The Role of Arrestin-1 Middle Loop in Rhodopsin Binding.

Arrestins preferentially bind active phosphorylated G protein-coupled receptors (GPCRs). The middle loop, highly conserved in all arrestin subtypes, is localized in the central crest on the GPCR-binding side. Upon receptor binding, it directly interacts with bound GPCR and demonstrates the largest movement of any arrestin element in the structures of the complexes. Comprehensive mutagenesis of the middle loop of rhodopsin-specific arrestin-1 suggests that it primarily serves as a suppressor of binding to non-preferred forms of the receptor. Several mutations in the middle loop increase the binding to unphosphorylated light-activated rhodopsin severalfold, which makes them candidates for improving enhanced phosphorylation-independent arrestins. The data also suggest that enhanced forms of arrestin do not bind GPCRs exactly like the wild-type protein. Thus, the structures of the arrestin-receptor complexes, in all of which different enhanced arrestin mutants and reengineered receptors were used, must be interpreted with caution.

Structural Basis of Arrestin Selectivity for Active Phosphorylated G Protein-Coupled Receptors.

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.

Lysine in the lariat loop of arrestins does not serve as phosphate sensor.

Arrestins demonstrate strong preference for phosphorylated over unphosphorylated receptors, but how arrestins "sense" receptor phosphorylation is unclear. A conserved lysine in the lariat loop of arrestins directly binds the phosphate in crystal structures of activated arrestin-1, -2, and -3. The lariat loop supplies two negative charges to the central polar core, which must be disrupted for arrestin activation and high-affinity receptor binding. Therefore, we hypothesized that receptor-attached phosphates pull the lariat loop via this lysine, thus removing the negative charges and destabilizing the polar core. We tested the role of this lysine by introducing charge elimination (Lys->Ala) and reversal (Lys->Glu) mutations in arrestin-1, -2, and -3. These mutations in arrestin-1 only moderately reduced phospho-rhodopsin binding and had no detectable effect on arrestin-2 and -3 binding to cognate non-visual receptors in cells. The mutations of Lys300 in bovine and homologous Lys301 in mouse arrestin-1 on the background of pre-activated mutants had variable effects on the binding to light-activated phosphorylated rhodopsin, while affecting the binding to unphosphorylated rhodopsin to a greater extent. Thus, conserved lysine in the lariat loop participates in receptor binding, but does not play a critical role in phosphate-induced arrestin activation.