Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

Upon binding of extracellular ligands, G protein coupled-receptors (GPCRs) initiate signalling cascades by activating heterotrimeric G proteins through direct interactions with the α subunit. While the lipid dependence of ligand binding has previously been studied for one class A GPCR, the neurotensin receptor 1 (NTS1), the role the lipid environment plays in the interaction of activated GPCRs with G proteins is less well understood. It is therefore of interest to understand the balance of lipid interactions required to support both ligand binding and G protein activation, not least since some receptors have multiple locations, and may experience different membrane environments when signalling in the plasma membrane or during endocytosis. Here, using the sensitive biophysical technique of microscale thermophoresis in conjunction with nanodisc lipid bilayer reconstitution, we show that in more native lipid environments rich in phosphatidyl ethanolamine (PE), the Gαi1 subunit has a ~4-fold higher affinity for NTS1 than in the absence of native lipids. The G protein-receptor affinity was further shown to be dependent on the ligand-binding state of the receptor, with potential indication of biased signalling for the known antagonist SR142948A. Gαi1 also showed preferential interaction with empty nanodiscs of native lipid mixtures rich in PE by around 2- to 4-fold over phosphatidyl choline (PC)/phosphatidyl glycerol (PG) lipid mixtures. The lipid environment may therefore play a role in creating favourable micro-environments for efficient GPCR signalling. Our approach combining nanodiscs with microscale thermophoresis will be useful in future studies to elucidate further the complexity of the GPCR interactome.

Original publication

DOI

10.1016/j.bbamem.2015.08.004

Type

Journal article

Journal

Biochim Biophys Acta

Publication Date

11/2015

Volume

1848

Pages

2889 - 2897

Keywords

G protein, GPCR, Membrane, Microscale thermophoresis, Nanodisc, Neurotensin receptor, Animals, Brain Chemistry, GTP-Binding Protein alpha Subunits, Gi-Go, Humans, Ligands, Lipid Bilayers, Lipids, Membrane Lipids, Nanotechnology, Phosphatidylcholines, Phosphatidylethanolamines, Phosphatidylglycerols, Protein Binding, Receptors, G-Protein-Coupled, Receptors, Neurotensin, Signal Transduction, Swine, Temperature, Thermodynamics