Generation of G alpha i knock-out HEK293 cells illuminates G alpha i-coupling diversity of GPCRs

G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein alpha subunit i (G alpha i) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the G alpha i subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all G alpha i subunits, which enables the measurement of GPCR-G alpha i coupling upon transient re-expression of a specific G alpha i subunit. We profile G alpha i-coupling selectivity across 11 GPCRs by measuring ligand-induced inhibitory activity for cAMP accumulation. The coupling profiles are then classified into three clusters, representing those preferentially coupled to G alpha z, those to G alpha o, and those with unapparent selectivity. These results indicate that individual G alpha i-coupled GPCRs fine-tune G alpha i signaling by exerting coupling preference at the G alpha i-subunit level. A HEK293 cell line lacking all G alpha i subunits is generated which enables the measurement of coupling between a GPCR and individual G alpha i subunits upon transient re-expression of a specific G alpha i subunit.