Membrane cholesterol modulates engagement of β-arrestin with the ghrelin receptor

The precise contribution of lipids to G protein-coupled receptor (GPCR) signaling remains to be elucidated. Of all the lipids, cholesterol likely plays a special role due to its abundance in different membrane compartments. Here, we assembled the ghrelin receptor GHSR into lipid nanodiscs containing different amounts of cholesterol and leveraged fluorescence spectroscopy to analyze its impact on receptor conformational dynamics and ability to interact with signaling partners. We showed that specific lipid:cholesterol interactions shift the receptor conformational equilibrium toward the active/active-like states, stabilizing the complex the GHSR forms with its cognate Gq protein. In contrast, while low cholesterol levels favored arrestin recruitment through an interaction with the receptor transmembrane core, its C-terminus and the lipid bilayer, increasing the cholesterol-to-phospholipid ratio in the nanodiscs was associated with dissociation of β-arrestin1 from both the membrane and the receptor core, resulting in the formation of a tail-only engaged complex. Taken together, these data highlight the multifaceted role of membrane lipid composition as a possible modulator of the arrangement of the complex the GHSR forms with G proteins and β-arrestins, and thus as a regulator of GHSR selectivity in desensitization, endocytosis, and signaling.