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

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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.