Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2.

Arrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

Molecular basis of ß-arrestin coupling to formoterol-bound ß1-adrenoceptor.

The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.