Identifying common G protein-coupled receptor interacting proteins independent of heterotrimeric G proteins and β-arrestins

The canonical paradigm of G protein-coupled receptor (GPCR) signaling recognizes G proteins, β-arrestins, and GPCR kinases as the primary transducers that shape GPCR signaling. However, multiple studies have demonstrated that other interacting proteins regulate GPCR function in addition to these pathways, termed GPCR-interacting proteins (GIPs). The atypical chemokine receptor 3 (ACKR3) does not couple to G proteins, and β-arrestins have been shown to be dispensable for many of its functions. Here, we employed proximity labeling to identify proteins that interact with ACKR3 in cells devoid of β-arrestins. Among conserved proteins from our and several similar GPCR-based proximity labeling experiments, we discovered components of the endocytic machinery and other putative interacting proteins that can interact with GPCRs independent of G proteins and β-arrestins. Notably, we discovered that the bone morphogenetic protein 2-inducible kinase (BMP2K) interacts with ACKR3 in a phosphorylation-dependent manner that is distinct from its interaction with β-arrestin. We demonstrate BMP2K's ability to interact with many different GPCRs with varying dependency on β-arrestin. Using molecular dynamic simulations, we discovered an asparagine-proline-phenylephrine (NPF)-binding motif in BMP2K critical for its interaction with GPCRs and determined that this motif is conserved across many other GIPs. These findings reveal an expanded network of GIPs that interact with GPCRs independent of G proteins and β-arrestins, highlighting alternative mechanisms by which GPCR signaling may be transduced beyond canonical paradigms.