RAMP3 determines rapid recycling of atypical

G protein-coupled receptors (GPCRs) exist in multiprotein complexes on the surface of cells to respond to a wide variety of extracellular stimuli such as neurotransmitters, migratory signals, hormones, light, and odors. In this study, we uncover and characterize an expanded repertoire of GPCRs that interact with receptor activity-modifying proteins (RAMPs) – a class of proteins that can modulate the type and consequences of extracellular signals on GPCRs.

Specifically, we find that RAMP interaction with chemokine GPCRs is essential for enabling these receptors to bind and degrade extracellular migration signals and thus establish gradients for directed cell migration. In the absence of these essential proteins, the sprouting process of blood vessels in the postnatal retina is dysfunctional.

Receptor activity modifying proteins (RAMPs) are unique transmembrane proteins that serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have previously been studied in the context of their effects on B-family GPCRs, the co-evolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions.



Using approaches based on bioluminescence resonance energy transfer and cell surface expression, we comprehensively screen RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and almost all chemokine receptors. Specifically, we identify a robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration.

Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) decreases adrenomedullin (AM) ligand availability without altering G-protein coupling. Instead, RAMP3 is required for rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles after AM or SDF-1/CXCL12 binding, thereby allowing the formation of dynamic spatio-temporal chemotactic gradients.

Therefore, genetic deletion of ACKR3 or RAMP3 in mice abolishes the directed cell migration of retinal angiogenesis. Thus, the RAMP association with members of the chemokine receptor family represents a molecular interaction to control the signaling and trafficking properties of the receptors.