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

Abstract

Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N-and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to ␤2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements.

Role of Receptor-attached Phosphates in Binding of Visual and Non-visual Arrestins to G Protein-coupled Receptors

“…Translated radiolabeled WT arrestins and K2A mutants (50 fmol) were incubated with 0.3 g of P-Rh* in 50 l in room light at 37°C for 5 min. The samples were cooled on ice, and bound arrestins were separated from free by gel filtration on 2-ml Sepharose 2B-CL columns as described (43). Bound arrestins eluted with rhodopsin-containing membranes were quantified by scintillation counting.…”

“…BRET measurements were described previously (18,43). The appropriate agonists (10 M carbamylcholine for M2R, 10 M isoproterenol for ␤ 2 AR, or 10 M quinpirole for D2R; 25 min at 37°C) were added prior to the addition of 5 M coelenterazine-h.…”

“…A. Javitch, Columbia University, New York, NY) were engineered using pGEM2-based constructs. Venus was amplified by PCR using a forward primer that adds EcoRI and AsiSI sites upstream of the start codon and a reverse primer that codes for a short spacer with the "SGLKSRRALDS" sequence and an inframe NcoI site as described previously (18,43). Venus was subcloned between the EcoRI and NcoI restriction sites.…”

“…In Vitro Transcription, Translation, Rhodopsin Preparation, and P-Rh* Binding Assay-In vitro transcription, translation, rhodopsin preparation, and P-Rh* binding assay were performed as described recently (43).…”

Conformation of receptor-bound visual arrestin

“…Interestingly, it contains four conformationally distinct monomers (chains A-D) showing the natural plasticity of the arrestin-1 molecule. For example, the "finger loop" (residues 67-79), which is highly conserved in the arrestin family (29) and has been identified as a key receptor-binding element in two subtypes (11,30), has two distinct conformations in the crystal structure, bent (chain D) and extended (chain A) (Fig. 3A).…”