Crystal Structure of Arrestin-3 Reveals the Basis of the Difference in Receptor Binding Between Two Non-visual Subtypes

Abstract

Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. Here we report the first crystal structure of arrestin-3, solved at 3.0Å. Arrestin-3 is an elongated two-domain molecule with the overall fold and key inter-domain interactions that hold free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and -3 we show that the presence of this loose structure correlates with reduced arrestin selectivity for activated receptor, consistent with a conformational change in this β-sheet upon receptor binding.

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

“…7A). Arrestin-3, because of structural peculiarities of the receptor-binding surface unique for this subtype (65), is the most promiscuous of the two nonvisual arrestins, demonstrating the least pronounced selectivity for active receptor over inactive and for phosphorylated over nonphosphorylated forms (60 -62, 65). Overexpression of GRK2 does not enhance the recruitment of arrestin-3 to ␤2AR regardless of activation (Fig.…”

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

“…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.…”

“…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.…”

“…In contrast, ubiquitously expressed arrestin-2 and -3 (5) apparently interact with the widely varied binding interfaces offered by other GPCRs. Crystal structures of arrestin-1 (16), -2 (17), -3 (18), and -4 (19) reveal a highly conserved characteristic two-domain fold shared by very few other proteins (20). In the basal state, the C-tail folds back onto the N-domain, forming a three-element interaction with ␤-strand I and ␣-helix I mediated by hydro-phobic side chains (21).…”

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

“…The four mammalian arrestin subtypes have greater than 50% amino acid conservation and a similar elongated two-domain structure in the basal state (4,6,14,(21)(22)(23)(24). The two conserved domains, the N-domain and C-domain, are connected through a hinge region (Fig.…”