Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2-Gβ1γ2 Complex

Abstract

Interactions between proteins and cell membranes are critical for biological processes such as transmembrane signaling, and specific components of the membrane may play roles in helping to organize or mandate particular conformations of both integral and peripheral membrane proteins. One example of a signaling enzyme whose function is dependent on membrane binding and whose activity is affected by specific lipid components is G protein-coupled receptor (GPCR) kinase 2 (GRK2). Efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by heterotrimeric Gβγ subunits but also on the presence of highly negatively charged lipids, in particular phosphatidylinositol 4',5'-bisphosphate (PIP2). We hypothesized that PIP2 may favor a distinct orientation of the GRK2-Gβγ complex on the membrane that is more optimal for function. In this study, we compared the possible orientations of the GRK2-Gβγ complex and Gβγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation vibrational and attenuated total reflectance Fourier transform infrared spectroscopic methods. Our results indicate that PIP2 affects the membrane orientation of the GRK2-Gβ1γ2 complex but not that of complexes formed with anionic phospholipid binding deficient mutations in the GRK2 pleckstrin homology (PH) domain. Gβ1γ2 exhibits a similar orientation on the lipid bilayer regardless of its lipid composition. The PIP2-induced orientation of the GRK2-Gβ1γ2 complex is therefore most likely caused by specific interactions between PIP2 and the GRK2 PH domain. Thus, PIP2 not only helps recruit GRK2 to the membrane but also "fine tunes" the orientation of the GRK2-Gβγ complex so that it is better positioned to phosphorylate activated GPCRs.

Figure 1

SFG and ATR-FTIR Amide I spectra for GRK2-Gβ₁γ₂ bound to phospholipid bilayers. (A) SFG specta for GRK2-Gβ₁γ₂ associated with a 9:1 POPC:PIP₂ lipid bilayer. (B) ATR-FTIR spectra for GRK2-Gβ₁γ₂ associated with a 9:1 POPC:PIP₂ lipid bilayer. (C) ATR-FTIR spectra for GRK2-Gβ₁γ₂ associated with a 9:1 POPC:POPG lipid bilayer. The circles and crosses are experimental data. The solid lines are the fitting results.

Figure 2

Possible orientations of GRK2-Gβ₁γ₂ on bilayers with various compositions. (A) Possible orientations of the complex using a combination of SFG $({\chi }_{zzz}^{(2)}/{\chi }_{xxz}^{(2)}=1.9\pm 0.2)$ and ATR-FTIR (dichroic ratio R^ATR =1.6 ± 0.2) measurements on a 9:1 POPC:PIP₂ lipid bilayer. (B) Possible orientations of the complex using a combination of SFG $({\chi }_{zzz}^{(2)}/{\chi }_{xxz}^{(2)}=2.2\pm 0.2)$ and ATR-FTIR (dichroic ratio R^ATR =1.5 ± 0.2) measurements on a 9:1 POPC:POPG lipid bilayer. These are identical to those calculated for the GRK2(K567E/R578E)-Gβ₁γ₂ and GRK2(K567A/R578A)-Gβ₁γ₂ complexes on a 9:1 POPC:PIP₂ lipid bilayer because they have identical SFG and ATR-FTIR ratios. In each panel, the effect of experimental errors is accounted for using a coloring scheme based on how well the calculated and experimentally measured quantities agree for each possible orientation, within specified error bars (±10%). If the calculated ${\chi }_{zzz}^{(2)}/{\chi }_{xxz}^{(2)}$ ratio or R^ATR does not match the experimental value within ±10%, a score of 0 is assigned and show in blue.^– The total score is calculated as the product of the scores for all individual criteria. A score of 100% indicates an exact match for all experimental measurements and show in red color.^– The dark areas indicate orientations of GRK2-Gβ₁γ₂ that are considered to be physically reasonable, according to previously defined criteria (e.g. the Gβ₁γ₂ component has a membrane anchor that imposes some constraints on the possible orientations of the associated GRK2-Gβ₁γ₂ complex). Physically obtainable orientations fall within the shadowed region.

Figure 3

Deduced possible membrane orientation of GRK2-Gβ₁γ₂ on (A) 9:1 POPC:PIP₂ lipid bilayer (twist=75°, tilt=15°), and (B) 9:1 POPC:POPG lipid bilayer (twist=100°, tilt=65°). (C) Deduced possible membrane orientation of Gβ₁γ₂ on 9:1 POPC:PIP₂ lipid bilayer (twist=94°, tilt=23°). GRK2 is colored blue with cyan helices, Gβ is yellow, Gγ is green, and the GRK2 α10 helix is red. The purple sphere indicates the geranylgeranyl site, and ruby spheres residues that bind anionic phospholipids. The plane of the membrane is shown as a blue-grey rectangle.

Figure 4

Membrane orientation of Gβ₁γ₂ as a function of lipid composition. (A) SFG Amide I spectra from Gβ₁γ₂ associated with a 9:1 POPC:PIP₂ lipid bilayer. (B) ATR-FTIR Amide I spectra from Gβ₁γ₂ associated with a 9:1 POPC:PIP₂ lipid bilayer. The symbols represent experimental data, and solid lines the fitting results. (C) The possible orientations of Gβ₁γ₂ determined by the combination of SFG $({\chi }_{zzz}^{(2)}/{\chi }_{xxz}^{(2)}=2.0\pm 0.2)$ and ATR-FTIR (dichroic ratio R^ATR =1.9 ± 0.2) measurements on 9:1 POPC:PIP₂ lipid bilayer. The dark region demarks physically allowed orientations for Gβ₁γ₂.