Modulation of the Interaction between Neurotensin Receptor NTS1 and Gq Protein by Lipid

Abstract

Membrane lipids have been implicated to influence the activity of G protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus insight gained from rhodopsin signaling may not be simply translated to other non-visual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin receptor 1 (NTS1) under defined experimental conditions, using self-assembled phospholipid nanodiscs prepared with the zwitter-ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), or a POPC/POPG mixture. A combination of dynamic light scattering and sedimentation velocity showed that NTS1 was monomeric in POPC-, POPC/POPG- and POPG-nanodiscs. Binding of the agonist neurotensin to NTS1 occurred with similar affinities and was essentially unaffected by the phospholipid composition. In contrast, Gq protein coupling to NTS1 in various lipid nanodiscs was significantly different and the apparent affinity of Gαq and Gβ1γ1 to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gβ1γ1 and neurotensin was crucially affected by the lipid type, with exchange rates higher by one or two orders of magnitude in POPC/POPG- and POPG-nanodiscs, respectively, compared to POPC-nanodiscs. Our data demonstrate that negatively charged lipids in the immediate vicinity of a non-visual GPCR modulate the G protein-coupling step.

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

“…As a control, PglC and MSP in the absence of phospholipids did not afford detectable particles, and it is likely that the protein components precipitated under detergent withdrawal conditions. We note that the measured polydispersities are somewhat high, which may indicate the presence of a population of aggregates, which has been documented previously with DLS measurements on nanodisc assemblies (33).…”

“…Undecaprenol was extracted from the leaves of the staghorn sumac (Rhus typhina) and phosphorylated using chemical or enzymatic methods (SI Text). Und-[ 33 P]P was prepared by using Streptococcus mutans kinase and [γ 33 P] ATP (46). UDP-diNAcBac was prepared using a chemoenzymatic approach exploiting PglF, PglE, and PglD of the pgl pathway (47).…”

“…Enzyme function in the nanodisc assemblies was assessed using isotopically labeled substrates (Und-[ 33 After 1 h, the reactions were quenched in chloroform:methanol (2:1, 1 mL) and the organic layer was washed with chloroform:methanol: 0.1 M aqueous potassium chloride (3:48:47, 3× 400 μL). The organic layer was dried and resuspended in chloroform:methanol (4:1, 100 μL) for analysis using NP-HPLC.…”

“…A variety of synthetic lipids have been used for nanodisc preparation, including variants of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), as well as Escherichia coli lipid extracts (25,(27)(28)(29). Diverse membrane proteins have been incorporated into nanodiscs and studies on these systems have revealed fascinating nuances of protein structure, protein oligomeric state, protein-protein interactions, substrate binding, and enzyme activity (30)(31)(32)(33)(34)(35)(36). In addition, particularly noteworthy for the current study, the effects of lipid composition on enzymes have been probed in a highly reproducible manner relative to other membrane mimetic systems (37,38).…”

GPCRs: Lipid-Dependent Membrane Receptors That Act as Drug Targets

“…The interaction of membrane lipids with these receptors is therefore an important determinant in their structure and function [36,[41][42][43][44]. In addition, it has recently been reported that the interaction between GPCRs and G proteins could be modulated by membrane lipids [45]. Importantly, the membrane lipid environment of GPCRs has been implicated in disease progression during aging [46].…”