Journal Article

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

Hoi, Kin Kuan, Warne, Tony, Liko, Idlir, Heine, Philipp, Wu, Di, Tate, Christopher G., Yen, Hsin-Yung, Song, Wanling, Hedger, George, Sansom, Mark S. P., Plückthun, Andreas, Robinson, Carol V., Carpenter, Byron, Horrell, Michael R., Lee, Yang

2018

Overview

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets 1 . 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)P 2 ) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P 2 binding. Endogenous lipids were also observed bound directly to the trimeric Gα s βγ protein complex of the adenosine A 2A receptor (A 2A R) in the gas phase. Using engineered Gα subunits (mini-Gα s, mini-Gα i and mini-Gα 12 ) 2 , we demonstrate that the complex of mini-Gα s with the β 1 adrenergic receptor (β 1 AR) is stabilized by the binding of two PtdIns(4,5)P 2 molecules. By contrast, PtdIns(4,5)P 2 does not stabilize coupling between β 1 AR and other Gα subunits (mini-Gα i or mini-Gα 12 ) 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)P 2 . Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gα i βγ complex in the presence of PtdIns(4,5)P 2 provide further evidence for a specific effect of PtdIns(4,5)P 2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P 2 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. Mass spectrometry-based assays are used to reveal specificity and structural determinants of lipid binding to class A G-protein-coupled receptors, and the effects of specific lipids on receptor coupling to G proteins.

• Publisher: Nature Publishing Group UK
• Subject: 101/58 / 631/45 / 631/535 / 82/80 / 82/83 / Humanities and Social Sciences / Letter / multidisciplinary / Science / Science (multidisciplinary)