The Differential Engagement of Arrestin Surface Charges by the Various Functional Forms of the Receptor

Abstract

G-protein-coupled receptor signaling is terminated by arrestin proteins that preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, we performed an extensive analysis of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicated that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. We identified several additional phosphate-binding elements in the N-domain and demonstrated for the first time that the active receptor preferentially engages the arrestin C-domain. We also found that the interdomain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high affinity receptor binding state.Arrestins regulate G-protein-coupled receptor signaling by binding to the activated phosphorylated receptor with high affinity to prevent its further interaction with G proteins. Receptor-bound arrestin can serve as an adaptor linking the complex to the internalization machinery of the coated pit through its binding to clathrin and AP2 (1) and as a scaffold through the formation of multiprotein signaling complexes with mitogen-activated protein (MAP) kinases and other partners (2). The fact that several of these proteins preferentially bind to the "active" receptor-bound form of arrestin (3) suggests that arrestin undergoes a significant conformation rearrangement upon receptor binding. This was initially proposed based on the observation that arrestin-receptor binding has an unusually large activation energy (4) and also by an increase of the sensitivity of receptor-bound arrestin to proteolysis (5). The crystal structure of arrestin (6) and numerous mutagenesis studies (7-10) support the idea that arrestin undergoes a significant conformational rearrangement upon binding. The molecular mechanism that governs arrestin-receptor interaction was first elucidated on the visual arrestin-rhodopsin model. Visual arrestin binds light-activated phosphorylated rhodopsin (P-Rh*) 3 with remarkable selectivity, whereas its binding to an equal amount of dark (inactive) phosphorhodopsin (P-Rh) or light-activated unphosphorylated rhodopsin (Rh*) is 10 -20 times lower (7). A model of sequential multisite binding was proposed to explain the selectivity of arrestin for P-Rh*. This model posits that arrestin has two "sensor" sites, detecting...

Opposing Effects of Inositol Hexakisphosphate on Rod Arrestin and Arrestin2 Self-Association

“…GPCRs (46)(47)(48), microtubules (14,15), and calmodulin (13) all bind to the same arrestin surface, suggesting that these partners could compete with IP6. The affinity of arrestins for their cognate receptors is subnanomolar (49,50), so that IP6 even at 100 μM does not significantly inhibit receptor binding (16,40).…”

Few Residues within an Extensive Binding Interface Drive Receptor Interaction and Determine the Specificity of Arrestin Proteins

“…Several positively charged residues interact with multiple receptor-attached phosphates (23,31,(57)(58)(59)(60). A least 10 exposed residues mediate receptor subtype-specific binding, contributing to receptor discrimination (Figs.…”

“…We used light-activated phosphorylated rhodopsin (P-Rh*) as a model receptor because both non-visual arrestins were shown to bind it specifically in an activation-and phosphorylation-dependent fashion (14 -16, 47). To determine the functional consequences of mutations and labeling of arrestin-2, we incubated purified spin-labeled arrestin-2 (25 M) with 250 M P-Rh* for 5 min at 37°C (23,51). Arrestin-2 bound to rhodopsin-containing membranes was pelleted by centrifugation.…”

“…The precise structural basis of receptor specificity of arrestin proteins remains to be elucidated. Recent studies of arrestin-1 identified an extensive receptor binding surface covering virtually all of the concave sides of both arrestin domains (22)(23)(24)(25)). Here we demonstrate that a similarly extensive surface of the most abundant non-visual subtype, arrestin-2, is involved in receptor binding and identify surprisingly few residues that play key role in receptor interaction and GPCR preference.…”

“…Mutations were introduced by PCR, as described (23). All mutant constructs were confirmed by dideoxy sequencing.…”