Crystal Structure of β-Arrestin at 1.9 Å Possible Mechanism of Receptor Binding and Membrane Translocation

AbstractBackground: Arrestins are responsible for the desensitization of many sequence-divergent G protein-coupled receptors. They compete with G proteins for binding to activated phosphorylated receptors, initiate receptor internalization, and activate additional signaling pathways.Results: In order to understand the structural basis for receptor binding and arrestin's function as an adaptor molecule, we determined the X-ray crystal structure of two truncated forms of bovine β-arrestin in its cytosolic inactive state to 1.9 Å. Mutational analysis and chimera studies identify the regions in β-arrestin responsible for receptor binding specificity. β-arrestin demonstrates high structural homology with the previously solved visual arrestin. All key structural elements responsible for arrestin's mechanism of activation are conserved.Conclusions: Based on structural analysis and mutagenesis data, we propose a previously unappreciated part in β-arrestin's mode of action by which a cationic amphipathic helix may function as a reversible membrane anchor. This novel activation mechanism would facilitate the formation of a high-affinity complex between β-arrestin and an activated receptor regardless of its specific subtype. Like the interaction between β-arrestin's polar core and the phosphorylated receptor, such a general activation mechanism would contribute to β-arrestin's versatility as a regulator of many receptors

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/1/embr201344.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/2/embr201344.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/3/embr201344-sup-0001.pd

Enhanced Mutant Compensates for Defects in Rhodopsin Phosphorylation in the Presence of Endogenous Arrestin-1

We determined the effects of different expression levels of arrestin-1-3A mutant with enhanced binding to light-activated rhodopsin that is independent of phosphorylation. To this end, transgenic mice that express mutant rhodopsin with zero, one, or two phosphorylation sites, instead of six in the WT mouse rhodopsin, and normal complement of WT arrestin-1, were bred with mice expressing enhanced phosphorylation-independent arrestin-1-3A mutant. The resulting lines were characterized by retinal histology (thickness of the outer nuclear layer, reflecting the number of rod photoreceptors, and the length of the outer segments, which reflects rod health), as well as single- and double-flash ERG to determine the functionality of rods and the rate of photoresponse recovery. The effect of co-expression of enhanced arrestin-1-3A mutant with WT arrestin-1 in these lines depended on its level: higher (240% of WT) expression reduced the thickness of ONL and the length of OS, whereas lower (50% of WT) expression was harmless in the retinas expressing rhodopsin with zero or one phosphorylation site, and improved photoreceptor morphology in animals expressing rhodopsin with two phosphorylation sites. Neither expression level increased the amplitude of the a- and b-wave of the photoresponse in any of the lines. However, high expression of enhanced arrestin-1-3A mutant facilitated photoresponse recovery 2-3-fold, whereas lower level was ineffective. Thus, in the presence of normal complement of WT arrestin-1 only supra-physiological expression of enhanced mutant is sufficient to compensate for the defects of rhodopsin phosphorylation

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were(13)C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring(13)C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp281(6.48)of the highly conserved SWLP motif and Trp327(7.55)adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp116(23.50)was identified

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Structural Basis of Arrestin Selectivity for Active Phosphorylated G Protein-Coupled Receptors

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs