Local membrane charge regulates β2 adrenergic receptor coupling to Gi
G protein coupled receptors (GPCRs) are transmembrane receptors that signal through heterotrimeric G proteins. Lipid modifications anchor G proteins to the plasma membrane; however, little is known about the effect of phospholipid composition on GPCR-G protein coupling. The {beta}2 adrenergic receptor ({beta}2AR) signals through both Gs and Gi in cardiac myocytes where studies suggest that Gi signaling may be cardioprotective. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study {beta}2AR-Gi interactions. To investigate the role of phospholipid composition on Gs and Gi coupling, we reconstituted {beta}2AR in detergent/lipid mixed micelles and found that negatively charged phospholipids (PS and PG) inhibit {beta}2AR-Gi3 coupling. Replacing negatively charged lipids with neutral lipids (PC or PE) facilitated the formation of a functional {beta}2AR-Gi3 interaction that activated Gi3. Ca2+, known to interact with negatively charged PS, facilitated {beta}2AR-Gi3 interaction in PS. Mutational analysis suggested that Ca2+ interacts with the negatively charged EDGE motif on the carboxyl-terminal end of the N helix of Gi3 and coordinates an EDGE-PS interaction. These results were confirmed in {beta}2AR reconstituted into nanodisc phospholipid bilayers. {beta}2AR-Gi3 interaction was favored in neutral lipids (PE and PC) over negatively charged lipids (PG and PS). In contrast, basal {beta}2AR-Gs interaction was favored in negatively charged lipids over neutral lipids. In negatively-charged lipids, Ca2+ and Mg2+ facilitated {beta}2AR-Gi3 interaction. Taken together, our observations suggest that local membrane charge modulates the interaction between {beta}2AR and competing G protein subtypes.