Regulation of GPCR Signaling by Sorting Nexins

Abstract

<b>Abstract ID 27670</b> <b>Poster Board 529</b> G protein-coupled receptor (GPCR) signaling is primarily regulated by GPCR kinases (GRKs) and β-arrestins. GRKs phosphorylate ligand-activated GPCRs, which promotes β-arrestin recruitment and binding. β-arrestin binding prevents G protein coupling while also targeting the GPCR for internalization via clathrin-coated pits. However, in some cases, β-arrestins are recruited to the receptor and promote its desensitization but fail to target it for internalization. For example, we show that ligand-stimulated internalization of the GPCR CXCR4 remains intact in HEK293 or HeLa cells that have been deleted of β-arrestins by CRISPR/Cas9 gene editing, despite the fact that β-arrestins are required for desensitization of CXCR4 signaling. Here, we report on an endocytic remodeling protein as a novel ligand-stimulated adaptor protein that mediates β-arrestin-independent GPCR internalization. This protein is sorting nexin 9 (SNX9), a protein that we previously showed by proximity labeling to be in the CXCR4 signaling network. We show that depletion of SNX9 by RNAi or knockout by CRISPR/Cas9 gene editing along with depletion of related protein SNX18 significantly decreased ligand-stimulated internalization of CXCR4 in HEK293 cells. In contrast, SNX9 and SNX18 depletion did not impact the internalization of GPCRs that require β-arrestins for internalization. Using bioluminescence resonance energy transfer (BRET) based cellular assays we show that SNX9 is recruited to CXCR4 following ligand stimulation at the plasma membrane. Through a combination of <i>in&nbsp;vitro</i> and cellular biophysical assays, we show that SNX9 interacts directly with the C-tail of CXCR4 in a phosphorylation-dependent manner. The determinants of this interaction do not overlap with previously identified determinants of the CXCR4/β-arrestin interaction. Further, we show that SNX9 is functionally relevant to CXCR4 signaling as SNX9 and SNX18 deficient cells show reduced desensitization to ligand stimulation compared with control cells. Here, we define a new internalization pathway for ligand-activated GPCRs that is dependent on the membrane remodeling protein SNX9. We provide evidence for the first time that SNX9 acts as an adaptor protein by binding directly to a phosphorylated GPCR and targeting the receptor for internalization. This represents a previously undescribed function of SNX9, which we propose is independent of its previously described role as an accessory protein during constitutive internalization. Our study expands our understanding of the mechanisms regulating GPCR signaling. Support/Funding Information: R01 GM106727