Mechanism of G protein-coupled receptor kinase binding and activation by G protein-coupled receptors

G protein-coupled receptors (GPCRs) are regulated through the process of desensitization, where the receptor becomes refractory to agonist stimulation. The first step in desensitization is phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs). GRK2 is one of seven mammalian GRKs and is the best characterized of the family. In order to define the mechanism of interaction between the receptor and GRKs, we studied the molecular determinants involved in GRK2 binding and how this binding plays a critical role for receptor phosphorylation as well as kinase activity. We also characterized the role of the extreme amino terminus of GRK2 in receptor phosphorylation. To determine the molecular interaction between GRKs and receptor, we used the α2A-adrenergic receptor (α2A-AR) as our model GPCR to identify regions that interact with GRK2. We generated glutathione S-transferase (GST) fusion proteins of various regions of the α 2A-AR and used them in direct binding assays with purified GRK2. Through these studies we identified four direct binding sites to GRK2; the second intracellular loop and three regions in the third intracellular loop of the α 2A-AR. Truncation and site-directed mutagenesis studies revealed that basic residues on the receptor are important for binding to GRK2. GRK2 utilizes a number of co-factors to mediate GPCR phosphorylation including acidic phospholipids and G protein subunits. In addition, GRK2 can be stimulated by binding to the agonist-occupied form of receptors. Disruption of GRK binding resulted in decreased activation of the kinase and attenuated phosphorylation of the receptor. The x-ray crystal structure of the inactive form of GRK2 has been solved, revealing an equilateral triangle formed by the three main domains: RGS, kinase and pleckstrin homology domain. Residues 1-29 were unstructured. The amino terminus of GRKs has been implicated in receptor phosphorylation. Here we further characterized the extreme amino terminus of GRK2, which suggested a novel role for this region in regulating kinase activity. Mutations in the first 10 residues of GRK2 impairs its ability to phosphorylate a receptor substrate. Upon further characterization of three mutants, D3K, L4A and D10A, we found that these mutants were severely impaired in β2-adrenergic receptor phosphorylation, however phosphorylation of a non-receptor substrate, tubulin, was not affected. The mutants were similar to the wild-type in their ability to bind to β2-adrenergic receptor, however they were not activated by the receptor. We generated a synthetic peptide of the first 14 amino acids of GRK2. A secondary structure prediction program and circular dichroism suggested that the peptide forms an amphipathic alpha helix. In vitro, increasing concentrations of the peptide inhibited receptor phosphorylation by full-length GRK2 and enhanced phospholipid binding of GRK2. Kinetic studies with the same peptide revealed non-competitive inhibition. Crosslinking experiments with the synthetic peptide and GRK2 suggested an intramolecular interaction between the amino terminus and the rest of GRK2. Taken together, these results suggest a novel role for the amino terminus of GRK2 in its binding and activation by GPCRs. We hypothesize that this intramolecular interaction is necessary to stabilize the extreme amino terminus of the kinase by binding to the RGS domain of GRK2 thereby causing a conformational change in the catalytic domain and modulating the kinase activity from inactive to the active state