Functional map of arrestin binding to phosphorylated opsin, with and without agonist

Abstract

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

C-edge loops of arrestin function as a membrane anchor

“…This mode of binding was captured in our MD simulations using an isolated C-domain of pre-active p44, and we anticipate that this interaction significantly stabilizes the high-affinity complex. Note that the different membrane anchor orientations for the pre-complex and high-affinity complex, which we predict based on our fluorescence data, is corroborated by a recent alanine scan mutagenesis study of arrestin-1 binding to inactive and active phosphorylated rhodopsin 41 .…”

Molecular Mechanisms of GPCR Signaling: A Structural Perspective

“…Comprehensive mutagenesis of every residue in the arrestin-1 revealed numerous elements affecting receptor binding, as well as an unexpected fact that there are arrestin-1 residues that differentially affect the binding to phosphorylated opsin that does or does not have bound agonist, all-trans-retinal [ 84 ], supporting the idea that receptor-bound arrestin can be in different “poses” [ 70 ] and conformations [ 61 ]. Mapping of the receptor footprint on arrestin using site-directed spin labeling/EPR [ 58 , 79 ], chimeras between rhodopsin-specific arrestin-1 and more promiscuous arrestin-2 [ 85 ], and site-directed mutagenesis [ 79 ] revealed that receptors engage a fairly large part of the concave sides of the two arrestin domains.…”

Arrestins: Introducing Signaling Bias Into Multifunctional Proteins

“…3,44 Experimental swapping of various elements 44 and individual residues 45 between arrestin-1 and nonvisual arrestin-2 identified a limited set of side chains that determine receptor preference. Mutagenesis of every residue in arrestin-1 46,47 coupled with analysis of the crystal structure of the arrestin-1 complex with rhodopsin 48,49 (Fig. 2) revealed several additional residues that play important roles in GPCR binding and/or directly contact the receptor, and therefore might contribute to receptor specificity.…”

“…Chimera construction, 38,44,89 site-directed mutagenesis, 45–47,50–52,90 peptide interaction studies, 91 EPR, 21,92 NMR, 93 and X-ray crystallography 48,49,56,94 all suggest that the receptors engage the concave sides of both arrestin domains (Figs. 1 and 2).…”