Recoverin Binds Exclusively to an Amphipathic Peptide at the N Terminus of Rhodopsin Kinase, Inhibiting Rhodopsin Phosphorylation without Affecting Catalytic Activity of the Kinase

Abstract

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.Light-dependent activation of rhodopsin in rod cells initiates a G protein-mediated signaling cascade that hyperpolarises the rod and inhibits glutamate release at its synaptic terminal. Inactivation of rhodopsin is essential for termination of this signaling event and for resetting rod cells to receive future stimuli. The inactivation process depends on phosphorylation of the C terminus of light-activated rhodopsin through the action of rhodopsin kinase (1, 2). The subsequent binding of arrestin to this phosphorylated peptide, and to cytoplasmic loops of the receptor, blocks access of the G protein, transducin, and prevents further signaling (3).Rhodopsin kinase is one of seven G protein-coupled receptor kinases (GRKs) 2 (4). These contain a common domain structure for the N-terminal 520 residues and a divergent C-terminal region (5). Rhodopsin kinase (also known as GRK1) shows many similarities to the more ubiquitous ␤-adrenergic receptor kinase (GRK2), sharing two major domains, the N-terminal regulator of G protein signaling (RGS) homology domain and the classical bilobal catalytic domain. The two kinases have 34% sequence identity, with 44% identity in the catalytic domain (6). The crystal structure of GRK2 in complex with G protein subunits, G␤␥, shows the structure of these major domains (7). The N-terminal RGS homology domain comprises nine ␣-helices, spanning residues 30 -185, and two additional ␣-helices from the C terminus of the protein (res...

Structural Basis for Calcium-induced Inhibition of Rhodopsin Kinase by Recoverin

“…Recoverin Binding to RK25 and Rhodopsin-Previous studies have shown that recoverin interacts with the N-terminal 25 residues of RK (called RK25) (19,20). To determine the atomicresolution structural interaction of recoverin bound to RK25 by NMR, we first developed a recombinant RK25 construct expressed in E. coli that produces milligram amounts of isotopically labeled RK25 and without interference from glutathione S-transferase or other fusion partners (see "Experimental Procedures").…”

“…Ca 2ϩ -bound recoverin interacts with RK exclusively at residues 1-25 (19), and deletion of these residues also abolishes the kinase interaction with light-excited rhodopsin (20). According to surface plasmon resonance measurements, the N-terminal RK peptide binds to recoverin with micromolar affinity and only in the presence of Ca 2ϩ (19,20). Recoverin is structurally similar to other NCS proteins (Fig.…”

“…The structure reveals that RK25 forms a long amphipathic ␣-helix, whose hydrophobic surface interacts with the hydrophobic groove of recoverin described above. We propose that recoverin binding to this N-terminal helix might block rhodopsin binding to the kinase active site, because deletion of the N-terminal RK residues abolishes interaction with light-excited rhodopsin (20). The structural interaction of RK25 with recoverin reported here is somewhat different from structures of target complexes seen previously with other EF-hand proteins like calmodulin (31), KChIP1 (26), calcineurin B (32), and troponin C (33) and may offer new insight into the structural basis of target specificity.…”

A Highly Conserved Cysteine of Neuronal Calcium-sensing Proteins Controls Cooperative Binding of Ca2+ to Recoverin

“…Under in vitro conditions, recoverin inhibits rhodopsin kinase (RK) 3 in a Ca 2ϩ -dependent manner resulting in extended activation of the visual pigment rhodopsin (3,4). Ca 2ϩ -bound recoverin binds the N-terminal helix of RK (5,6), an amphipathic helix also recognized by rhodopsin (7), and thus prevents phosphorylation of activated rhodopsin. When the Ca 2ϩ concentration is low, RK is released by recoverin and is then free to phosphorylate rhodopsin in a reaction that helps terminate the photoactivated state (8).…”

Structures of Rhodopsin Kinase in Different Ligand States Reveal Key Elements Involved in G Protein-coupled Receptor Kinase Activation

“…Conversely, how GRK1 recognizes an activated receptor is far less understood. The N-terminal 30 amino acids of GRK1 appear to be critical for receptor recognition, because the in vitro binding of Ca 2ϩ -recoverin to this region (17,18) or the binding of an antibody directed against GRK1 residues 17-34 (19) inhibits receptor phosphorylation yet has no effect on GRK1-mediated phosphorylation of peptide substrates.…”