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 nonvisual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin (NT) 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 NT 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 Gβ(1)γ(1) to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gβ(1)γ(1) and NT was crucially affected by the lipid type, with exchange rates higher by 1 or 2 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 nonvisual GPCR modulate the G-protein-coupling step.

Fig. 1

Preparation of NTS1-nanodiscs. (a) The reconstitution reactions were carried out using MSP1D1, cholate-dissolved lipids, and NTS1_f. After detergent removal, nanodiscs without NTS1_f were separated from NTS1_f-nanodiscs by immobilized metal affinity chromatography exploiting the H10 tail of the receptor fusion protein. Purified NTS1_f-nanodisc was treated with Tev protease prior to ligand binding and nucleotide exchange experiments, to generate the NTS1-nanodisc. (b) Samples of the reconstitution and purification steps were analyzed by SDS-PAGE (4 μg protein/lane). Lane 1: Perfect Protein Markers (15-150 kDa) from Novagen; lane 2: MSP1D1; lane 3: purified NTS1_f; lane 4: mixture after detergent removal by Bio-Beads; lane 5: Talon column flow-through containing nanodiscs without NTS1_f; lane 6: Talon resin eluate containing purified NTS1_f-nanodiscs; lane 7: NTS1-nanodiscs after treatment of NTS1_f-nanodiscs with Tev protease. The SDS-gel shown here was from a reconstitution reaction using POPC. Reconstitutions with POPC/POPG and POPG gave similar results.

Fig. 2

DLS analysis of nanodiscs. (a) DLS autocorrelation functions for empty-nanodiscs reconstituted with POPC (blue), POPC/POPG (green) and POPG (red) are best modeled in terms of contributions from two discrete species corresponding to empty-nanodiscs (R_h,1) and traces of aggregates (R_h,2). The autocorrelation function is shown along with the best-fit two species model and corresponding residuals. (b) DLS autocorrelation functions for NTS1_f-nanodiscs prepared using POPC (blue), POPC/POPG (green) and POPG (red) are best modeled in terms of a paucidisperse species. The autocorrelation function for a batch of NTS1_f-containing POPC-MSP1D1 nanodisc is shown along with the best-fit using a quadratic cumulant and corresponding residuals. Data for these preparations of the POPC/POPG and POPG nanodiscs practically superimpose.

Fig. 3

Sedimentation velocities analysis of nanodiscs. (a) Sedimentation velocity c(s) distributions obtained in SEDFIT for empty-nanodiscs reconstituted with POPC (blue), POPC/POPG (green) and POPG (red) based on interference data. (b) c(s) distributions obtained in SEDFIT for NTS1_f-nanodiscs reconstituted with POPC (blue), POPC/POPG (green) and POPG (red) based on interference data. Besides the major species, a small contribution is observed at lower and higher S values, possibly representing small amounts of free MSP1D1 or empty-nanodiscs, and aggregated NTS1_f-nanodiscs, respectively. Data were collected at 40 krpm and 10.0°C.

Fig. 4

Nucleotide exchange at Gαq requires a near authentic receptor C-terminus. GDP/³⁵S-GTPγS exchange experiments were conducted with POPC-nanodiscs containing NTS1_f in the presence and absence of Tev protease. Increased ³⁵S-GTPγS binding was observed after cleaving off the C-terminal affinity tag. Two similar experiments in duplicates were performed for each test; one representative experiment is shown.

Fig. 5

Pharmacological properties of NTS1 in nanodiscs. (a)-(c) Saturation binding experiments: ³H-NT binding to NTS1 incorporated into nanodiscs prepared using POPC (a), POPC/POPG (b), and POPG (c). A one-site binding equation was used for curve fitting. (Inset) Scatchard transformation with B/F = bound/free. (d)-(f) Homologous competition and agonist-stimulated activation of Gαq: ³H-NT/NT competition experiments in the absence of G protein are shown for NTS1-nanodiscs prepared with POPC (d), POPC/POPG (e), and POPG (f) [● (grey circles), total NT added (x axis) vs. total ³H-NT displaced (right y axis). Agonist-stimulated activation of Gαq: ³⁵S-GTPγS binding was recorded in response to the indicated amounts of NT in the presence of Gαq and Gβ₁γ₁ [■, total NT added (x axis) vs. total ³⁵S-GTPγS bound (left y axis)]. Data were best fit to equations with a Hill slope of 1. Note that the nucleotide exchange reactions proceeded for 120 min, 45 min and 15 min in the case of POPC-, POPC/POPG- and POPG-nanodiscs, respectively. One representative experiment for each is shown.

Fig. 6

Gαq (a) and Gβ₁γ₁ (b) saturation of NTS1-catalyzed GDP/GTPγS exchange. ³⁵S-GTPγS binding was measured in reactions containing NTS1-nanodiscs with POPC (blue), POPC/POPG (green) and POPG (red), respectively. The fractional contribution of non-catalyzed nucleotide exchange at a given Gαq or Gβ₁γ₁ concentration was estimated in the absence of NT and subtracted from total ³⁵S-GTPγS binding. Normalization of the data to catalyzed ³⁵S-GTPγS bound (cpm) (nM NTS1 protein)⁻¹ min⁻¹ [Gαq (c) and Gβ₁γ₁ (d) saturation] emphasizes the effect of POPG on the nucleotide exchange reaction. One representative experiment for each is shown.