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.

The finger loop as an activation sensor in arrestin.

The finger loop in the central crest of the receptor-binding site of arrestins engages the cavity between the transmembrane helices of activated G-protein-coupled receptors. Therefore, it was hypothesized to serve as the sensor that detects the activation state of the receptor. We performed comprehensive mutagenesis of the finger loop in bovine visual arrestin-1, generated mutant radiolabeled proteins by cell-free translation, and determined the effects of mutations on the in vitro binding of arrestin-1 to purified phosphorylated light-activated rhodopsin. This interaction is driven by two factors, rhodopsin activation and rhodopsin-attached phosphates. Therefore, the binding of arrestin-1 to light-activated unphosphorylated rhodopsin is low. To evaluate the role of the finger loop specifically in the recognition of the active receptor conformation, we tested the effects of these mutations in the context of truncated arrestin-1 that demonstrates much higher binding to unphosphorylated activated and phosphorylated inactive rhodopsin. The majority of finger loop residues proved important for arrestin-1 binding to light-activated rhodopsin, with six mutations affecting the binding exclusively to this form. Thus, the finger loop is the key element of arrestin-1 activation sensor. The data also suggest that arrestin-1 and its enhanced mutant bind various functional forms of rhodopsin differently.