Anionic phospholipids determine G protein coupling selectivity at distinct subcellular membrane compartments

G protein-coupled receptors (GPCRs) activate different signaling pathways through coupling to heterotrimeric G proteins consisting of four types of G alpha (Gα) subunits and the beta gamma (ýy) subunits. The C-terminal regions of Gα proteins is thought to determine coupling selectivity. However, there are several reports indicating that some receptors are promiscuous and can couple to multiple Gα proteins. The precise mechanism promoting promiscuous coupling is not fully understood. Here we show that anionic phospholipids such as phosphoinositide 4,5 bisphosphate (PI(4,5)P2) promote electrostatic interactions between anionic lipid head groups and positively charged amino acids in the transmembrane 4 (TM4) region of beta adrenergic receptors (βARs) to prime receptor coupling to the cognate Gαs and non-cognate Gαi proteins. The promiscuous coupling can only occur at the plasma membrane, a membrane compartment enriched in PI(4,5)P2, while receptors only couple to cognate Gαs proteins in compartments that are enriched in less negatively charged phosphoinositides such PI4P at the Golgi membranes. We took advantage of the rapamycin dimerization system to rapidly and inducibly deplete PI(4,5)P2 by recruiting a phosphatase to the plasma membrane and found that in the absence of anionic phospholipids, βARs couple only to their cognate Gαs protein. Finally, we found that mutating βARs PI(4,5)P2 binding motifs or depleting PI(4,5)P2 results in enhanced βARs-mediated cAMP response. Together, these findings reveal a role for anionic phosphoinositides in regulating GPCR activity at different subcellular compartments.