Conformational Changes in β‐arrestin1: The Importance of β‐arrestin1’s N‐domain

Abstract

β-arrestins are multifunctional adaptor proteins that regulate seven transmembrane spanning receptor (7TMR) desensitization, internalization and have been shown to initiate alternate signaling pathways. Studies have shown that arrestins undergo a conformational change upon binding activated, phosphorylated 7TMRs. Accordingly, we have determined conformational changes in β-arrestin1 using limited tryptic proteolysis and MALDI-TOF MS in the presence of a phosphopeptide from the the human V2 vasopressin receptor (V2Rpp) or an unphosphorylated peptide (V2Rnp). V2Rpp binds specifically to β-arrestin1 causing significant conformational changes whereas V2Rnp does not alter the conformation. Activation of β-arrestin1 is demonstrated by the release of its C-terminus and disruption of its polar core, indicated by increased accessibility of R393 and R285, respectively. We further demonstrate a functional consequence of β-arrestin1 activation by enhanced clathrin binding. A striking difference in the conformational changes observed in β-arrestin1 when compared to β-arrestin2 is the marked protection of the N-domain. The data are consistent with previous models for arrestin activation and support the idea that the N-domain is responsible for recognizing phosphates in 7TMRs. This study represents the first direct evidence that the “receptor-bound” conformations of β-arrestins1 and 2 are different.