Mechanism Insight Into Conformational Change Of ?-arrestin-1 By Genetic Code Expansion And ¹⁹F-NMR

G-protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular signalling cascades primarily through G proteins or arrestin-mediated pathways. G proteins transduce signals by regulating the levels of second messengers,whereas arestins recruit distinct downstream proteins to either desensitize receptors or initiate their own signalling pathways. Recently, significant conformational changes in arrestin have been observed following specific phosphopeptide binding or the formation of a receptor/arrestin complex. For example,the crystal structure of the V2-vasopressin receptor carboxy-terminal—phosphopeptide (V2R-phosphopeptide(V2Rpp))/?-arrestin-1 complex revealed that the binding of V2Rpp induced the rotation of the amino domain of ?-arrestin-1 with respect to its C-terminal domain. In another study, data obtained by electron microscopy (EM) and hydrogen-deuterium exchange mass spectrometry studies revealed increased dynamics in both the N- and C-terminal domains of ?-arrestin-1 after ?2-adrenergic receptor (?2AR)/?-arrestin-1 complex formation. These results suggest that the structural plasticity of ?-arrestin underlies their important cellular functions.However,the precise mechanism by which arrestin conformation is determined based on the receptor binding remains uncertain. How are these conformational rearrangements of arrestin coupled to diverse and precise receptor signalling controls?Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific ?-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface,?-arrestin-1 'reads'the message in the receptor phospho-C-tails and distinct phospho interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops,different phospho-interaction patterns induce distinct structural states of ?-arrestin-1 that are coupled to distinct arrestin functions. Whereas GRK2-phosphopeptides(GRK2pps) interact with ?-arrestin-1 within a phospho-binding sequence of the 1-4-6-7 pattern and promote clathrin recruitment, the GRK6-phosphopeptide(GRK6pp) interacts with ?-arrestin-1 through a different 1-5 sequence and provides the SRC signalling order. The revealed ability of arrestin to read and translate various phosphorylation patterns through its phosphate-binding concave surface contributes to their diverse functions.The phosphate-binding sites along the N terminus of ?-arrestin-1 are arranged in a shape that is similar to the holes in a flute and arrestin moves differently according to the instructions of the phospho-receptor 'fingers'. Whereas a receptor-encoded 1-4-6-7 pattern determines the specific ?-arrestin-1 conformation for clathrin recruitment, a different receptor 'tune' 1-5, provides the SRC signalling order. The combination of all phosphate-binding sites in a single arrestin allows the expression of more than 1,000 patterns (210-1=1,023) that allow it, in theory, to produce a plethora of arrestin conformations,facilitating numerous downstream protein interactions.