The Active Conformation of β-Arrestin1 DIRECT EVIDENCE FOR THE PHOSPHATE SENSOR IN THE N-DOMAIN AND CONFORMATIONAL DIFFERENCES IN THE ACTIVE STATES OF β-ARRESTINS1 AND -2

Abstract β-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that β-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and β-arrestin2, these studies are not representative of conformational changes in β-arrestin1. Accordingly, in this study, we determine conformational changes in β-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V2 vasopressin receptor (V2Rpp) or the corresponding unphosphorylated peptide (V2Rnp). V2Rpp binds specifically to β-arrestin1 causing significant conformational changes, whereas V2Rnp does not alter the conformation of β-arrestin1. Upon V2Rpp binding, we show that the previously shielded Arg393 becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg285 becomes more accessible, and this residue is located in a region of β-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate “activation” of β-arrestin1, and we also show a functional consequence of the release of the C terminus of β-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of β-arrestin1 in the presence of V2Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in β-arrestin1 when compared with β-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the “receptor-bound” conformations of β-arrestins1 and 2 are different.