ACKR3 Proximity Labeling Identifies Novel G protein- and ß-arrestin-independent GPCR Interacting Proteins.

The canonical paradigm of GPCR signaling recognizes G proteins and ß-arrestins as the two primary transducers that promote GPCR signaling. Recent evidence suggests the atypical chemokine receptor 3 (ACKR3) does not couple to G proteins, and ß-arrestins are dispensable for some of its functions. Here, we employed proximity labeling to identify proteins that interact with ACKR3 in cells devoid of ß-arrestin. We identified proteins involved in the endocytic machinery and evaluated a subset of proteins conserved across several GPCR-based proximity labeling experiments. We discovered that the bone morphogenic protein 2-inducible kinase (BMP2K) interacts with many different GPCRs with varying dependency on ß-arrestin. Together, our work highlights the existence of modulators that can act independently of G proteins and ß-arrestins to regulate GPCR signaling and provides important evidence for other targets that may regulate GPCR signaling.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of ß-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.