling specificity, we proposed a set of chimeric G<jats:sub>s</jats:sub>α where the COOH-terminal five amino acids are replaced by those of other Gα proteins and used these to dissect the potential Gα linked to a given GPCR. Unlike G<jats:sub>q</jats:sub>α, G<jats:sub>12</jats:sub>α, and G<jats:sub>i</jats:sub>α outputs, compounding the signals from several Gα members, the chimeric G<jats:sub>s</jats:sub>α proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Gα coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G<jats:sub>q</jats:sub>signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G<jats:sub>i</jats:sub>pathways. Dissecting the potential Gα coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Gα subclass.</jats:p>