SUMMARY The phosphorylation of agonist-occupied G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry, electron microscopy, mutagenesis, molecular dynamics simulations and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C-terminus of the β2AR with the GRK5 RH bundle subdomain, membrane-binding surface and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs.

This review is provided in recognition of the extensive contributions of Dr. Robert J. Lefkowitz to the G protein-coupled receptor (GPCR) field and to celebrate his 75th birthday. Since one of the authors trained with Bob in the 80s, we provide a history of work done in the Lefkowitz lab during the 80s that focused on dissecting the mechanisms that regulate GPCR signaling, with a particular emphasis on the GPCR kinases (GRKs). In addition, we highlight structure/function characteristics of GRK interaction with GPCRs as well as a review of two recent reports that provide a molecular model for GRK-GPCR interaction. Finally, we offer our perspective on some future studies that we believe will drive this field.

Recoverin is an EF-hand Ca2؉ -binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca 2؉ -dependent manner. It undergoes a Ca 2؉ -myristoyl switch when Ca 2؉ binds to EF-hand 2 and 3. We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca 2؉ binding. EF-hand 2 and 3 display different properties and serve different functions. Binding of Ca 2؉ to recoverin is a sequential process, wherein EFhand 3 is occupied first followed by the filling of EFhand 2. After EF-hand 3 bound Ca 2؉ , the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes. In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold. We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.G-protein-coupled receptor kinases provide desensitization of G-protein-coupled receptors by phosphorylation of serine and threonine residues at their cytoplasmic C terminus (1). A wellknown system represents the light absorbing pigment rhodopsin and the corresponding kinase, rhodopsin kinase, or GRK-1, which phosphorylates (and thus desensitizes) photobleached rhodopsin. Arrestin then binds to phosphorylated rhodopsin and thereby stops any further activation of the G-protein transducin (2, 3). Illumination causes the decrease in the concentration of the intracellular transmitters of excitation and adaption, cGMP and cytoplasmic [Ca 2ϩ ], respectively. The decrease in cytoplasmic [Ca 2ϩ ] is sensed by Ca 2ϩ sensor proteins such as recoverin (4 -6; for a recent review, see Ref. 7). Recoverin or the amphibian orthologue S-modulin (8, 9) inhibit rhodopsin kinase at high levels of free [Ca 2ϩ ], thereby relieving inhibition when the Ca 2ϩ level decreases. The Ca 2ϩ

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.