Conformational Changes in the Phosphorylated C-terminal Domain of Rhodopsin during Rhodopsin Arrestin Interactions

Abstract

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helixloop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.Following activation by a variety of sensory stimuli, such as hormones, neurotransmitters, or light, G-protein-coupled receptors (GPCRs) 1 are deactivated by multiple phosphorylations and subsequent binding of a regulatory protein arrestin (1, 2). Deactivation of the active receptor is obligatory and ensures the quantum character of the response to an extracellular signal. GPCR kinases (GRKs) and arrestin proteins are receptor-specific but share universal mechanisms of action, with prototypical rhodopsin kinase and visual arrestin involved in the termination of visual signal transduction (3, 4). Quenching of the photoresponse in the retinal photoreceptor cells proceeds through phosphorylation of multiple serine and threonine residues at the C terminus of light-activated rhodopsin by GRK1 and high affinity binding of visual arrestin. In vitro studies have implicated all seven serine and threonine residues within the Rh-(334 -343) C-terminal stretch as possible substrates of the phosphorylation reaction. The question of the exact number and position of residues phosphorylated in vivo, however, remains uncertain possibly due to the inherent difficulties of controlling dephosphorylation, different kinetics of phosphorylation/dephosphorylation at individual residues, possible contribution of kinases other than GRK1, and other secondary factors. Either Ser-334 (5) or Ser-343 (6) is phosphorylated initially. The majority of studies provides strong evidence, however, that multiple phosphorylation is required for reproducible deactivation (5-8). In order to circumvent the current uncertainty about the exact sequence of phosphorylation events, we have used a model synthetic peptide phosphorylated at all sev...

Arrestin Binds to Different Phosphorylated Regions of the Thyrotropin-Releasing Hormone Receptor with Distinct Functional Consequences

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Structure of Bovine Rhodopsin in a Trigonal Crystal Form

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Structure of the Third Intracellular Loop of the Vasopressin V2 Receptor and Conformational Changes upon Binding to gC1qR

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