The Rhodopsin-Arrestin-1 Interaction in Bicelles

G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G-protein-mediated signaling. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Under physiological conditions, the rhodopsin-arrestin-1 interaction occurs in highly specialized disk membrane in which rhodopsin resides. This membrane is replaced with mimetics when working with purified proteins. While detergents are commonly used as membrane mimetics, most detergents denature arrestin-1, preventing biochemical studies of this interaction. In contrast, bicelles provide a suitable alternative medium. An advantage of bicelles is that they contain lipids, which have been shown to be necessary for normal rhodopsin-arrestin-1 interaction. Here we describe how to reconstitute rhodopsin into bicelles, and how bicelle properties affect the rhodopsin-arrestin-1 interaction

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Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide

The functions of G-protein coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein coupled receptor kinases (GRKs), and the arrestins(1). G proteins mediate activation of second messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors(2), and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization(3). Arrestins activated by interaction with phosphorylated receptors can also mediate G protein-independent signaling by serving as adaptors to link receptors to numerous signaling pathways(4). Despite their central role in regulation and signaling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here, we report the crystal structure of β-arrestin1 in complex with a fully phosphorylated 29 amino acid carboxy-terminal peptide derived from the V(2) vasopressin receptor (V(2)Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin1(5). To capture this active conformation, we utilized a conformationally-selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin1. The structure of the β-arrestin1:V(2)Rpp:Fab30 complex shows striking conformational differences in β-arrestin1 compared to its inactive conformation. These include rotation of the amino and carboxy-terminal domains relative to each other, and a major reorientation of the “lariat loop” implicated in maintaining the inactive state of β-arrestin1. These results reveal, for the first time at high resolution, a receptor-interacting interface on β-arrestin, and they suggest a potentially general molecular mechanism for activation of these multifunctional signaling and regulatory proteins