A142V GRK4γ increased RH‐kinase domain separation is dependent on interaction with the plasma membrane
Authors: Yeon Jae Kyung , Botello‐Smith Wesley M. , Luo Yun Lyna , Andresen Bradley T. 
Published: June 2021

A142V GRK4γ is associated with essential hypertension and transgenic mice harboring A142V GRK4γ are hypertensive. Initial studies indicate that the A142V polymorphism increases GRK4γ activity, but the mechanism underlying how a change from alanine to valine within the regulators of G protein signaling homology (RH) domain increases the kinase activity remains unknown. Recent data indicates that electrostatic interactions hold together the RH and kinase domain (KD) of GRK5 in a closed conformation. The RH‐KD must separate/open for GRK5 to be active. As all GRK4 family members (GRK4, 5, and 6) have a similar structure and show similar electrostatic interactions between the RH and KD, it is expected that opening of the RH‐KD is a family‐wide biochemical mechanism mediating activity. Therefore, we hypothesize that A142V GRK4γ displays greater RH‐KD separation than wild‐type (wt) GRK4γ. To examine domain separation, we utilized explicit solvent molecular dynamics simulations (MD) of A142V and wt GRK4γ. Simulations were run for 1 μs in a water box, and two distinctions were immediately clear. First, A142V GRK4γ opened less than wt GRK4γ, 3% versus 8% of the total simulation time were in the open conformation, respectively. Second, the carboxyl terminus of A142V GRK4γ sampled three unique spaces that the carboxyl terminus of wt did not sample. As the carboxyl terminus of GRK4γ is palmitoylated, this suggests that A142V GRK4γ will initially bind to the plasma membrane in orientations not shared by wt GRK4γ. Utilizing steered MD the top four most frequented carboxyl terminus locations were anchored to the membrane with a palmitate on cysteine 532. For wt GRK4γ there were only two unique carboxyl terminus locations; whereas, A142V GRK4 had four unique carboxyl terminus locations. The resulting MD simulations followed by ward clustering to examine contacts with the membrane indicate that wt GRK4γ converged to two unique poses with three simulations in one pose and one in a unique pose; whereas, each A142V GRK4γ simulation resulted in a unique pose. One A142V GRK4γ pose that began as a unique insertion point mimicked the pose of the three wt GRK4γ simulations. The percentage of time that GRK4γ displayed RH‐KD separation increased upon binding to the membrane. 15% and 27% of the simulations were in the open state in wt and A142V GRK4γ, respectively. Future studies can be designed to examine how the lipid membrane contributes to GRK4γ RH‐KD separation, and biologically confirm that increased frequency of domain opening is the mechanism underlying increased A142V GRK4γ activity. Since GRK5 must be in an open conformation to phosphorylate GPCRs, these data begin to explain the increased activity of A142V GRK4γ and suggest how to regulate GRK4γ activity.

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This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.