Each rhodopsin molecule binds its own arrestin

Abstract

Arrestins (Arrs) are ubiquitous regulators of the most numerous family of signaling proteins, G protein-coupled receptors. Two models of the Arr-receptor interaction have been proposed: the binding of one Arr to an individual receptor or to two receptors in a dimer. To determine the binding stoichiometry in vivo, we used rod photoreceptors where rhodopsin (Rh) and Arr are expressed at comparably high levels and where Arr localization in the light is determined by its binding to activated Rh. Genetic manipulation of the expression of both proteins shows that the maximum amount of Arr that moves to the Rh-containing compartment exceeds 80%, but not 100%, of the molar amount of Rh present. In vitro experiments with purified proteins confirm that Arr "saturates" Rh at a 1:1 ratio. Thus, a single Rh molecule is necessary and sufficient to bind Arr. Remarkable structural conservation among receptors and Arrs strongly suggests that all Arr subtypes bind individual molecules of their cognate receptors.

Fig. 1.

The extent of light-dependent Arr translocation is determined by the Arr/Rh expression ratio. (A) Mice with the indicated genotypes were dark-adapted overnight (DARK) or exposed to 2,700 lux for 1 h (LIGHT). One eyecup was fixed and processed for Arr immunohistochemistry (9). The positions of the outer (OS) and inner segments (IS) and of the outer nuclear layer (ONL) are indicated. (B) The proportion of Arr localized in the OS was quantified by the intensity of Arr immunostaining (green) in 10 images per animal from 3–5 animals per genotype per light condition. Means ± SD are shown. The data for light- and dark-adapted mice were analyzed separately by one-way ANOVA with genotype as a main factor. Statistical significance of the differences is indicated above the corresponding bars: ∗, P < 0.001; ∗∗, P < 0.0001. (C) Typical Western blots for Arr and Rh (Rh) in mice with the indicated genotypes and corresponding standards.

Fig. 2.

Rh “saturation” with Arr is achieved at equimolar binding. (A) Purified bovine Arr (142, 286, 572, and 858 pmol) was incubated for 5 min in the light with (+P-Rh*) or without (−P-Rh*) 10 μg (261 pmol) of phosphoRh in 25 μl of 50 mM Mops-Na, pH 7.2, 100 mM NaCl. Rh with bound Arr was pelleted through a 50-μl cushion of the same buffer with 0.2 M sucrose. Equal aliquots of the sample before centrifugation (Load) (1/14), Pellet (1/4), and supernatant (Sup) (1/4) were subjected to SDS/PAGE. Arr was visualized by Coomassie blue staining. (B) The absolute amount of Arr and Rh in the pellet was measured by quantitative Western blot. Arr binding is plotted as a function of the Arr/Rh molar ratio in the sample. Means ± SD of two experiments are shown. The analysis of the binding data (by using GraphPad Prizm) yields Bmax (saturation) at 0.99 ± 0.08 mol/mol.