Abstract
β-Arrestins are key regulation proteins for G protein-coupled receptors (GPCRs) signaling. Experimental evidence suggests that β-arrestins undergo conformational changes concomitant with binding to activated, phosphorylated GPCRs. We developed a mass spectrometry-based structural proteomic assay to monitor conformational changes associated with the activation of β-arrestins. This assay utilizes synthesized phosphopeptides mimicking phosphorylated C-terminal tails of GPCRs to activate β-arrestins. The activation-dependent conformational changes of β-arrestins are revealed using limited proteolysis coupled with both SDS-PAGE and mass spectrometry analysis. As an in vitro β-arrestin activation assay, this mass spectrometry-based structural method can be adapted as a simple but useful tool to study the nature and extent of conformational changes of β-arrestins downstream of different receptors as well as β-arrestin conformations associated with different functions, such as desensitization, internalization, and signaling.
Published Version
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