PtdIns(4,5)P₂ stabilizes active states of GPCRs and enhances selectivity of G-protein coupling

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets¹. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P₂ binding. Endogenous lipids were also observed bound directly to the trimeric Gα_sβγ protein complex of the adenosine A_2A receptor (A_2AR) in the gas phase. Using engineered Gα subunits (mini-Gα_s, mini-Gα_i and mini-Gα₁₂)², we demonstrate that the complex of mini-Gα_s with the β₁ adrenergic receptor (β₁AR) is stabilized by the binding of two PtdIns(4,5)P₂ molecules. By contrast, PtdIns(4,5)P₂ does not stabilize coupling between β₁AR and other Gα subunits (mini-Gα_i or mini-Gα₁₂) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P₂. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gα_iβγ complex in the presence of PtdIns(4,5)P₂ provide further evidence for a specific effect of PtdIns(4,5)P₂ on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P₂ forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.