Identifying β‐arrestin1 conformational states by a non‐GPCR binding partner
Authors: Zhuo Ya , Klug Candice , Marchese Adriano 
Published: April 2020

The G protein‐coupled receptor (GPCR) C‐X‐C motif chemokine receptor 4 (CXCR4), and its cognate chemokine CXCL12, play essential roles in hematopoiesis, cardiogenesis, neurogenesis and immune responses. In addition, CXCR4 signaling contributes to several aspects of cancer progression and other diseases. Despite its importance CXCR4 signaling remains poorly understood. Because of the critical involvement in human cancers, a comprehensive understanding of CXCR4 signaling is of great therapeutic significance. The present study is focused on a novel CXCR4 signaling pathway thatregulates chemotaxis via the adaptor protein β‐arrestin1 (βarr1). β‐arrestins typically bind to activated GPCRs to negatively regulate signaling by promoting desensitization and internalization, but β‐arrestins also positively promote signaling by interacting with non‐GPCR binding partners. It is relatively well known how β‐arrestins interact with activated GPCRs to promote their internalization and desensitization, however very little is known how β‐arrestins interact with non‐GPCR binding partners to initiate signaling. Previously, our lab showed that βarr1 interacts directly with the adaptor protein STAM1 (signal transducing adaptor molecule) to promote CXCR4‐dependent activation of focal adhesion kinase (FAK), however the mechanism remains unknown. Here, we tested the hypothesis that STAM1 induces a unique conformational signature in βarr1 that facilitates activation of FAK. To test this, we employed Site‐Directed Spin Labeling (SDSL) Electron Paramagnetic Resonance (EPR) spectroscopy. We employed double electron‐electron resonance (DEER) spectroscopy to determine if STAM1 induces a unique conformational signature on βarr1 by incorporating spin labels on selected double cysteine mutants of βarr1 and measuring distances between spin labeled residues upon STAM1 or GPCR binding. STAM1 binding induced similar conformational changes on βarr1 in one double cysteine labeled mutant, but not on two others, as compared to a GPCR. These data suggest that βarr1 undergoes a unique conformational change upon STAM1 binding, in contrast to the well‐established conformational change that occurs upon GPCR binding. To determine if this could be explained by the fact that STAM1 and GPCRs bind to different sites on βarr1 we employed CW(continuous wave)‐EPR spectroscopy to map the STAM1 binding surface on βarr1 by monitoring spectral line‐shape changes of selected reporter sites in βarr1 in the presence or absence of STAM1. Spin labels attached to selected sites on the front surface of βarr1 did not show any side chain immobilization when bound to STAM1, suggesting that STAM1 does not bind to the front surface. In contrast, we identified two residues located on the back surface of βarr1 that showed significant spin label immobilization when bound to STAM1, suggesting STAM1 binds to this surface of βarr1. As GPCRs and other non‐GPCR binding partners typically bind to the front surface of β‐arrestins, our data suggest that STAM1 binds to the back surface on βarr1, which is likely linked to discrete βarr1 conformational dynamics. We propose that STAM1 induces a unique βarr1 conformational signature, which is required for CXCR4‐dependent activation of FAK and chemotaxis.

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GM106727 06A1