Structural and Biochemical Analysis of G Protein-Coupled Receptor Kinase Activation and Small Molecule Inhibitor Selectivity

Abstract

Cellular signaling through G protein-coupled receptors (GPCRs) is essential for most physiological processes, ranging from vision, taste, and smell to immune, neurological, and cardiovascular functions. In order to remain prepared to receive future signals and to protect the cell from the toxic effects of prolonged signaling, the duration and strength of GPCR-mediated signal transduction pathways must be tightly regulated. GPCR kinases (GRKs) drive signal termination by selectively recognizing and phosphorylating the C-terminal tails and intracellular loops of GPCRs that are actively signaling. GRK-mediated phosphorylation leads to the recruitment of arrestin, decoupling from G proteins, and GPCR internalization into the cytosol. The mechanism of GRK activation is complex and not fully understood. It is well-established that activated GPCRs allosterically increase GRK activity 100- to 1000-fold, but many questions remain about the structural determinants of GRK engagement with GPCRs and how this interaction stabilizes a fully activated kinase domain conformation. The mechanisms underlying these processes are important to understand because aberrant signal transduction is commonly found in human diseases. In heart failure, compromised β-adrenergic receptor (βAR) signaling and elevated levels of GRK2 and 5 lead to decreased cardiac output even in the presence of high circulating levels of the signaling molecules, adrenaline and noradrenaline. Given the elevated expression and activity of GRK2 and 5 in heart failure, GRK inhibition has exciting potential for the treatment of heart failure. In addition, there is a need for GRK-selective chemical probes that can be used to elucidate the distinct roles that GRK2 and 5 play in the progression of heart failure. With the overall goal of determining structural features that contribute to GRK activation and small molecule inhibitor specificity in the ATP-binding site, combined biochemical, pharmacological, and structural methods were utilized to probe GRK function. To assess GRK activation, steady-state kinetic analyses centered around 1) a region on GRK2 predicted to be involved in GPCR engagement and 2) an intramolecular interface in GRKs identified using principal component analysis of all available GRK crystal structures were performed. Results from these experiments suggest that GPCR docking sites may differ among the GRKs, and an intramolecular GRK interface distal to the active site contributes to stabilization of the activated kinase domain conformation in what we interpret to be an intermediate state on the path to full GPCR-mediated GRK activation. To understand the molecular determinants of small molecule inhibitor specificity, several GRK2–Gβγ∙inhibitor complexes were crystallized and structurally characterized. Structure-activity relationships were assessed for two libraries of GRK2-selective inhibitors and one library of GRK5-selective inhibitors. These studies resulted in highly potent and selective GRK2 inhibitors and covalent GRK5 inhibitors, and the structural analyses indicate subtle but significant differences in the ATP-binding sites of GRK2 and 5 that can be utilized in future rounds of GRK inhibitor development and optimization.