PIP(2) stabilises active states of GPCRs and enhances the selectivity of G-protein coupling

G protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets 1. Despite detailed structural information however the effects of lipids on the receptors themselves, or on their downstream coupling to G proteins, are ill defined. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We demonstrate preferential binding of phosphatidylinositol 4,5 bisphosphate (PIP(2)) over related lipids and confirm hotspots for PIP(2) binding on the intracellular face of the receptors. Endogenous lipids were also observed bound directly to the entire adenosine A(2A) receptor (A(2A)R) trimeric Gα(s)βγ protein complex in the gas phase. Recruiting engineered Gα subunits (mini-G(s, i, 12)) 2 we found that the β(1)AR: mini-Gα(s) complex is stabilised by the binding of two PIP(2) molecules. By contrast PIP(2) did not stabilise coupling between β(1)AR and other Gα subunits (mini- Gα(i, 12)) or a high affinity nanobody. Other endogenous lipids that bound to receptors showed no effect on coupling, highlighting the specificity of PIP(2). Potential of mean force calculations and increased GTP turnover of the activated neurotensin receptor: trimeric Gα(i)βγ complex in the presence of PIP(2) further support the specific effect of PIP(2) on coupling. Together our results identify key residues on cognate Gα subunits that mediate simultaneous PIP(2) bridging interactions between basic residues on class A receptors, which do not correspond with those on class B GPCRs. By uncovering the effects of lipid modulation of receptors we highlight consequences for understanding function, G protein selectivity and drug targeting of class A GPCRs.