Rhodopsin kinase structure: different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase

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Singh, Puja, 1979-

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G protein coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins and downregulation. The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension. How GRKs recognize and are activated by GPCRs are not known, in part because the critical N-terminus and the kinase C-terminal extension were not resolved in GRK2 and GRK6 structures. The long-term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1), which represents a model system for studying phosphorylation-dependent desensitization of activated GPCRs. Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide-ligand binding states. One of the (Mg²⁺)₂·ADP·GRK1 structures is the most high-resolution structure (1.85 Å) of a GRK to date. In one (Mg²⁺)₂·ATP·GRK1 structure, almost the entire N-terminal region (residues 5-30) is observed. In addition, different segments of the kinase C-terminal extension are ordered in the various nucleotide-bound structures. Together, these two elements form a putative receptor-docking site adjacent to the hinge of the kinase domain. Based on these structures, a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase. Two novel phosphorylation sites were also identified at the N-terminus. The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS) homology domain of GRK1 was assessed using site-directed mutagenesis. In addition to mediating interaction with activated GPCRs, the N-terminus of GRKs also forms a binding site for calcium sensing proteins. Although its physiological significance is debated, the structures of these complexes could lend further insights into the conformation of the N-terminus of GRKs. The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes. Finally, the RH domain of GRK2 binds G[alpha subscript q], G[alpha]₁₁, and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors. The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆, a member of G[alpha subscript q] family.