The RAMP3 proximity interactor MYO6 determines spatiotemporal signaling of the GPCR CLR in lymphatic endothelial cells

Receptor activity–modifying protein 3 (RAMP3) heterodimerizes with the G protein–coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) to form the receptor for the prolymphangiogenic peptide adrenomedullin (AM). RAMP3 allosterically regulates CLR to coordinate signaling, internalization, and endosomal recycling after stimulation with AM. Here, we used proximity proteomics to identify components of the RAMP3 interactome that regulated CLR and characterized how the interactome changed in response to AM. We identified unconventional myosin VI (MYO6), an actin-based molecular motor, as a proximal RAMP3-CLR interactor. In primary human lymphatic endothelial cells, the knockdown of MYO6 inhibited the AM-stimulated activation of the G protein Gα s and the kinases ERK and AKT but enhanced cAMP accumulation, thus increasing the activity of the kinase PKA and the transcription factor CREB. Furthermore, MYO6 inhibition blocked AM-dependent RAMP3-CLR internalization and trafficking to Rab5-positive endosomes, suggesting that signaling endosomes in primary lymphatic endothelial cells inhibit cAMP generation. Last, knockdown of MYO6 affected AM-mediated adherens junction remodeling and migration. Thus, identifying components of the RAMP3-CLR interactome has revealed a spatiotemporal regulator of AM signaling in lymphatic endothelial cells.