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 GRK2-Gβγ and Gβγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation (SFG) vibrational and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic methods. Our results indicate that PIP2 affects the membrane orientation of GRK2-Gβ1γ2, but not that of complexes form 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.

G Protein-Coupled Receptor Kinase 2 (GRK2) as a Potential Therapeutic Target in Cardiovascular and Metabolic Diseases

“…Similar to the already discussed inhibitory effect caused by disruption of the interaction between GRK2 and Gβγ subunits, the GRK2 K567E/R578E mutation, which eliminates anionic phospholipid binding, also ablates receptor phosphorylation in cells (Carman et al., 2000). In fact, efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by Gβγ subunits but also on the presence of phosphatidylinositol 4′,5′-bisphosphate (PIP2), a highly negatively charged lipid that not only helps recruit GRK2 but also orients the GRK2-Gβγ complex so that it is better able to phosphorylate activated GPCRs (Yang et al., 2016). This result is in contrast to what occurs with the GRK5 isoform, which adopts similar orientations on lipid bilayers whether or not they contain PIP2 (Yang et al., 2013).…”

Gβγ Pathways in Cell Polarity and Migration Linked to Oncogenic GPCR Signaling: Potential Relevance in Tumor Microenvironment

“…For instance, in the case of GRK-2 (G protein-coupled receptor kinase-2), one of the few Gbg effectors known at the structural level in complex with the heterodimer (Tesmer et al, 2005), GRK-2 is first recruited to the membrane by Gbg. Then, the spatial orientation of GRK-2 is fine-tuned by lipid second messengers that fix the pleckstrin-homology (PH)-domain in a position that orients the catalytic domain in the proper conformation for access to its substrates and regulators (Yang et al, 2016). This general mechanism may also be effective for other cytosolic Gbg-effectors that integrate PIP3 and Gbg signaling, such as P-Rex1.…”

Noncanonical Roles of G Protein-coupled Receptor Kinases in Cardiovascular Signaling

“…Following receptor activation and dissociation of the heterotrimeric G-proteins, GRK2 and GRK3 associate with membrane bound G βγ through their C-terminal pleckstrin homology domain, thereby facilitating their membrane translocation (26–28). The pleckstrin homology domain then further orients GRK2 at the site of GPCR action via association with membrane phospholipids such as PIP2 (29, 30). The critical interaction of GRK2 and GRK3 with Gβγ subunits is further affected by the expression and localization of different β and γ subunit isoforms (31–33) that may partially account for the receptor selectivity of these GRKs.…”