Role of sorting nexin adaptor proteins in GPCR regulation

Abstract

Signaling by the G protein-coupled receptor CXCR4 is essential to a number of developmental and physiological processes, including stem cell mobilization, organ development, and immune responses. In addition, CXCR4 signaling has been linked to disease, including cancer and certain immune disorders. Despite this, the mechanisms governing CXCR4 signaling remain poorly defined. Typically, upon binding to its cognate ligand CXCL12, CXCR4 signaling occurs via G protein- and β-arrestin-dependent pathways. Although G protein-dependent CXCR4-mediated signaling is well understood, β-arrestin1-dependent signaling remains poorly understood. With this in mind, we performed proximity biotin labeling by ascorbic acid peroxidase (APEX2) fused to β-arrestin1 in order to identify proteins that are part of the β-arrestin1 interaction network downstream of CXCR4. Labeled proteins from cells treated with vehicle or CXCL12 for 5 or 60 minutes were selectively purified and identified by mass spectrometry. Several proteins known to be part of the b-arrestin network downstream of CXCR4 including STAM1 and FAK were identified. Several other proteins of biological relevance were identified that were enriched in cells treated with CXCL12 for 5 or 60 min. Orthogonal biochemical experiments verified that two of these proteins, WAVE2 and SNX9, interact with β-arrestin1. We selected SNX9 for further study of its functional role in CXCR4 signaling. SNX9 (sorting nexin 9), and related proteins SNX18 and SNX33, are multifunctional adaptor proteins best known for their role in clathrin-mediated endocytosis of multiple cell surface receptors. Whether they mediate endocytosis of GPCRs remains unknown. To address this, we transfected HEK293 cells stably expressing epitope-tagged CXCR4 with siRNA against SNX9, SNX18, and SNX33, and examined agonist-induced endocytosis of CXCR4 by whole cell ELISA. Simultaneous transfection with siRNA against SNX9 and SNX18 significantly reduced CXCR4 endocytosis when compared with control siRNA or when cells were transfected with individual siRNA against SNX9, SNX18 or SNX33. These data provide evidence for the first time for a role of members of the SNX9-family in GPCR endocytosis, likely by serving as adaptors for clathrin-mediated endocytosis. Additional work is needed to conclusively demonstrate that SNX9 functions as an adaptor protein for CXCR4 endocytosis and to determine the mechanism by which this occurs. To accomplish this we will use a combination of biochemical, molecular, imaging and biophysical approaches. In addition, we will investigate how broadly SNX9-family members impact CXCR4 endocytosis across multiple cell types and also address the impact of the SNX9-family on GPCR endocytosis in general.