Dynamics of Arrestin-Rhodopsin Interactions

Abstract

In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.Rhodopsin activity is controlled by a multistep process whereby activated rhodopsin is phosphorylated on its C terminus by rhodopsin kinase, leading to the binding and quenching of rhodopsin activity by visual arrestin. Visual arrestin belongs to the arrestin superfamily, which includes rod and cone arrestins as well the ␤-arrestins (1). The ␤-arrestins, which are involved in the inactivation of agonist-occupied G-proteincoupled receptors (for review, see Ref. 2), have an additional role not seen in visual arrestins whereby they direct receptor endocytosis by acting as a clathrin or AP-2 adaptor; e.g. Refs. 3 and 4. Visual arrestin is an important model system not only for understanding the visual response but also for understanding the broadreaching mechanism for controlling cellular signal transduction cascades mediated by G protein-coupled receptors.The crystal structures of rod, cone, and ␤-1 arrestin are remarkably similar. All show a structure consisting of two cupshaped domains formed from a seven-stranded ␤ sandwich (5-7). These structures, of the unbound state, have provided an important framework on which to base predictions for the binding interaction between arrestin and light-activated phosphorylated rhodopsin (Rho*-P). 4 Interestingly, in the crystal structures of visual arrestin, Loop V-VI (residues 68 -78 in bovine visual arrestin (7)) is seen in two primary conformers, one in which Loop V-VI is extended away from the main bulk of the protein (Fig. 1, ␣ conformer) and one with the loop folded into the N-terminal domain of the protein (␤ conformer). Several groups have identified that Loop V-VI has an important role in receptor binding. Experimental evidence shows (i) this loop is a vital component of arrestin that directs receptor preference (8), (ii) insertion of a peptide into this loop can either directly block or be used to competitively block binding to Rho*-P (9, 10), (iii) a fluorescent label at residue 72 in this loop apparently buries itself into the rhodopsin interface upon binding (10, 11), and (iv) spin labels in this loop have reduced ...

Structural Determinants of Arrestin Functions

“…73,86,109 In addition, the movement and/or structural rearrangement of the “finger loop” in the central crest of the receptor-binding side was also reported. 83,131 Both polar core and the three-element interaction clearly support the basal conformation in all arrestin structures, 18–20,22 so their destabilization by the phosphates was consistent with the idea that global rearrangement is necessary for receptor binding. Partial destabilization of the interface between the two domains enhanced arrestin binding to inactive receptor, 132 again suggesting that arrestin conformation must change upon receptor binding.…”

“…18,19 Multiple residues in this loop were shown to be immobilized upon receptor binding in both arrestin-1 73 and -2. 71 Previous studies using fluorescent labels 131 and NMR 83 suggested that this loop extends and forms an α-helix upon receptor binding. Indeed, this loop was found to move in the direction of the receptor, although not as much as previously proposed, 131 and the data were consistent with its helical conformation in receptor-associated arrestin-1.…”

“…71 Previous studies using fluorescent labels 131 and NMR 83 suggested that this loop extends and forms an α-helix upon receptor binding. Indeed, this loop was found to move in the direction of the receptor, although not as much as previously proposed, 131 and the data were consistent with its helical conformation in receptor-associated arrestin-1. 72 However, hypothetical movement of the two arrestin domains relative to each other, which was proposed to improve the fit between arrestins and GPCRs, 30 turned out to be very small, clearly insufficient to significantly reduce the size of the receptor-binding arrestin surface.…”

Arrestin-Rhodopsin Binding Stoichiometry in Isolated Rod Outer Segment Membranes Depends on the Percentage of Activated Receptors

“…"Cysless" arrestin has been previously shown to retain wild-type function (32,33), and we verified that our mutant construct was functionally identical to native arrestin by centrifugal pulldown, light scattering, and extra Meta II analyses (data not shown). Single cysteine substitutions I72C, S344C, and A366C (see Fig.…”

“…Labeling of these introduced cysteine residues with IANBD was performed as described previously for bimane (31). The concentration and labeling efficiency were determined using molar extinction coefficients of 0.025 M Ϫ1 cm Ϫ1 at 500 nm for IANBD (as reported by the manufacturer) and 0.02076 M Ϫ1 cm Ϫ1 at 278 nm for arrestin (33). When bound to the protein, IANBD was found to contribute no absorbance at 278 nm, and all mutants were labeled at near 100% efficiency.…”