Modeling the Structural Differences between Wild-Type and Polymorphic G Protein-Coupled Receptor Kinase 4 Gamma

Abstract

Polymorphisms within GRK4 (R65L, A142V, and A486V) are associated with human hypertension. Furthermore, transgenic mice studies indicate that R65L and A486V induce salt-sensitive hypertension and A142V induces hypertension, whereas transgenic mice harboring wild-type GRK4γ are normotensive. These data suggest that the polymorphisms fundamentally alter the structure/function of GRK4γ; current theory suggests that polymorphic GRK4γ is hyperactive. As the structure of GRK4γ is not known, we modeled GRK4γ from GRK6 (2ACX) using SWISS-MODEL. Full-length GRK4γ was generated via patching using CHARMM, and explicit solvent simulations were run until stable. We then tested the hypothesis that adding polymorphisms associated with hypertension would alter the overall structure of GRK4γ; 90 nanoseconds explicit solvent simulations were performed for each mutation along with wild-type GRK4γ. Structurally, each polymorphism is unique compared to wild-type; however, A142V shows the most drastic changes including an altered affinity for ATP, lower interactions between the kinase domain and RH domain, and a drastically altered C-terminal location. The carboxyl-tail is palmitoylated, so its orientation dictates the orientation at membranes and thus the accessible cytosolic portion of GRK4γ. Additionally, there were multiple changes culminating in altering the solvent-exposed surface of each polymorphism with relatively few changes around the ATP-binding pocket. The marked differences in A142V GRK4γ compared to wild-type likely explain the observed biochemical alterations and hypertension; whereas, the less extreme changes in R65L and A486V likely explain their less severe phenotypes. In conjunction with biochemical data, the models suggest that altered protein-protein interactions, due to novel surfaces on GRK4γ, are responsible for the polymorphism-derived phenotypes not altered kinase activity.