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 helix-loop 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.
Highlights
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]
The fully phosphorylated form was chosen because multiple phosphorylation was observed in vivo [6, 8], and both serine and threonine residues have been shown to be required for activation of the action of arrestin (5, 24 –26). 7-phospho-Rh-(330 –348) (7PP) inhibited phototransduction from photoactivated unphosphorylated rhodopsin in the presence of arrestin, based on the phosphodiesterase assay [27]
As predicted from the previous data, we show here that in direct competition experiments, binding of transducin to lightactivated unphosphorylated rhodopsin in membranes is inhibited by addition of arrestin and 7PP together but not arrestin or 7PP alone (Fig. 1)
Summary
Following activation by a variety of sensory stimuli, such as hormones, neurotransmitters, or light, G-protein-coupled receptors (GPCRs) are deactivated by multiple phosphorylations and subsequent binding of a regulatory protein arrestin [1, 2]. 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. NMR studies using model peptides with various numbers of phosphates [12] and full protein [13] showed little ordering of this region of rhodopsin upon phosphorylation. We posed the question whether the phosphorylated C terminus of rhodopsin is passive bait for arrestin or if it is directly involved in shaping the cytoplasmic surface of the receptor into a conformation associated with termination of a signaling state. We used high resolution proton NMR to study the dynamics of the model synthetic peptide, representing the fully phosphorylated region of rhodopsin Rh-(330 –348), 7PP, in solution and in an arrestinbound state. The bars represent average data and S.E. from five independent experiments
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